History of DNA Sequencing and Research

DNA sequencing technology has evolved very rapidly since its inception in the 1970s, and continues to evolve and grow today. This paper will review the major innovations and developments in sequencing technology and briefly summarize their methodologies.
The first group that was able to sequence DNA was the team of Allan Maxam and Walter Gilbert (Maxam and Gilbert). This was a first generation sequencing reaction, and was developed in 1976-1977. This method uses purified DNA and relies on chemical modification of DNA bases (like depurination of adenine and guanine using formic acid and methylation using hydrazine or dimethyl sulfate). The 5′ end is radioactively labeled so that it can be visualized in a gel, and then fragments of modified DNA are electrophoresed. Autoradiography can then be used to visualize the sizes of each DNA fragment. The maximum read length for this technique was approximately 100 bases long.

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The next major innovation in DNA sequencing was the Sanger dideoxy chain termination method. This was developed in 1977 by Frederick Sanger (Sanger, Nicklen, and Coulson), and became much more popular than Maxam and Gilbert’s method. Sanger sequencing is a synthesis reaction and uses dideoxy nucleotides to randomly terminate synthesized strands of DNA. The DNA strands that had been terminated with ddNTPs originally were run in 4 different lanes (one for each ddNTP) and were radiolabeled so that they could be visualized with autoradiography. Later innovations made Sanger sequencing even easier when each dideoxynucleotide was labeled with different fluorescent dyes. As such, sequences could be run on a single gel in a single lane. This method was the most popular way of sequencing DNA for many years, and was prevalent until about 2004. While read length was initially about 100 base pairs long, Sanger sequencing now has a read length of about 800 to 1000 base pairs long when run in capillary gels.
With the start of the human genome project, it was necessary to find ways to sequence DNA much more quickly and more cost-effectively than had been done previously. This led to the development of so-called “second generationâ€Â DNA sequencers. It also allowed for the use of smaller samples for sequencing.
One of the first major automated platforms was the Roche 454 (Margulies et al.). This utilizes pyrosequencing, which is a synthesis type sequencing reaction. This also uses emulsion PCR on beads. When a dNTP is incorporated, it releases a pyrophosphate (PPi). ATP sulfurylase is present in the reaction mix, and when PPi is released, converts it to ATP, which can activate luciferase and the emission of light. The Roche 454 can measure the amount of light given off and relate it to the number of nucleotides that have been incorporated. One problem with this type of sequencing is that it can be difficult to accurately characterize sequences of the same nucleotide in a row as the intensity of the pyrophosphate peak given off does not have a linear relationship with the number of homopolymers present. The read length for 454 is approximately 250 base pairs long, and the error mode tends to have indels.
The next major second gen sequencer is the Illumina Solexa platform (Bennett). The chemistry of this platform is that it utilizes reversible terminators and sequences by synthesis. A flow cell is covered with DNA oligonucleotides that are complementary to adaptor sequences that have been ligated to the ends of fragmented genome pieces. As the genome fragments are streamed across the surface of the flow cell, they will randomly bind and go through multiple cycles of denaturation and extension, which creates clusters of clones. After these clusters have been generated, they are loaded into a sequencer which measures fluorescent signals as single nucleotides are incorporated by taking a picture and noting the location of fluorescence. Read lengths are about 26-50 bases on average, and the types of errors that are typically present tend to be SNP errors.
Another important second generation sequencer is the ABI-SOLiD (Sequencing by Oligonucleotide Ligation and Detection) sequencing platform (Valouev et al.). This is another sequencing by synthesis reaction, but unlike Illumina and 454, which use polymerases, this uses ligases. After using emulsion PCR on beads to create clonal clusters, primers base pair to a known adapter sequence that has been ligated to the genomic DNA. Differently labeled probes competitively base pair to the sequencing primer, and sequencing goes through several cycles in which different primers are used each time to bind to positions offset by a single nucleotide each time. DNA bases are added in groups of two in this method. Average read lengths for this technique are on average about 35 base pairs long.
The next second generation sequencing technique is Ion Torrent, which is a sequencing by synthesis technique (http://www.iontorrent.com). When nucleotides are added to a growing DNA chain, pyrophosphate and a hydrogen ion are released. Ion Torrent takes advantage of this by measuring the pH of the reaction mix after flooding a DNA strand with the four bases (one at a time) to determine sequences. One major advantage of this technique is that it doesn’t require a high-cost camera set-up to measure incorporation events. However, because it indirectly measures nucleotide addition through changes in pH, it has difficulty with accuracy in calling sequences of homopolymers, resulting in indel errors (like pyrosequencing). Average read lengths using this technique are about 200 base pairs long.
A more recent innovation is the Helicos-True Single Molecule Sequencing (tSMS) technique (Thompson and Steinmann). It is somewhat similar to Illumina sequencing in that it also uses fragmented DNA, adaptors, and fluorescently labeled dNTPs, but there is no amplification step. This helps eliminate issues with GC bias, which tend to affect amplification steps and can cause errors in base calling. Average read length is greater than 25 base pairs.
Pacific Biosciences’ SMRT technology (Single Molecule Real Time sequencing) immobilizes a DNA polymerase at the bottom of a well and is a sequencing by synthesis technique (Eid et al.). Fluorescently labeled phosphate groups in dNTPs are added to the reaction mix and as the base is added to the growing DNA strand, the machine can measure the light that is given off (each base is labeled with a different fluorescent molecule). The major advantage of this technique is that it can sequence very long reads (more than 1000 bp!) which is very important in de novo sequence assembly. In addition, PacBio can also measure methylation of DNA sequences based on the kinetics of addition of base pairs (using the observation that modified base pairs tend to take longer to incorporate into a DNA strand). Furthermore, this technique can also potentially use a single molecule of DNA, which reduces any GC bias that occurs due to amplification.
The final technique that will be discussed here is nanopore sequencing (Stoddart et al.). The idea behind this is that DNA may be threaded through a nanopore one base at a time. As it’s fed through, the sequencer can measure the change in current as it passes through (which will vary based on what base is moving through the pore). Thus, the sequence can be determined straight from the DNA without the need for modifications or reagents. In addition, because this can be done on a single molecule, there is again no need for amplification and thus no possibility of any GC bias in base calls.
 

Mitochondrial Dna And Genetic Evidence Biology Essay

Introduction:
The Out of Africa model, also referred to as the African origins, total replacement, Noah’s ark or Eve model is one model suggesting the origins of humankind. This model hypothesizes that the evolution of the modern humankind from their archaic ancestors occurred in one place at the one time. It suggests that modern humans arose as a new species about 150,000 years ago and that this took place in Africa. It was after this speciation event that the modern humans moved out of Africa, replacing all non-African archaic populations. Africa was identified as the origin of Homo sapiens because of the high genetic diversity among Africans. It is much higher than the genetic diversity of other populations around the world. The further away, geographically, from Africa the less genetically diverse the populations are. The last regions to be settled, for instance South America and the Pacific Islands, have the lowest genetic diversity.

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This review will focus on the evidence obtained from mitochondrial DNA and Y-chromosomal DNA. Both mtDNA and Y-chromosomal DNA are non-recombinant and their inheritance is easier to analysis than for other parts of the genome. MtDNA is only inherited through the maternal line and can therefore be used to determine the female lineage. Analysis of mtDNA revealed a series of population bottlenecks and a progressive loss of diversity moving away from East Africa. The Y-chromosome is passed from father to son and can be used to determine the male lineage. The Y chromosome does not undergo recombination because it is so different from the X chromosome that they don’t swap information. This means that the Y-chromosome passed on is the same in father and son (unless it undergoes mutation) making it useful for studying the male lineage. Mutations of both mtDNA and Y-chromosomal DNA accumulate at a fairly constant rate over time, making them useful for estimating the time of human population splits. Mitochondrial DNA is also a very good indicator of migration routes and range expansion due to its high distribution and variation.
The first lineage to branch off from mitochondrial eve is the L0 haplogroup. The L1, L2 and L3 haplogroups are all descendant of this L0 lineage and are largely confined to Africa. L3 subdivided into the macro haplogroups M and N. These are the lineages found outside of Africa with a low frequency in Africa. The Y-chromosomal haplogroup DE is limited to Africa. Haplogroup F originated in either North Africa or in South Asia. If it originated in North Africa it would indicate a second out of Africa migration.
There are two possible scenarios for modern human’s dispersal out of Africa. The first suggests a single migration in which only about 150 people left Africa by crossing the Red Sea. The second possibility is that there were two migrations out of Africa. Haplogroup M left by crossing the Red Sea, travelling along the coast to India taking the Southern route. Haplogroup N is thought to have followed the Nile from East Africa, headed north and crossed into Asia via the Sinai Peninsula in Egypt.
Historical Background:
Charles Darwin was one of the first to propose the idea that the ancestor of the modern human originated in Africa. In his book “The Descent of Man” he proposed that all living organism originated from a common ancestor and he outlined his views that man descended from apes. He stated that “in each great region of the world the living mammals are closely related to the extinct species of the same region. It is, therefore, probable that Africa was formerly inhabited by extinct apes closely allied to the gorilla and chimpanzee; and as these two species are now man’s nearest allies, it is somewhat more probable that our early progenitors lived on the African continent than elsewhere. But it is useless to speculate on this subject, for an ape nearly as large as a man, namely the Dryopithecus of Lartet, which was closely allied to the anthropomorphous Hylobates, existed in Europe during the Upper Miocene period; and since so remote a period the earth has certainly undergone many great revolutions, and there has been ample time for migration on the largest scale”. Here he is saying that if his theory of common descent was correct and that man really did descend from apes then it would be likely that man originated in Africa as Africa was the region inhabited at that time by apes.
Mitochondrial Eve and Y-chromosomal Adam:
Mitochondrial eve is the matrilineal most recent common ancestor, estimated to have lived about 200,000 years ago. All living people’s mitochondrial DNA is descended from hers. She was thought to have lived in East Africa and her discovery supported the theory that all modern humans originated in Africa and migrated from there.
Y-chromosome Adam is the patrilineal most recent common ancestor, estimated to have lived between 90,000 to 60,000 years ago. He was also believed to have originated in Africa.
The original paper supporting the Out of Africa theory was written by Cann et al in 1987. In which they found evidence that the MRCA lived in Africa about 200,000 years ago. They studied mitochondrial DNA from one hundred and forty seven people between five different populations, African, Asian, Australian, Caucasian and New Guinean. They found that out of the one hundred and forty seven mtDNA mapped, 133 were distinct from each other. Using the parsimony method they constructed a tree relating the 133 types of human mtDNA and the reference sequence:
Figure 1: Genealogical tree for 134 types of human mtDNA. The tree accounts for the site differences observed between restriction maps of these mtDNAs with 398 mutations. No other order of branching tested is more parsimonious than this one. This order of branching was obtained by ignoring every site present in only one type of mtDNA or absent in only one type and confining attention to the remaining 93 polymorphic sites. The computer programme produces an unrooted network which was converted into a tree by placing the root (arrow) at the midpoint of the longest path connecting the two lineages. The numbers refer to mtDNA types found in more than one individual.
(both figure and text taken from Cann et al, 1987)
This is a tree of minimum length. On this tree there are two primary branches, one composed of Africans only and the other composed of all five populations studied. From this tree it was suggested that Africa was the source of the human mitochondrial gene pool. This is because two of the primary branches lead solely to African mtDNAs and the second branch also leads to African mtDNAs. The common ancestor a must be of African origin in order to minimise the number of migrations that occurred. This tree also indicates that every population except for Africa must have multiple origins. For example, mtDNA type 49 is New Guinean but its nearest relative is not New Guinean and is in fact Asian. New Guinea seems to have been colonised by at least seven maternal lineages. This seems to be the same for all other populations apart from Africa. By assuming that human mitochondrial DNA sequence divergence accumulates at a constant rate they were able to work out that the common ancestor, ‘Mitochondrial Eve’ of all surviving mtDNA types existed 140,000 to 290,000 years ago. The mtDNA results do not show when the migrations out of Africa took place. Nuclear DNA studies carried out based on polymorphic blood groups, red cell enzymes and serum proteins showed that differences between racial groups are smaller than within and that the largest gene frequency differences are between Africans and other populations. This supports the Out of Africa theory because it suggests that the human nuclear gene pool also originated in Africa. (Cann et al, 1987)
The Genetic Evidence:
The technique used to deduce the colonization pattern of the world is coalescence. This theory is a population genetics model based on the genealogy of gene copies and favours the Out of Africa theory. It describes the characteristics of the joining of lineages back in time to a common ancestor.This lineage joining is referred to as coalescence. The theory provides a way of estimating the expected time to coalescence and establishing the relationships of coalescence times to population size, and age of the most recent common ancestor. This theory makes use of the fact that genetic drift over time will result in the extinction of lineages. This means that any sample of DNA markers will coalesce to a common ancestor when looking backward from the present day generation. The limitation of this theory is that all genetic variation coalesces to the MRCA and as a result the population history before this MRCA is unknown. Genomic phylogenetics reconstruction is necessary to assume the dispersal routes of early modern humans.
Mitochondrial DNA evidence:
A study was carried out by Ingman et al describing the global human diversity in humans based on analyses of the complete mtDNA sequence of 53humans of varied origins. They created a neighbour-joining phylogram on complete mtDNA sequences:
Figure 2: Neighbour joining phylogram based on complete mtDNA genome sequences (excluding the D-loop). The population origin of the individual is given at the twigs. Individuals of African descent are found below the dashed line and non-Africans above. The node marked with an asterisk refers to the MRCA of the youngest clade containing both African and non-African indivdulals.
(Both figure and text taken from Ingman et al, 2000)
In this tree, the three deepest branches lead to exlusively African mtDNAs and the fourth deepest branch contains both African and non-African mtDNA. The deepest branch provides excellent support for the origin of human mtDNA in Africa. The amount of mtDNA sequence diversity among Africans is more than double that of non-Africans. This suggests that ther is a longer genetic history for African mtDNA than for non-African mtDNA. The “star” shaped phylogeny of the non -African sequences suggest a population bottleneck. This is more than likely associatd with the colonisation of Euroasia from Africa, in which the previous populations are replaced with the modern human’s dispersal into Euroasia.
The figures below show the mtDNA mismatch distributions for Africans and non-Africans The mtDNA from the non-Africans show a bell-shaped distribution , indicating a recent population expansion. The mtDNA from individuals of African origin show a ragged distribution, indicating a constant population size.
Figure 3: Mismatch distributions of pairwise nucleotide differences between mtDNA genomes (excluding the D-loop) a) African; b) Non-African.
(Both figure and text taken from Ingman et al, 2000)
The initial Homo sapiens population dynamics and dispersal routes remain poorly understood. The mtDNA phylogeny can be collapsed into two sister branches L0 and L1’2’3’4’5’6 (L1’5). The L1’5 group is more widespread and has given rise to almost all mtDNA lineages found today. The non-African genetic diversity being formed from two subclades of the L3 branch, M and N. Some of the L clades show significant phylogeographic structure in Africa, such as the localization of L1c1a to Central Africa and L0d and L0k to the Khosian people.(Behar et al, 2008)
Analysis of the complete mtDNA sequences of Khosian people suggests the divided from other modern humans no later than 90,000 years ago. This reveals evidence for the existence of an early maternal structure in the history of Homo sapiens. L0abfk split over 133,000 years ago. Since this split the expansion of L0d, L0k, L0abf and L1’5 clades have progressed in an uneven way. L0d and L0k localized in South Africa, giving rise to the Khosian people and L0abf and L1’5spread all over the world giving rise to all non-Khosian populations. These maternal southern and eastern populations remained isolated from each other for a long period of time. This isolation suggests the formation of small, independent populations in Africa instead of the previously thought uniform spread of modern humans. (Behar et al, 2008)
Mitochondrial DNA L haplogroups:
Single nucleotide polymorphism studies have shown that human mitochondrial DNA can be classified into groups of related haplotypes.
An early paper by Chen et al analysed mitochondrial DNA variation in Africa, revealing continent specific groups of mtDNA haplotypes (haplogroups). There is an HpaI site gain at nucleotide pair (np) 3592 which is found in sub-Saharan populations with a low frequency in populations which have been known to have mixed with Africans. The mtDNA that contain the HpaI site at np 3592 form the most divergent mtDNA haplogroups in the world. Continent specific polymorphisms characterize mtDNAs from European, Asian and Native American populations. These continent specific polymorphisms have a high frequency in one continental population and are specific to either European, Asian or Native American populations. These mutations took place after the genetic separation of the ancestral population that formed the modern human ethnic groups. The oldest and the largest haplogroup in each continent is usually the one that is the most divergent. All the mtDNAs associated with the HpaI site gain at np 3592 all come from the same common ancestor. These cluster in the L haplogroup. This haplogroup is subdivided into theL0, L1, L2, L3, L4, L5 and L6 sub-haplogroups by additional polymorphisms. The L haplogroup and L1 and L2 sub- haplogroups are said to be of ancient origin due to their dominance in sub-Saharan populations. The ages of these haplogroups were determined from the assumption that nucleotide substitution accumulates at a constant rate. The age of haplogroup L is between 98,000 and 130,000 years, haplogroup L1 is between 86,000 and 113,000 years and haplogroup L2 is between 59,000 and 78,000 years. Comparison of the sequence divergence of the L haplogroup determined that the African haplogroup is the most divergent. The approximate ages for the continent specific haplogroups agree with the theory that all modern humans have a common ancestor from an ancestral population in Africa. These ages also agree with the suggested times of dispersal and migration of the modern human populations into the other continents. The age of the haplogroup L could indicate that this haplogroup originated before modern humans dispersed from Africa. However, the haplogroups L1 and L2 were not carried from Africa by the modern human populations that migrated to the Middle East and Asia. Instead another haplogroup must have participated in this migration. There are mtDNAs that do not contain the HpaI site gain in np 3592. These were found in sub-Saharan populations and suggest that there were some mtDNAs without the 3592 HpaI site that originated in Africa. They are widely distributed in sub-Saharan populations and most likely have an ancient African origin. These mtDNAs are similar to mtDNAs in Europe and Asia and seem to be the only mtDNAs carried out of Africa by migration of the modern humans. They gave rise to the non-African modern human populations and are now know to be haplogroup L3. This paper exhibits data that confirms that there was a high sequence divergence within Africans compared to the rest of the world thereby supporting the Out of Africa Theory. There is less sequence divergence in Asians than in Africans. Native American populations have the lowest values of sequence divergence. (Chen et al, 1995)
The minimum coalescence age for modern humans has been estimated to be between 156,000 and 169,000 years before present. Analysis of the L haplogroup has been carried out in order to find those sub-haplogroups involved in the migration of modern humans out of Africa. The L0 haplogroup is the earliest descendant of mitochondrial Eve and is a sister group to the L1 haplogroup. L0 is subdivided into L0a, L0d, L0f and L0k. L0a is thought to have originated in Eastern Africa and is dominant in Ethiopia. The idea that east Africa is the most likely region for L0a variation is further supported by the phylogeny of the L0 clade. L0d and L0k originated in Southern African. L0f is rare and confined to East Africa. The relationship between L0d and L0k is still uncertain.
The first ancient split from this into L1b/c occurred over 120,000 years ago. The L1 haplogroup is divided into L1b and L1c. L1b is common in Western Africa and L1c is frequent among central African Bantu speakers. See figure__ for the relationship between these two haplogroups. FIG. 3.-Phylogenetic tree of mtDNA genomes (excluding the d-loop) obtained by maximum likelihood Bayesian analysis.
The split into the L2 lineage occurred in Africa over The L2 lineage is divided into two sub-clades L2a1 and L2b. A mutation at np12693 characterizes the L2a1 clade. Ethiopian L2a1 sequences contain mutations at the np 16189 and the np 16309. L2a1c contains mutations at np 16209, 16301 and 16354. L2a1a has a mutation at np 16286. L2a1a is found mostly in South-Eastern Africa.
The split into the L3 sub-clade occurred over 59,000 years ago in Africa. The most frequent of the L3 sub-clades is the L3f haplogroup. This haplogroup seems to be confined to East Africa. However, there is an occurrence of variations of this clade in West Africa indicating an early dispersal of the L3f1 lineages. L3f1 is characterized by two mutations in its coding region. The L3 haplogroup is subdivided into three clades, L3i, L3x and L3w. Haplogroup L3i contains a transition at np 7645. It was also found to occur within a sister group of W haplogroup lineages in Eurasia. The L3x haplogroup is characterized by transitions at nps 6401, 13708 and 16169. This haplogroup is very frequent among Ethiopians, especially among the Oromos. It can be sub divided into two clades, L3x1 and L3x2. These two clades are confined to the Horn of Africa and the Nile Valley. The L3w haplogroup contains substitutions at nps 15388 and 16260. This haplogroup is confined to East and North-eastern Africa. L3b and L3e haplogroups are found in West Africa and Bantu-speaking populations in South-east Africa. The L3d haplogroup is mostly found in Western Africa. It is divided into the two sub-clades L3d1 and L3d2. The L3d1 sub clade has a high frequency in South-East Africa. L3d2 is characterised by transcriptions at nps 15358 and 16256. These occur in Western Africa. Ethiopian L3d2 lineages contain a transition at np 16368 and this is not found anywhere else in Africa. The L3 clade is more related to Eurasian haplogroups than to African clusters of the L1 and L2 haplogroups.
L4 is an early branch from L3. It is divided into two sub-clades by three coding and three control region markers. Substitutions at nps 195, 198, 7376, 16207 and 16260 characterise the L4a1 haplogroup. L4g was previously named L3g but it was found to share ancestral character states at nps 769 and 1018 with haplogroup L4a. It is mostly found in Ethiopia. L4a and L4g have high haplotype frequencies and sequence diversity in Ethiopians.The L5 haplogroup is divided into L5a and L5b. L5a is found almost exclusively in East Africa. L5 b on the other hand is spread through Southern Africa.The L6 haplogroup contains six coding transitions and one control region transition. This haplogroup is thought to have originated in East Africa. It is a sister clade of the L2, L3 and L4 are all frequent there, giving support to this theory.
The mtDNA tree splits at its core layers into branches that carry exclusively African sequences and just one, L3, which the Africans share with the rest of the world. All non-African mtDNA lineages are derived from just two branches, M and N, branching from the root of the L3 haplogroup. These also give rise to a number of sub-clades specific only to African populations. The N haplogroup gives rise to a daughter clade, R, which is also a founder of extant non-African populations. The first informative split in the mtDNA tree with regards to phylogeny occurs at the level of L3/M, N, R clades. The next informative split in the mtDNA tree distinguishes all major continents excluding America beneath the M, N and R founders.
The M and N Haplogroups:
The M1 haplogroup has a high frequency in Ethiopia. It has two subclades, M1a and M1b. M1a contains a transition at np 16359. It can be found in Near Eastern, Caucasus and in European populations. The M1b group is smaller and confined to East Africa. Both M1a and M1b are rare in North Africa. Another clade, M1c, is present in Northern Africa, the Canary Islands and the Near East. This clade is characterized by a transition at np 16185.
The N (preHV) haplogroup is the most frequent in Ethiopian lineages. This lineage occurs in populations in the Near East, Southern Caucasia and North Africa.
Y-chromosomal DNA evidence:
The Y chromosome Consortium (2002) tree was updated in a paper by Karafet et al in 2008. This tree identifies the 18 major clades, A to R, in the Y chromosome tree. There are five paragroups that were not based on a derived character and they represent the interior nodes of the tree. There are 243 different mutational events that give rise to 153 non recombining Y chromosome haplogroups. The C and FT haplogroups were united by the P143 mutation. These haplogroups contain lineages that are not usually found in sub-Saharan Africa. The C-FR chromosome must have been carried out of Africa early on in the dispersal out of Africa. The IJ clade is joined by seven mutations and the NO clade is joined by six mutations. The M lineage is joined to two K haplogroups by the P256 marker into the M super clade.
Diagram p4 from the revised Y chromosome haplogroup tree.
Two mutations, M91 and P97, identify Clade A. This clade is one of the most base haplogroups on the Y-chromosome tree and is almost entirely confined to Africa, being most frequent in Khosian, Ethiopian and Sudanese populations. Clade B is characterized by four mutations and is also almost completely restricted to Africa, mostly confined to sub-Saharan Africa with the highest frequencies in Pygmy populations. The C haplogroup is identified by five mutations. It has not been found in African populations and may have an originated in Asia after the dispersal of modern humans out of Africa. Haplogroup D is defined by two mutations. This haplogroup is also thought to have originated in Asia as it has not been found anywhere else. These lineages are found almost completely in Central Asia and Japan with a low frequency in Southeast Asia and the Andaman Islands. Clade E is identified by 18 mutations and is the most mutationally diverse Y chromosomal haplogroup. These are found mostly in Africa with moderate frequencies in the Middle East and low frequencies in Central and South Asia. The FT clade is defined by 25 mutations. The F* paragroups has a low frequency in India. The G clade is identified by two mutations and is divided into two subclades, G1 and G2. This clade is mostly present in the Middle East, the Mediterranean and the Caucasus Mountains. Haplogroup H is characterized by one mutation and is divided into two subclades, h1 and H2. This group is almost exclusive to the Indian subcontinent. Clade I is characterised by six mutations and is sub-divided into two subclades, I1 and I2. This clade represents two of the major European Y chromosome haplogroups with clade I1 being found mostly in Northern Europe and clade I2 is widespread in Eastern Europe and the Balkans. Clade J is defined by three mutations and is divided into two major subclades, J1 and J2, and also contains a paragroup J*. These lineages are found at high frequencies in North Africa, the Middle East, Europe, Central Asia, Pakistan and India. Haplogroup K is defined by the derived state at four sites and the ancestral state at the mutations that characterize the L, M, NO, P, S and T lineages. There is a paragroup K* and four different lineages characterized by five mutations. The K1 haplogroup is found at a low frequency in India and the K2, K3 and K4 haplogroups are found in Oceania, Indonesia and Australia. The L haplogroup is characterized by six mutations and the majority of this haplogroup is found in India, with the L haplogroup also being present in the Middle East, Asia, Northern Africa and along the Mediterranean coast. The M superclade contains 19 internal mutations. This lineage is confined to Oceania and eastern Indonesia. The N haplogroup is defined by 10 mutations and is restricted to Northern Eurasia. Clade O is defined by four mutations and is a major haplogroup in East Asia. It is also found at a low frequency in Central Asia and Oceania. Haplogroup contains the Q and R lineages. Clade Q is characterized by the M242 mutation and is distributed in North Eurasia with a high frequency in some Siberian groups. It is also found in Europe, East Asia and the Middle East and is the major lineage in native Americans. Cade R is defined by eight mutations and is the major y chromosomal lineage of Europeans. Clade S is defined by three mutations and is mostly found in Oceania and Indonesia. Clade T is identified by six mutations and is divided into two subclades found at a low frequency in Africa, Europe and the Middle East.
The two primary splits in this tree lead to the A and B haplogroups, both of which are restricted to Africa. These are genetically diverse and have sub-haplogroups geographically distinct from each other. The remainder of the deep structures of the phylogeny are characterized by three sub-clusters that coalesce at the root of the CR-M168 node. These represent all the African haplogroups and all the non African haplogroups. There is a shared presence of the De haplogroup in Africa and Asia. The C haplogroup is a non African haplogroup and is widely distributed in East Asia, Oceania and North America. The haplogroup F-M89 is another non African cluster that is distributed all around the world. The F* and H haplogroups are restricted to Asia, the I haplogroup in Europe and the J haplogroup in the Middle East.
Apart from the A and B haplogroups all other Y chromosome haplogroups descend from one ancestral node, CDEF which is defined by the mutations M168 and M294. This node is split into the C, DE and F haplogroups and these make up the majority of African and non African affiliated chromosomes. Due to the fact that the A and B haplogroups originate in Africa it was proposed the CDEF node also originated in Africa. An African origin of the DE haplogroup was supported with the detection of the DE* chromosome in Nigeria and by the recognition of the D-M174 haplogroup.
See figure8d page 555 from Underhill
It was proposed that two independent founder types D and CF evolved out of Africa (see figure above) The common ancestry of C and F founder types was supported by a single mutation, implying the diversification of CF from DE was shortly followed by they split of C from F. Although the D and E haplogroups share a common ancestry there is a geographic distance existing between the two of them. The D haplogroup is widely distributed in Asia and the E haplogroup is frequent in Africa. This suggests long term isolation and extinction of descendants in the area between Africa and Asia.
Upon analysis of the Y chromosome it is clear that North Africa is genetically similar to the Middle East and there is a clear genetic difference between North-Western Africa and Sub-Sahara Africa and Europe. The lineages most prevalent to North Africa are absent in both Europe and sub-Saharan Africa. E3b2 is most common in North Africa, R1b is common in Europe and E3a is common in many sub-Saharan areas. This suggests that there was limited gene flow between North Africa and Sub-Saharan Africa and Europe. E3b2 is rare outside of North Africa and the other dominant haplogroup J* in North Africa reaches its highest frequency in the Middle East indicating that there was gene flow between these two populations. It has been proposed that the J haplogroup originated in the Middle East. The M35 lineage is thought to have originated in East Africa due to its high frequency and diversity there. It is thought to have given rise to the M81 lineage, E3b2, that is found in North Africa. (Arredi et al, 2004)
Exodus from Africa:
The migration out of Africa is thought to have occurred over 100,000 years ago and is believed to have led to the later colonization of the rest of the world. The first evidence of the existence of modern humans outside of Africa has been dated to over 80,000 years ago. However, this was an isolated incidence and is thought to represent an early offshoot that has since died out. Successful migrations are believed to have occurred between 45,000 and 75,000 years ago. There are two scenarios describing modern human’s dispersal from Africa. The first suggests a single migration event took place. This theory proposes that only about 150 people left Africa crossing the red sea. This is because only the descendants of one lineage, L3, are found outside Africa. The M and N haplogroups are rare in Africa and seem to have arrived recently. This may be a result of mutations in the L3 haplogroup arising in East Africa just before the dispersal out of Africa or may have arisen shortly after the migration from Africa. The second scenario suggests a multiple dispersal model. This indicates that the M haplogroup crossed the Red Sea, travelled along the coast and arrived in India and the N haplogroup headed North, trailing the Nile and crossed into Asia through the Sinai Peninsula in Egypt. This group divided and went in several different directions. Some went east into Asia and others went to Europe. This scenario might clarify why the N haplogroup is predominant in Europe and the M haplogroup is absent.
Mitochondrial evidence for the dispersal from Africa:
Mitochondrial DNA analysis of present day African lineages points to a rapid population growth in the ancestral African population. Studies revealed a peak in African populations about 80,000 years ago with similar peaks in Asia and Europe somewhere between 60,000 and 40,000 years ago. This evidence shows a rapid increase in the African population much earlier than in Europe or Asia indicating expansion in Africa due to dispersion from a small population to other parts of the continent. There was an expansion of the L2 and L3 mitochondrial lineages about 80,000 and 60,000 years ago.
Population diversity among African populations:
There seems to be limited haplotype sharing among northern, eastern and Sub-Saharan Africans. Some haplotypes are common in one area but missing from the others. Chromosomes with the PN2 T and DYS271 A alleles are common in both northern and eastern Africa. These have been divided into different haplotypes, one of which bears the M81 mutation and is present in some Northern African populations and absent in Eastern African populations. There has been a population expansion in Northern Africa suggested by the age and the high frequency of the M81 haplotypes in north-western Africa. The spread of haplotypes 22 and 24, both of which contain the DYS271 allele, has erased pre-existing genetic differences among different regions in sub-Saharan Africa. Haplotypes 22, 24 and 41 have an extremely high frequency in Sub-Saharan Africans. It is thought that haplotype 41 was involved in the expansion of Bantu-speaking populations from western Africa into southern Africa. This is supported by the fact that the variance of haplotype 41 is much higher in the central western Africa than in southern Khosians. This is also true for the 22 and 24 haplotypes.
An Eastern African origin:
The oldest remains of modern humans were found in eastern and southern Ethiopia and have been dated to over 160,000 years ago. Eastern Africa is thought to be the origin of the earliest migrations of modern humans out of Africa. The M haplogroup has been found in high frequencies in Ethiopia and Asia. The presence of the ‘Asian’ mtDNA haplogroup M is unique to Ethiopia. These two regions have a different variation o
 

DNA Tranlession Polymerase in Prokaryotic Cells

DNA Tranlession Polymerase in prokaryotic cells: History, structures and function

DNA is one of the most important part of the cell that gives cell integrity and character. This part of the cell can be exposed to different kinds of damages that may put the cell’s integrity in jeopardy. The only part of the cell that has this ability to be repaired is DNA. Basically repairing should be done due to a reasonable reason. Repairing the other macromolecules are not profitable. For example, if a defective protein forms, the protein can be simply be replaced by another one. But defects in DNA can cause problem in the whole cell organisms and the character of cell [1]. Usually the whole repairing process is happening fast, although there are defects that persist against this process. The repairing process is done by special polymerases and the whole process of DNA repair is called “translesion DNA synthesis” (TLS) [2].
DNA can be damaged due to different reasons, such as base modification, elimination or addition of nucleotides, crosslinking of DNA strands and breakage of phosphodiester backbone [1]. These reasons can be due to some environmental conditions such as radiation or insertion of certain chemicals in to the body or due to malfunction of polymerases and enzymes in cellular process, such as putting wrong nucleotide in the DNA strand chain [1]. Up to now, it is known that there are three translesion DNA polymerases (TLS polymerases) in E. coli and about fifteen polymerases in eukaryotes that can run this process [2].
History
For the first time it was in the early 1940s, that it was found agents causing mutational changes such as ionizing and radiation of UV, interact with cells and can damage their genome [3]. Also it was found that these cells can survive and recover from theses damages [4] and the term DNA repair was found. “DNA repair is a biochemical term that defines biological processes during which alterations in the chemistry of DNA (DNA damage) are removed and the integrity of the genome is restored” [3].

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The first DNA repair mechanism to be discovered was enzymatic photo reactivation (EPR) [3]. This process is referred to the elimination of cyclobutane pyrimidine, which are generated by UV radiation and can block both DNA replication and transcription, from the genome [4]. This reaction can be catalyzed by photoreactivating enzyme in a reaction that needs a visible range light. The second mechanism found was excision repair [3]. This mechanism is referred to DNA damages cut out from genome that leaves some gaps in DNA duplex. These gaps are repaired by a non-semiconservative mode of DNA synthesis called repair synthesis [5].
By the end of the 1970s, it was known that cells are using various mechanisms for DNA repair process that focus around two basic principles: the excision of base damage or its direct reversal such as EPR [3]. In the mid1970s Miroslav Radman proposed a new hypothesis called SOS hypothesis [5, 6]. TheSOS hypothesis proposes an overall response to DNA damage in which thecell cycleis stopped andDNA repairis induced. Genetics experiments demonstrated that main players involved in damage-induced mutagenesis are lexA, recA along with umuD and umuC [2]. LexA cleavage from recA* and also umuD cleavage that form umuD’ use the same mechanism and is an absolute requirement for SOS mutagenesis. For showing that, E. coli because of its simple structure was used as a model for translesion DNA synthesis and mutagenesis.
Later Harrison Echols proposed another model and suggested that in order to help the replication process against the lesions it is possible to reduce the fidelity of proteins so when DNA replication process is stopped at a location of unrepaired DNA damage, certain SOS-regulated genes can encode proteins that interact with the hindered replication process in a manner that reduces their fidelity [3]. In the late 1980s and early 1990s, it was demonstrated that Echols genes are in fact specialized low-fidelity DNA polymerases that enhance low-fidelity replication across the lesion, the so-called translesion DNA synthesis (TLS) [3]. Their highly reduced fidelity allows the replicative bypass of sites of DNA damage, but with a high chance of combining incorrect nucleotides [5].
Early TLS models and PolV
Bridges and Woodgate were the first ones who defined the function of Umu proteins during UV-induced TLS in 1985 [7]. According to them, TLS happened in two steps. In the first step Pol III add a nucleotide opposite the first (3′) T of a T-T cytidine diphosphate diacylgelycerol (CPD). Bounding a RecA protein to the template proximal to the lesion is a requirement for this step. In the second step, Pol III interacts with UmuDC proteins to incorporate another nucleotide at the second (5′) T of the cytidine diphosphate diacylgelycerol (CPD). At least one of these two steps are non-WC, causing a mutation targeted at the site of the CPD [2]. Figure 1 shows the process schematically.
Another model was proposed by Echols and Goodman in 1990 [3]. In this model they proposed that when Pol III encounters a template lesion, its holoenzymes (Pol III core, beta sliding clamp, gamma-clamp-loading complex) are completely blocked. This process follows by the assembly of a damage localized nucleoprotein complex involving RecA, UmuC, UmuD′, SSB, and Pol III holoenzyme, a mutasome, to copy past a template lesion [2]. The fact that RecA* simplifies the cleavage of UmuD to UmuD’ was used in this model [8]. Later, it was demonstrated that it was actually a dimeric UmuD2 that is cleaved to UmuD’2 and that next interacts with UmuC to form a stable complex of UmuD’2C [9]. This complex was named as Pol V in 1999 by Tang et al. [10]. It can be said that genome replication done on undamaged DNA by Pol III is rapid and error-free [11], the TLS process carried out by mutasome is slow and error-prone [2].
“A key feature of the mutasome model is the assembly of RecA* on ssDNA proximal to the lesion (Fig. 1). When a replication fork encounters a lesion, an uncoupling of leading-and lagging-strand synthesis may ensue. Then, one of the TLS Pols can replace Pol III on the β- clamp and copy the damaged DNA” [2]. For both leading and lagging strands it be easily seen that RecA* can be assembled on the form of template strand, proximal to lesion. If the lesion occurred in the leading strand, RecA filaments can be formed on a region of ssDNA that is created by DNA unwinding by DnaB helicase downstream from the lesion but if lesions exist in lagging strand ssDNA is present as a result of Okazaki fragment synthesis [2].
Schlacher and Goodman [12] showed RecA* act in trans form on a non-template ssDNA strand and this transactivation of PolV by RecA* to perform TLS happens in-vitro. And this lead to the PolV mutasome model of TLS (Fig. 1). Jiang et al. [13] demonstrated this new PolV form as PolV Mut = UmuD2’C-RecA-ATP. PolV Mut has this ability to copy both damaged and undamaged DNA (e.g. performs TLS) when RecA* is not present [2]. So, the straight role of RecA* in SOS-mediated TLS is to transfer a RecA molecule from the 3′-filament tip with a molecule of ATP to convert into Pol V Mut, that can cross a different number of DNA lesions on its own. (Fig. 1) [2, 14]. PolV Mut can have two conformations. One is activated form that can copy DNA, the other one is deactivated form that is unable to copy the DNA. The activation of PolV Mut is depended on the location of RecA-ATP bond to the polymerase subunits UmuD2′ and UmuC [13]. By representing the RecA* again, the deactivated form of PolV Mut can be activated. In this case, the old RecA-ATP is substituted by a new RecA-ATP from the 3′-filament tip [13]. This type of switching on and off is unique to this kind of polymerase and has not been seen in other types of polymerases. This method can be useful specially preventing the undamaged DNA to go under mutation in E.Coli, and give the cell this power to activate Pol V Mut whenever replication fork have stuck at DNA template damage site [2].
PolII and PolIV in E.Coli TLS
PolII discovered in 1970[15]. At first, it was thought that mutation is non-informative in PolII [16]. Pol II has an activity isolated from UV-irradiated cells that has this ability to replicate past abasic template lesions [2, 17]. This polymerase has some responses to UV radiation and this activity derives from that [2]. By purification it was proved that the induced lesion-copying protein was Pol II [17]. In 1980, Kenyon and Walker [18] discovered a DNA damage-inducible gene called dinA that can encode PolII. Also, one of the features of PolII is bypassing N2-deoxyguanosine-acetyl aminofluorene (AAF) adducts, this behavior can be error-free and produces 2-frame shift mutations [19]. “AAF adducts are of family of aromatic amides that induce frame shift mutations within GpC sequences, such as the NarI sequences” [19]. These adducts are able to increase the GC dinucleotide loss in NarI sequence (CGCGCC) by 107 times when they are bound to the G in middle of sequence [20]. PolII and PolV can complement each other, but it does not mean that their activities are functionally unneeded [21]. As Pham et al. [21] mentioned PolV job is to copy UV-damaged DNA in an error-prone manner in TLS. But Pol II is able to copy chromosomal DNA in an error-free replication process.
Kenyon and walker also introduced another gene called dinB gene that can be induced by cellular SOS response to DNA damage [18]. For many years, the function of this gene was unknown. After some year Ohmori et al. [22] found other gene, dinP gene, in the same section that dinB gene was found and Wagner et al. showed that they are able to encode Y-family DNA PolIV [23]. This kind of polymerases like other polymerases used in TLS are not crucial for life. Their role is to bypass certain N2-dG adducts (such as N2-furfuryl-dG) in an error-free manner [2]. Kumari et al. demonstrated they can copy past N2-N2-guanine interstrand cross-links in a high fidelity manner [24].
Regulation of TLS polymerases
Different polymerases have this ability to traverse an extensive range of DNA lesions but this ability may cause in reducing the fidelity during replicating the undamaged DNA. Usually cells have several mechanisms to check and control the TLS polymerases because except PolII, all of them has this potential to delete errors made when duplicating an undamaged DNA [2].
Usually no regulation is needed for PolII. Because it has high fidelity derived by high intrinsic 3′-5′ exonucleolytic proofreading. On the other hand, the Y-family polymerases such as PolIV and PolV are exo-nuclease deficient and needed to be controlled [2].
PolV activity can be regulated by many proteins and many ways. First as said before the UmuD’ should be activated by UmuD. All UmuD, UmuC and UmuD’ proteins are all exposed to degradation by Lon and ClpXP protease. RecA* that forms PolV Mut can interact with UmuD2’C complexes and active them. The PolV Mut itself activity can be enhanced by binding to the β-clamp [2].
As Wagner et al. showed the PolIV activity can be stimulated by protein interaction with RecA, UmuD and β-clamp [25].
Although the main mechanisms of DNA repair by various polymerases are known now, more studies can be conducted on E.Coli cells to find more details about the regulation and side reactions happening in this process. E.Coli cells as simple cells are an appropriate model to analyze these functions. Jarosz et al. proposed well questions about the future studies on Y-family DNA polymerases [26]:
“(1)How do Y-family polymerases gain access to an appropriate primer terminus and how is their action coordinated with that of replicative polymerases?
(2)How do protein–protein interactions regulate the activity of Y-family polymerases?
(3)Are there families of cognate lesions for each different Y-family polymerase?
(4) Can mutations introduced by Y-family polymerases be corrected by exonucleolytic proofreading in trans?”
Different polymerases act in different paces after the damaged. For example PolII is induced immediately after DNA damage but PolV is induced about 50 min after the damage [21]. An area of interest could be study on how they can be regulated to be induced in shorter time.
References

Horton, R. H., Moran, L. A., Perry, M. D., Rawn, D. J. and Scrimgeour, G. K. (2006)Principles of biochemistry. 4th edn. United States: Pearson Education (US).
Goodman, M. F. and Woodgate, R. (2013) ‘Translesion DNA Polymerases’,Cold Spring Harbor Perspectives in Biology, 5(10). doi: 10.1101/cshperspect.a010363.
Friedberg, E. C. (2008) ‘A brief history of the DNA repair field’,Cell Research, 18(1), pp. 3–7. doi: 10.1038/cr.2007.113.
Hollaender, A. and Duggar, B. M. (1938) ‘The effects of sublethal doses of monochromatic ultraviolet radiation on the growth properties of bacteria’,Journal of Bacteriology, 36(1): 17-37.
Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T. DNA Repair and Mutagenesis. Washington DC, ASM Press, 2005
Friedberg EC. Correcting the Blueprint of Life. An Historical Accounting of the Discovery DNA Repairing Mechanisms. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1997.
Ruiz-Rubio, M., Woodgate, R., Bridges, B. A., Herrera, G. and Blanco, M. (1986) ‘New Role for Photoreversible Pyrimidine Dimers in Induction of Prototrophic Mutations in Excision-Deficient Escherichia coli by UV Light’,Journal of Bacteriology, 166(3): 1141-1143.
Burckhardt, S. E., Woodgate, R., Scheuermann, R. H. and Echols, H. (1988) ‘UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA.’,Proceedings of the National Academy of Sciences, 85(6), pp. 1811–1815. doi: 10.1073/pnas.85.6.1811.
Woodgate, R., Rajagopalan, M., Lu, C. and Echols, H. (1989) ‘UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD’,Proceedings of the National Academy of Sciences, 86(19), pp. 7301–7305. doi: 10.1073/pnas.86.19.7301.
Tang M, Shen X, Frank EG, O’Donnell M, Woodgate R, Goodman MF. UmuD2′C is an error-prone DNA polymerase, Escherichia coli, DNA pol V. Proc Natl Acad Sci. 1999; 96:8919–8924.
Johnson A, O’Donnell M. Cellular DNA replicases: Components and dynamics at the replication fork. Annu Rev Biochem. 2005; 74:283–315
Schlacher K, Goodman MF. Lessons from 50 years of SOS DNA-damage-induced mutagenesis. Nat Rev Mol Cell Biol. 2007; 8:587–594
Jiang Q, Karata K, Woodgate R, Cox MM, Goodman MF. The active form of DNA polymerase V is UmuD2′C-RecA-ATP. Nature. 2009; 460:359–363.
Dutreix M, Moreau PL, Bailone A, Galibert F, Battista JR, Walker GC, Devoret R. New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis. J Bacteriol. 1989; 171:2415–2423.
Knippers R. DNA polymerase II. Nature. 1970; 228:1050–1053.
Foster PL, Gudmundsson G, Trimarchi JM, Cai H, Goodman MF. Proofreading-defective DNA polymerase II increases adaptive mutation in Escherichia coli. Proc Natl Acad Sci. 1995; 92:7951–7955.
Bonner CA, Randall SK, Rayssiguier C, Radman M, Eritja R, Kaplan BE, McEntee K, Goodman MF. Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA. J Biol Chem. 1988; 263:18946–18952.
Kenyon CJ, Walker GC. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci. 1980; 77:2819–2823.
Napolitano, R., Janel-Bintz, R., Wagner, J. and Fuchs, R. P. P. (2000)All three SOS-inducible DNA polymerases (Pol II,Pol IV and Pol V) are involved in induced mutagenesis, The EMBO Journal, 19(29), pp. 6259-6265.
Koffel-Schwartz, N., Verdier, J.-M., Bichara, M., Freund, A.-M., Daune, M. P. and Fuchs, R. P. P. (1984) ‘Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli’,Journal of Molecular Biology, 177(1), pp. 33–51
Pham, P., Rangarajan, S., Woodgate, R. and Goodman, M. F. (2001) ‘Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli’,Proceedings of the National Academy of Sciences, 98(15), pp. 8350–8354.
Ohmori H, Hatada E, Qiao Y, Tsuji M, Fukuda R. dinP, a new gene in Escherichia coli, whose product shows similarities to UmuC and its homologues. Mutat Res. 1995; 347:1-7.
Wagner J, Gruz P, Kim SR, Yamada M, Matsui K, Fuchs RPP, Nohmi T. The dinB gene encodes a novel Escherichia coli DNA polymerase, DNA Pol IV, involved in mutagenesis. Mol Cell. 1999; 4:281–286.
Kumari A, Minko IG, Harbut MB, Finkel SE, Goodman MF, Lloyd RS. Replication bypass of interstrand cross-link intermediates by Escherichia coli DNA polymerase IV. J Biol Chem. 2008; 283:27433–27437.
Wagner J, Fujii S, Gruz P, Nohmi T, Fuchs RP. The β clamp targets DNA polymerase IV to DNA and strongly increases its processivity. EMBO Rep. 2001; 1:484–488.
Jarosz, D. F., Beuning, P. J., Cohen, S. E. and Walker, G. C. (2007) ‘Y-family DNA polymerases in Escherichia coli’,Trends in Microbiology, 15(2), pp. 70–77.

 

Isolation and Characterization of Onion DNA

The experiment was about the isolation and characterization of DNA. The DNA was isolated from the onion. The mass of the isolated DNA was 15.11 g. The purity of isolated DNA was estimated by calculating the ratio based from the absorbance at 260nm and 280nm resulted to 0.671 meaning more protein was absorbed. Meanwhile in denaturation of DNA, the initial absorbance at 260 nm was 1.304 higher than the absorbance at 260 nm after heating which was 1.095.
INTRODUCTION
Deoxyribonucleic acid (DNA) is the genetic material in humans and all other organisms. DNA isolation is the removal of DNA from the cell which it normally resides. Isolation is the removal of DNA from the cell in which it normally inhabits. (1)
Onions are used since it contains little amount of starch which allows the DNA to be more visible. The filtrate is made up of onions treated with salt, distilled water and detergent collectively called as lysis solution. DNA purification is done by enzymatic degradation of contaminating proteins with ethanol. A spectrophotometer is used in determining the concentration and purity of the proteins. (2)

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MATERIALS AND METHODS
Isolation of DNA from Onion
The peeled onion bulb was chopped and measured homogenized. The sample was placed in a blender added with an ice-cold lysis solution then for 45 seconds at low speed. Meanwhile, the lysis solution used was prepared beforehand by mixing 5.00 ml of liquid detergent, 5.00 ml of 0.500M EDTA, 10.0 ml of 50% Na Cl solution, and 80 ml of distilled water and placed in an ice bath. After homogenizing, the sample was filtered through the cheesecloth and the collected filtrate was placed in a 250-ml beaker. A 10.0 ml of 5% pepsin solution was added to the filtrate and placed on an ice bath for 10 minutes with occasional stirring. Ice cold 30.0 ml of 95% ethanol was pipette to the side of the beaker containing the sample and stand for 10 minutes on ice bath. Once the DNA precipitates appeared at the interface of the solution, the DNA was already ready for isolation. The spooled DNA was transferred immediately to a pre-weighed 100-ml beaker to determine the mass and percent yield of the sample. The isolated DNA was added with 10.0 ml of 95% ethanol then covered with aluminum foil and refrigerated in preparation for the next laboratory procedure.
Characterization of DNA
Little amount of DNA sample was placed in a test tube added with 1.00 ml of 20% TCA followed by heating the sample for 10 minutes in water bath with 1.00 ml distilled water. A 2.00 ml of diphenylamine solution was added then heat again in a water bath for 10 minutes. The color change was observed and the absorbance of the sample from 400 nm to 700 nm was scanned to determine the wavelength of maximum absorption. Mean while, little amount of the DNA sample was placed in a separate test tube filled with 5.00 ml distilled water and scanned to read the absorbance at 260 nm then at 280 nm. After determining the A260/A280 value, the sample was heated to boil for 5 minutes and read the absorbance adain at 260 nm.
RESULTS AND DISCUSSIONS
The mass of the raw sample gathered from onion is 30.4 g. After homogenization and adding of pepsin solution and ethanol, DNA precipitates were became visible and transferred to another beaker. The isolated DNA measures 23 g.
The calculated percentage yield was quite high. However, still some sources of error was done while conducting the experiment, the sample with DNA precipitates was disturbed while transferring the DNA. The accumulated DNA precipitates is enough for the next procedure which is characterization.
Heat denaturation of DNA, causes the double helix structure to unwind and form single stranded DNA. Thus, the bases unstacked and can absorb more light causing an increase after denaturation. But based on the results gathered, the initial absorbance at 260 nm was 1.304 then was decreased after heating which was 1.095. The calculated percent increase in absorbance was 8%. This error is maybe, due to the heating process. The DNA acquired was quite greater and was not totally heated afterwards causing double helix structure not to unwind and form a single stranded DNA.
The filtrate gathered from this experiment was made of onions and lysis solution. Onion was used in this study due to low starch content, allowing the DNA to be more visible considering the onion as one of the best source of DNA. (4)
The used of lysis solution was to separate the DNA from extra cell components and to keep the location in which the DNA will not be tainted. The NaCL provides NA+ ions that will obstruct the negative charge as of phosphate ends of DNA. Permitting these ends to come nearer so they can precipitate out of a cold solution. The detergent causes the breaking down of the cell membrane by emulsifying the cell proteins and lipids. Also, disrupting the polar connections that collectively holds the cell membrane. The complexes formed with these lipids and proteins causes the precipitate out of solution. Meanwhile, the purpose of EDTA is to chelates metal ions. (5) A Pepsin solution was used for purification via enzymatic degradation.
DNA is polar due to its extremely charged phosphate backbone which makes it soluble in water. Thus DNA is insoluble in ice cold ethanol, as a result when the cold ethanol was added, it causes stable ionic bonds to form and precipitate the DNA.
Heating the sample is the one responsible for the formation of the observed color of DNA with diphenylamine. When the DNA is heated with acid, the 2-deoxyribose is converted to w-hydroxylaevulinic aldehyde, which reacts with the compound diphenylamine. Through this, a blue-colored compound supposed to produce. In our sample the color observed was green possibly because of the DNA concentration.
The ratio of absorptions at 260 nm vs 280 nm is frequently used to evaluate DNA contamination of protein solutions. The nucleic acids, DNA and RNA, absorbs at 260 nm and proteins absorb at 280 nm. Based on the results, the rate ratio of absorptions at 260 nm vs 280 nm is 0.671. Since proteins absorb light at 280 nm, the ratio is low meaning there is a lot of protein absorbed at 280nm.
 

DNA Extraction From Chicken Liver

Deoxyribonucleic acid (DNA) is the hereditary material in humans and almost all other organisms. Nearly every cell in a person’s body has the same DNA. Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA).

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The information in DNA is stored as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences.
DNA bases pair up with each other, A with T and C with G, to form units called base pairs. Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral called a double helix. The structure of the double helix is somewhat like a ladder, with the base pairs forming the ladder’s rungs and the sugar and phosphate molecules forming the vertical sidepieces of the ladder.
An important property of DNA is that it can replicate, or make copies of itself. Each strand of DNA in the double helix can serve as a pattern for duplicating the sequence of bases. This is critical when cells divide because each new cell needs to have an exact copy of the DNA present in the old cell.
The extraction of DNA from cells and its purification are of primary importance to the field of biotechnology and forensics. Extraction and purification of DNA are the first steps in the analysis and manipulation of DNA that allow scientists to detect genetic disorders, produce DNA fingerprints of individuals, and even create genetically engineered organisms that can produce beneficial products such as insulin, antibiotics, and hormones. 
Once the DNA has been isolated, it is essential to accurately determine its concentration for subsequent manipulation such as cloning or sequence determination.
To quantify the amount of DNA that extracted by using spectrophotometry.
The aims of this experience is to:

To use the properties of DNA to isolate long strands of DNA from liver cells.
To determine the yield of DNA isolated from a given amount of tissue.
To examine the light absorbing properties of purified DNA.
To examne the relationship between the concentration of a DNA solution and the absorbnce at 595nm of DNA-diphenylamine solution.
To generate a standrad curve relating DNA concentraton with the absorbance of DNA-diphenylamine solutions.
To use a standard curve to determine the concentration of an unknown DNA solution.

Materials and Methods
As per lab manual.
Results
Firstly, the chicken liver cell homogenate is treated with a salt solution such as NaCl and a detergent solution containing the compound SDS (sodiumdodecyl sulfate). These solutions break down and emulsify the fat & proteins that make up a cell membrane. Finally, ethanol is added because DNA is soluble in water. After adding ethanol a relatively clear aqueous will be produced, the first layer is the milky solution that is the aqueous phase with DNA, the middle layer is the solid (precipitate proteins). The bottom layer is a clear solution (organic). The DNA can be spooled (wound) on a stirring rod and pulled from the solution at this point. The amount of DNA solution we got is 5.4ml.Than we put the DNA solution in 2ml tube (1.041g).
The total weight of DNA solution and tube is 1.106g. The amount of DNA we got is 1.106-1.041g = 0.065g.
Next we prepare 4 standard tubes by adding TE buffer (ml) to the DNA standard solution (ml). And also added to each of the 3 samples of my DNA. The total DNA (mg) is recorded in the table 1. The observed colour change of 4 standard tube and my 3 samples are recorded in table 2 and 3. We pipette the DNA samples and each standards tubes into separate wells of a 96 well microtitre plate. We measured the absorbance at 595nm of the DNA-diphenylamine solutions using the plate reader. Our results are shown in the graph with the used of the reading of table 4. Form the graph we find that the concentration of undiluted DNA is 0.23×2=0.46mg/ml.
Discussion and Conclusions
For this experiment we determinate the yield of the DNA isolate from given amount of tissue is:
1g -> 63mg
0.065g -> 4.095mg (wet weight of the DNA to dry weight)
3ml -> 4.095mg
5.4ml -> 7.371mg (DNA in the entire aqueous phase is collected)
3. 4ml -> 7.371mg
5.3ml -> 9.767mg
The final calculation of the dry DNA is 9.767mg/g liver.
For the experiment we examine that the light absorbing properties of purified DNA. The wavelength is range 220-300nm. The wavelength of the DNA is 260nm.
We also calculated that the yield of DNA per g of liver from Lab 2 is:
The amount (mg) of DNA contain => 0.46×1.5=0.69mg
Aqueous from lab 1 = 5.4mg
0.69/2 =0.345mg
(0.345×5.4)/3 = 0.621mg
The final value in mg of dry DNA/g liver is: 0.621mg/g.
In the end of the experiments, we managed to complete our objectives. In summary, we learn that the alcohol can causes DNA to precipitate, or settle out of the solution, leaving behind all the cellular components that aren’t soluble in alcohol. As alcohol is less dense than water, so it floats on top forming two separate layers. We also learn that the advantage of spectrophotometry is that diphenylamine only reacts with DNA more accurate as RNA would not be determined. The disadvantage of spectrophotometry is that it always requires standard solution. The advantage of calculating of yield by its weight is that it does not require standard solution. The disadvantage of calculating of yield by its weight is that it is less accurate as RNA is counted in.
 

Role of Telomeres in Eukaryotic DNA Replication

GENETICS OF AGING

In this essay I am going to investigate the importance of telomeres, their role in eukaryotic DNA replication, the importance of telomerase and shelterin complexes, the action of telomerase and most importantly how does telomere shortening cause the onset of Dyskeratosis congenita. ‘Dyskeratosis congenita is a rare syndrome of premature aging that was recognized as a clinical entity nearly a century ago’ (Masood A Shamas, 2011). It leads to a diverse range of phenotypes depending on the mode of inheritance and which of the main 6 genes associated with telomere maintenance and elongation have mutated.

Firstly, and probably the biggest, most exciting concept concerning genetic aging is the theory of telomere shortening and the action of telomerase. Telomeres are DNA protein complexes which are situated at both ends of both chromosomes. ‘They play a crucial role in protecting our genome from nucleolytic degradation, unnecessary recombination, repair, interchromosomal fusion and therefore preservation of the genetic information found with in our genome.’(Masood A Shamas 2011). Telomeres found in humans consist of a sequence of multiple repeats of TTAGGG and this can be repeated up to 3,000 times reaching a total of 15,000 bases long. The most important role this ribonucleoprotein plays is during DNA replication. This is due to eukaryotic chromosomes being linear, which unlike prokaryotic chromosomes, means that DNA located at the very end of the chromosome cannot be replicated.

 During the process of DNA replication, one of the two new strands called the leading strand (grey strand as seen on the diagram) is being made continuously at the replication fork. Whilst the second strand called the lagging strand (blue strand as seen on the diagram) is being produced from many small pieces called Okazaki fragments, each of which begins with its own RNA primer (pink as shown on the diagram). During prokaryotic DNA replication, on the lagging strand the RNA primer will be removed by the 5’-3’ exonuclease activity of DNA polymerase and then DNA polymerase will replace the missing bases by complementary base pairing. And the nicks between the Okazaki fragments are joined by the enzyme ligase.  However during eukaryotic division there is no way to start the Okazaki fragment at 5’ end of the chromosome, this is because the primer would fall beyond the chromosome end, and is therefore placed as much as 70-100 nucleotides away from the end. Also when the RNA primer required to make the last Okazaki fragment is removed DNA polymerase can no longer fit the gap and therefore cannot replace the missing bases by complementary base pairing. This therefore results in shortening of one 5’ end of each daughter DNA molecule, and with DNA replicating at a rate of up to 50 bases per second, repeated replication results in shorter and shorter DNA molecule and if this is not corrected eukaryotes would become extinct as their DNA would become destroyed, as a result eukaryotes have evolved a mechanism to preserve the ends of their chromosomes. This mechanism involves , the action of the enzyme telomerase and shelterin, a protein complex, that work together to add telomeric repeats to the chromosome ends to try and reverse this process of DNA shortening during replication. The action of telomerase and shelterin occurs particularly on quickly dividing cells such as germline, stem and hematopoietic cells, but is not present or found in small numbers on somatic cells.

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The TERC and TERT genes contain the information in the form of base sequences for transcription and translation of the two principal proteins that make up telomerase, hTR and Htert. Htr is an RNA molecule. It contains the RNA sequence which acts as a template for the synthesis of complementary DNA (telomeric repeats), which is added by telomerase to the telomeres( chromosome ends). Whilst Htert’s job is to add new DNA segments to the chromosome ends.

Dyskerin, is another essential protein, coded for by the DKC1 gene, its function is to bind to Htr and secure the telomerase complex.

In addition to this, shelterin plays a protective role, sheltering the telomeres from the cells DNA repair process. If it wasn’t for the action of shelterin, the DNA repair process would think of the telomeres as unusual breaks in DNA sequence and would try to join the ends together or trigger apoptosis. The main protein in shelterin complex is coded for by the TIF2 gene.

 Furthermore, telomerase is a very exclusive protein complex in the way that it has present DNA polymerase activity whilst also an RNA sequence which provides a template for the synthesis of telomeric repeat DNA, this type of molecule is known as an RNA- dependent DNA polymerase. Firstly, hybridization occurs between a section of the RNA sequence on the telomerase and the 3’ overhang of DNA forming a single stranded overhanging RNA sequence.  Next the DNA polymerase activity of telomerase comes into action synthesising a complementary DNA strand to the RNA template of telomerase, it will then move to the end of the newly made strand, this sequence of events will keep repeating until telomerase has achieved its objective and detaches from the DNA strand. DNA primase will then make an RNA primer as close to 3’ end of overhanging DNA as possible, DNA polymerase is then implemented to add nucleotides by complementary base pairing in the region between the RNA primer and the 5’ end of the original DNA. However, as I mentioned above when the RNA primer is added to the 3’ end it is not added at the very end but as close to the end as possible, therefore leaving a very small section of the 3’ end still singley stranded. In conclusion although telomerase adds hundreds of bases to the end of the 3’ end during DNA replication the ends of the chromosomes will still shorten, although a lot more slowly thanks to the action of telomerase.

This continuous reduction in telomere size caused by the RNA primer not being placed right at the end of the chromosome causes ‘senescence, apoptosis, or oncogenic transformation of somatic cells’( Masood A Shamas, 2018), thus disturbing the health and lifespan of an individual, this is why shorter telomeres have been found to correlate with a high frequency of disease and an overall shorter lifespan. This highlights one of the reasons why your likelihood of developing a disease or illness increases with age and one example of this that I found particularly fascinating through doing my research is that of, dyskeratosis congenita. Dyskeratosis congenita, is a genetic disease which is caused by poor maintenance of telomeres, reduced telomere length and shows phenotypes of premature ageing. This disease affects cells rapidly dividing cells more harshly, therefore these cells suffer the consequences of telomere shortening causing notable symptoms: with nail dystrophy, bone marrow failure (where sufferers produce an inadequate number of red blood cells leading to bleeding and bleeding of the skin) pigmentation of the skin, oral leukoplakia, being the most common.

This leads me on to how does dyskeratosis congenita arise. Firstly as I explained above TERC plays an essential role in the reverse transcription process carried out by telomerase allowing telomeric repeats to be added to DNA during replication. Considering the parts of TERC complex more closely, we see the box H/ACA domain plays a key role, its role is to make sure the maturation and stability of the TERC complex is complete and thus the overall regulation of telomerase is carried out correctly. In mammals this domain is found to contain four protein subunits: Gar 1, dyskerin, Nop10 and Nhp2 and mutations within the Nop 10 (a substitution of base sequences from cytosine to thymine associated with autosomal inheritance) , Nhp2 (three single nucleotide polymorphisms, associated with autosomal inheritance ) and dyskerin 1(point mutations associated with X linked recessive inheritance) genes have been proven to cause dyskeratosis congenita . Furthermore, mutations in the TERT ( associated with autosomal dominant and recessive inheritance  )and DKC1 genes have been found to cause dysfunction of telomerase whilst mutations in the TINF2 gene( associated with autosomal dominant inheritance  ) have been found to cause mutations in the shelterin complex also leading to dyskeratosis congenita.  With all these genes being involved in the maintenance and elongation of telomeres, when mutated it proves rapid shortening of telomeres is the underlying mechanism of this disease. 

Furthermore with regards to inheritance patterns dyskeratosis congenita is found to also undergo an X linked recessive pattern, along with autosomal dominant and autosomal recessive. The X linked recessive pattern is induced by mutations on the DKC1 gene found on the distal portion ( more specifically band 28) of the long arm of the X chromosome ( Xq28). The DKC1 gene contains instructions for the synthesis of the dyskerin protein. As mentioned above dyskerin binds to Htr RNA molecule and stabilizes the telomerase complex whilst also playing a key role in ribosome synthesis. Point mutations are widley seen on the DKC1 gene which include either insertion or deletion of only one amino acid, leading to a frameshift mutation and the wrong amino acid encoded for, this is known as a missense mutation.

As these mutations are situated on the X chromosomes, this has big implications when it comes down to inheritance, as it explains to us why the X linked recessive disorder is more common in males as opposed to females. Firstly, as this is an X linked recessive disorder if only one X chromosome contained the mutation the female will only be a carrier of the disease, which is very common but if both the X chromosomes in females carry the mutated DKC1 gene symptoms of the disease will then arise, which is quite rare. However on the other side of the coin, as males have only a single X chromosome, if this chromosome contains the mutated DKC1 gene, and the offspring inherit this modified gene, they will be carries of the disease. Therefore only one mutated copy of the gene per cell in males is enough to generate the disease, whereas females must contain two mutated copies of the gene per cell to suffer from the disease.

 Furthermore, another piece of evidence supporting this X linked recessive mutation is the fact that all the daughters of a male with the condition will all be carriers, whereas it is impossible for his sons to be carriers. This is because males with the mutation on X chromosome only pass their Y chromosome on to their male offspring not their X chromosome, proving sons will never become carriers.

In conclusion, I have outlined the main features of telomeres, there function in eukaryotic DNA replication , the action of telomerase and how shortening telomeres can have life threatening consequences. Another aspect that I found whilst researching is how sufferers of dyskeratosis congenita have a pre disposition to cancer, something which seems paradoxical due to cells lacking telomeres but however is extremely common. Unfortunately I didn’t have time to outline this in my essay but is a grave consequence of dyskeratosis congenita and furthermore shows how shortening telomeres can have even more catastrophic effects on humans than we may think.

 

Bibliography

URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370421/ 

Article Title:Telomeres, lifestyle, cancer, and ageing

Website Title: Telomeres, Lifestyle, cancer and ageing

Date Accessed: 18 Mar. 2018

In-Text: Masood A Shamas

Published: 14 Jan 2011

Author: Masood A. Shammas, Harvard (Dana Farber) Cancer Institute, Boston, Massachusetts, USA;

URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933587/

Article Title:Telomerase and the ageing process

Website Title:  Telomerase and the ageing process

Accessed :18 Mar. 2018

In-Text: Hornsby 2007

Published: 30 Mar 2007

Author:Peter J. Hornsby, Department of Physiology and Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Texas;

URL:https://en.wikipedia.org/wiki/Dyskeratosis_congenita

Article Title: Dyskeratosis Congenita

Website Title: en.wikipedia.org

Accessed:18 Mar. 2018

In-Text: (En.wikipedia.org, 2018)

Published: 3 Mar. 2018

Author: Collective Authors

URL:https://ghr.nlm.nih.gov/condition/dyskeratosis-congenita#inheritance

Article Title :Dyskeratosis Congenita

Website Title: Genetics Home Reference

Author: Genetics Reference

Accessed : 18 Mar. 2018

In- Text: (Reference, 2018)

Published : 13 Mar 2018

Author: Unknown

URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269008/

Article Title :The genetics of Dyskeratosis Congenita

Website Title: The genetics of Dyskeratosis Congenita

Accessed : 18 Mar. 2018

In- text: Philip J Mason and Monica Besslera, 2011

Published: December 2011

Author:Philip J Mason Division of Hematology, Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania

and Monica Besslera Internal Medicine, University of Pennsylvania

URL:https://www.ncbi.nlm.nih.gov/gene/7012

Article Title:  TERC telomerase RNA component [Homo sapiens (human)] – Gene – NCBI

Article Title: ncbi.nlm.nih.gov

Accessed : 18 Mar. 2018

In- text : (Ncbi.nlm.nih.gov, 2018)

Published: 10 Mar 2018

Author: Unknown

URL :https://rarediseases.org/rare-diseases/dyskeratosis-congenita/

Article title: Dyskeratosis Congenita – NORD (National Organisation for Rare Diseases)

Website Title: NORD (National Organization for Rare Diseases). (2018)

Accessed : 18 Mar. 2018

In-text: (NORD ( National Organisation for Rare Disorders),2018)

Published: 2008

Author: NORD gratefully acknowledges the following for assistance in the preparation of this report: Monica Bessler, MD, PhD, Philip J. Mason, PhD, and David B. Wilson, MD, PhD, Departments of Internal Medicine and Pediatrics, Washington University School of Medicine.

Article Title: Dyskeratosis congenita mutations in the H/ACA domain of human telomerase RNA affect its assembly into a pre- RNP

Author: Trahan C, Dragon F

Published : February 2009

Date accessed: 18 Mar. 2018

Amplification of DNA by the Polymerase Chain Reaction & DNA Electrophoresis

Abstract:

In the polymerase chain response, it was consolidated with gel electrophoresis to find the “Alu gene” in DNA using transposons. Polymerase Chain Reaction utilizes certain elements to imitate certain duplicates of a DNA arrangement to help give the gel more “Alu squences” to be resolved simpler. Transposons take into consideration the most widely recognized Alu quality to be perceived using the gel electrophoresis. Each human might possibly contain a chromosome containing the alu quality and some may or may not even have one chromosome that contains the genome. The Polymerase Chain Reaction combined with gel electrophoresis takes into consideration the specific gene that will be identified and in our personal samples to be either homozygous/heterozygous or negative/ positive. 

Introduction:

“Polymerase chain response”, also known as PCR, utilizes DNA polymerase to reproduce another strand of DNA from a supplement format. The response needs the accompanying response blend parts: Deoxynucleotides (ddNTPs), Chromosomal DNA, and Taq DNA polymerase; and additionally reverse and forward primers. The thermocycler was required to begin the procedure through the means of tempering, expansion and denaturing. Denaturing utilizes warmth to isolate DNA strands, toughening places the forward and switch preliminaries to set up the expansion stage and the taq DNA polymerase prolongs the strands of the DNA in the expanded stage.

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The bits of DNA are “transposons” that can supplant itself in somewhere else in the genome. It is where it can recreate and basically reorder itself into another DNA. “Double-stranded DNA reinsert elsewhere in the genome, i.e., the classic “cut-and-paste” transposons” (Cédric) The accessible and the most available transposons are Alu components which are in the 300bp range. ALu genes are a standout amongst the most copious found in the “human genome at roughly 1 million” (Prayel).

The technique in which DNA strands are assessed by length is obtained by using “Gel Electrophoresis”. Every model is put into a well in the agrose gel where it is subjected to electrical current to ultimately have the DNA strands move towards the anode or positive charge bearing. “Shorter strands can push ahead on account of the manner in which that the gel contains sugar fibers that can obfuscate longer strands and its movements” (Heuer).

As every human is different, some may or may not have the alu gene. Some may even be carriers of the alu gene (Hetrozygous), maybe they have obtained the alu gene from only one of their parents and not the other. Whereas other that are homologous negative, have not received the alu gene from either one of their parents.

Methods:

Everyone in the lab will take an example of their cheek cell and break it down through using the PCR method by utilizing a toothpick and scratching within the sides of the cheek/mouth. Once done, put the finish toothpick that you scratched your cheek cells with. Use a microcentrifuge to obtain .3 ml and mix it with 100 uL of the buffer extraction and whirl it around. Name and top the test tube to exchange to the warm cycler at fifty-five degrees celsius for an hour. Towards the finish of the cycle, for approximately ten minutes, it will be at ninty-five degrees Celsius to inactivate the protein. Recoup and ensure the test tube is legitimately named.

Start to set up the PCR by marking a tube “alu” with your initials and add .2 microliters. Next, add the following: 7.5 uL of distilled water, 1.25 uL of forward primer, 1.25 uL of the reverse primer, 2.5 uL of all the cheek cell genomic DNA, and 12.5 uL of the “Master Mix”. Each table will set up a control .2 microliters of the PCR tube and name it “C” with their initials and instead of adding the genomic cheek cell DNA will be adding 2.5 uL of distilled water. Set the examples in the thermocycler and sit patiently for the teacher to begin it for thirty-five cycles. Record the temperatures and times through one finish cycle 3 times. The denaturing step was set at ninty-five degrees Celsius for an entire hour, and for 60 seconds at sixty-two degrees Celsuis was the annealing step and the seventy degrees Celsius for 60 seconds was also for the extending step.

Proceed forward and obtain the sample, utilizing the class size, make sure the class samples can fit the considerably even # of gels. With the fourteen-well comb put 1.5 percent of agarose gel and give it time to solidify totally. Setting the gels in the electrophoresis chamber closest the dark anodes put 1X TBE and expel the brush. In the most remote left and right segments embed bp ladder of 6 ul. Now, in everyone’s 20 microliter sample, add “four uL” of the dye and from the mixture put “five uL” in the gel. Run the gel for half-an-hour at 120 volts. Once completed, take the gel to the region specified by your TA to put the gel in an UV light box and to see the photo of what is shown.

Results:

The outcomes of this lab demonstrated that various models had particular results in that some were either negative or positive for the gene of the alu. And some could actually be carriers of the gene, and be “heterozygous”.  Only four students that were on the left side will be are area of interest for this result section.

As for the markers that were outside wells, “seven and one”, the well four was the control. And well “one and three” showed the alu gene negative homozygous meanwhile for well 4, there was a “alu gene” that was positive homozygous. Also, nothing was showed on the gel for well two and well 3. None of the models were seen to be heterozygous subject to the undeniable social occasions that could be surely watched.

By utilizing the gel electrophoresis, the photo shows the distinctive alu gene pieces. Wells “two through six” were one gathering of four understudies that will be inspected further and wells “nine through twelve” was another gathering of three understudies. Wells “one, seven, eight and thirteen” contained every single marker while “four and ten” were the controls of the examination. Sadly, the personal sample had no alu gene shown on the gel. Wells “two and four” contained groups at the 400bp implying that the examples were negative homozygous for the gene of the alu in the two chromosomes. Well “six” contained another example in which was positive homozygous for the gene of the alu in the two chromosomes.

Discussion:

As for the “Polymerase Chain Reaction”, the results can be better furthered and can be bettered improved through the utilization of the recently created “microfluidic systems”, in which multiple, various reactions can occur simultaneously at once. The Real-time “Polymerase Chain Reaction”, PCR are can be finished in about an hour or less, or, in other words, way faster than the ordinary PCR that has been utilized in this lab. Through the utilization of the “real-time PCR”, it allows for a quicker diagnosis of infectious disease that would otherwise be fatal if it is not figured out in a timely manner (Ahrberg).

The flaws that could have occurred for this lab are infinite and could have occurred at any given time during the duration of the experiment. For instance, the measurements could have been off and as a result, skew the whole outcome of the experiment. Or a simple mistake in the beginning of the lab, not taking enough cheek cells on the toothpick. This may have been because there was a lack of scraping correctly on the sides of the insides of the mouth. As a result, the lack of cells on the toothpick couldn’t be used for the PCR to see the alu genes.

As for the sample that was personal, the photo shows nothing on wells three. Wells two and five had contained samples that showed homogenous negative for the gene of the alu. Meanwhile, for the well six, it showed that the sample was indeed positive homogenous for the gene of the alu. The bands that were faint were not shown because it was impossible to legitimately have the capacity to tell which tests were homozygous or heterozygous for the gene of the alu for those that were faint in color.

Conclusion:

The hypothesis was not ready to be tried in light of the fact that the individual sample was not found or done inaccurately. In any case, utilizing the first sample one, the outcomes were valid that the example would be a homozygous negative for the gene of the alu. The identification of the gene depended entirely upon the individual samples on whether or not each sample had a chromosome for the alu gene. “Polymerase Chain Reaction”, PCR is imperative since it recreates DNA strands using distinctive parts. PCR is precise in separating the DNA strand than reconstructing another supplement.

References:

Feschotte, Cédric, and Ellen J. Pritham. “DNA Transposons and the Evolution of Eukaryotic Genomes.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, 2007,

Ahrberg, C D, et al. “Polymerase Chain Reaction in Microfluidic Devices.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, 5 Oct. 2016

Alberts, Bruce. Essential Cell Biology. New York, NY: Garland Science Pub, 2017. Textbook.

Prayel, Leslie A. “Functions and Utility of Alu Genes.” Nature News, Nature Publishing Group, 201

Heuer, Tim. “PROTEIN GEL ELECTROPHORESIS.” Oligo-DT Cellulose Columns, 1998, 

 

Questions:

SECTION B:  PCR Amplification of Alu Sequences

Q1) Proteins are denatured at high temperatures. So how are we able to perform PCR at these high temperatures using DNA copying enzymes?

We were able to do this because it had a thermostable enzyme called Taq polymerase, in which could not denature at high temperatures.

Q2) What was the annealing temperature that was programmed into our thermocycler? Why was this temperature chosen?

The temperature was sixty two degrees celsius, and the reasoning behind that was because the primers had to start synthesizing DNA at the low temperatures.

Q3) If the sequences of the PCR primers are;

5’-GGA TCT CAG GGT GGG TGG CAA TGC T-3’

5’-GAA AGG CAA GCT ACC AGA AGC CCC AA-3’

Calculate the melting temperature of each, based on the observation that for every A or T add 2oC, and for each C or G add 4oC?   Show your calculations.

For the 1st strand we had to do:

–          Guanine/Cytosine=15

–          Adenine/Thymine=10

–          15×4 +10×2=80 C

For the 2nd Strand we had to do:

-Guanine/Cytosine=14

– Adenine/Thymine=12

-14×4 +12×2=80 C

Q4) If the extension temperature was eliminated altogether from the thermocycler, what would happened to our Alu PCRs? [Hint: the size of your fragments is important]

The alu PCRs will would become shorter because during the extensions stage, the DNA will become extended so that they don’t shrink. This is to ensure that the final product once done will display the alu genes, showing whether it’s is a either a negative/ positive for the heterozygous or homozygous.

 SECTION C:  Electrophoresis of Amplified DNA

Comprehension Questions:

Q1. What is a transposon?

A transposon, in essence, is a piece of “Deoxyribonucleic Acid”, DNA that that can replace itself in the DNA sequence.

 

Q2. How common are transposons in the human genome?

Interesting to note, Transposons are common in the human as there are approximately over a million of them in your very genome.

Q3. How does a transposon “jump” from one site in the genome to another?

They jump from site to site in the genome by a method that is called, “copy and paste”. This helps replicate itself using the mRNA and then cut out DNA pieces and incorporates itself inside.

Q4. What is a polymorphism?

Polymorphism is variation in the DNA’s sequence in which is a common in the population.

Q5. How can PCR be used to determine the Alu genotype of an individual?

 

PCR is utilized to determine the alu genoype of an individual by the use of gel electrophoresis. By using gel electrophoresis, the alu genotype of a person can be seen in the band length and therefore show whether a sample is positive or negative for the alu gene.

Q6. How is Taq DNA polymerase different from DNA polymerase found in E. coli?

Taq DNA polymerase is different from DNA polymerase found in E. Coli in that , the Taq is a lot quicker in the breakdown of the DNA by using high temperature.

Q7. What is the role of the forward and reverse primers used in PCR?

One of the main roles of the “forward” and “reverse” primers allows the DNA’s target reason to replicate.
 

Transfer of a DNA Fragment from pMB to Puc19

Subcloning of the PstI fragment from pMB into pUC19

1. introduction

      DNA should never be treated of considered as a static molecule since those molecules have a significant dynamic movement. It is constantly changing, and one mechanism by which this change is brought about is recombination.

     Recombination is the ability to transfer a specific gene from one organism to another resulting in offspring differ from either parent. It is a DNA re-arrangement between

two molecules of DNA having homologous sequences. The main objective of doing recombinant DNA technology is often to create a beneficial product or commercial ones.

      Recombination provides the tools through which genetics information is reformed. This, of course, provides an evolutionary advantage to eliminate the unwanted

mutations and to maintain the spreading of the desired mutations. Moreover, it has a significant role in conferring each individual a unique group of genetic information. Recombination must be taken place between precisely corresponding sequences to ensure no loss or addition for any base pair.

After the recombination has been done, in order to identify the recombinant vector from non-recombinant ones, two techniques must be used (Bolivar et al, 1977). First, growing the E. coli on the antibiotic-containing medium, which contains ampicillin. This one used to distinguish the recombinant bacteria from the non-recombinants. That can be determined easily since recombinant bacteria carry plasmid that has the ampicillin-resistance gene (AmpR), and in which can grow on ampicillin based medium, however, the non-recombinants cannot grow on the growth media since they have no resistance to the ampicillin (Smalla, Jechalke and Top, 2015). The problem, not all the plasmids transformed into cells may contain the inserted gene. Some of the cells do have the plasmid but without the inserted gene.

The second technique is the LacZ a-complementation (Fig1), which is a screening technique that allows the detection and distinguishes recombinants bacteria with insert DNA fragment from the one with only the plasmid but without the inserted gene by using blue/white selection to identify recombinant plasmid clones (Tolmachov, 2009).

 β-galactosidase is a protein produced by E. coli strain that carries the lacZ gene. In its active state, if bacteria are grown on an agar plate that has a substrate known as X-gal. when β-galactosidase is produced, X-gal is hydrolyzed(Tolmachov, 2009). Consequently, they produce an insoluble blue pigment. However, when Insertion of foreign DNA from pMB into the plasmid at the MCS region, which is located within the lacZα sequence (which can be cut by restriction enzymes),that would cause insertional inactivation(Smalla, Jechalke and Top, 2015). That would create a non-functional β-galactosidase enzyme, 

thereby it will stop the gene that produces α-peptide from working. Consequently, in cells that contain the plasmid with an insert in LacZα sequence, no functional β-galactosidase will form (Smalla, Jechalke and Top, 2015).

Therefore, The E.coli that has plasmid with no insertion appears blue in color since LacZ is functioning while the recombinant ones with insertion foreign DNA appear white. Consequently, it’s easy to pick the desired recombinant colonies from the culture.

 

 

 

 

 

 

1.1 Aim of the experiment

Subcloning experiment: transfer a DNA fragment from pMB to Puc19. Then detect the bacterial clones carrying the recombinant plasmid by using the insertional activation system.  Lastly, successful subcloning demonstrated by using restriction digest to confirm that the bacterial clones do contain recombinant plasmids.

2. Results:

Ampicillin

Kanamycin

Tetracycline

DH5

pUC19

+

+

pMA

+

+

pMB

+

XL1-blue

+

In this experiment, five different bacterial samples (Table1) have been tested for antibiotic resistance against three different antibiotics (Ampicillin, Kanamycin, Tetracycline). They are commonly used as selective agents in various cloning vectors.

       For the first antibiotic Ampicillin, only bacterial who contain Puc19 plasmid and pMB were resistant to Ampicillin, and the rest showed no growth. these suggest that pUC19 and pMB have the antibiotic-resistant gene in their plasmid (AmpR) against Ampicillin.

       However, for the Kanamycin, only bacterial that contain pMA plasmid were able to grow; these suggest that pMA plasmid has Kanamycin resistant gene.

       Lastly, for the Tetracycline antibiotic, bacterial who contain Puc19, pMA, and XL1-blue plasmids were able to grow. these suggest that bacteria that contain Puc19, pMA and XL1-blue plasmids have the antibiotic-resistant gene for Tetracycline

 

 

 

 

 

 

 

 

2.2. The double digests of pMA and pMB:

 

*Because of poor gel preparation (overheated and that increased the concentration of the gel) gel image has been obtained from group 4, everything below will be explained based on the date that has been obtained by group 4.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FIGURE  3. Restriction endonuclease digestions and electrophoretic separation of fragments of pMA and pMB

with a ruler to indicate the precise size of the fragment in centimeter. (refer to Fig 2, for the legend, since they share the same legend)

 on the left side, as marked from lane 1-7, it shows endonuclease digestions and electrophoretic separation of fragments of the plasmid pMA. On the right side as marked from lane 8-14 it shows endonuclease digestions and electrophoretic separation of fragments of the plasmid pMB. those abbreviation has been used to identify each restriction endonuclease enzyme as following

B: BamHI, E: EcoRI, P: PstI, X: XhoI and M: DNA marker

 

 

 

 

*cm

Bands

(lengths of the bands in kilo base pairs.)

2.65

10 kbp

2.75

8 kbp

2.9

6 kbp

3.09

5 kbp

3.2

4 kbp

3.38

3.5 kbp

3.59

3 kbp

3.72

2.5 kbp

4

2 kbp

4.5

1.5 kbp

5.25

1 kbp

5.88

0.75 kbp

6.7

0.5 kbp

7.8

0.25 kbp

FIGURE 4. measurement of DNA marker with a ruler. the y-axis represent the DNA marker measurement, while x-axis represent the ruler measurement in centimeter.

 

 

 

 

 

 

 

 

 

 

2.3. Restriction maps of pMA and pMB.

Table 3. Shows what has been electrophoresed on each lane from lane 8-14 (Fig 2) and what Restriction enzymes had been used on pMB plasmid, and the Size of the fragment that has been obtained. the numbers denote the lengths of the digestion products (fragments) in base pairs.

Table 2. Shows what has been electrophoresed on each lane from lane 1-7 (Fig 2.) and what Restriction enzymes had been used on pMA plasmid, and the Size of the fragment that has been obtained. the numbers denote the lengths of the digestion products (fragments) in base pairs.

 

lane

Digest performed on PMB

Size of fragment obtained

8

EcoRI +  XhoI

3100 bp, 1900 bp

9

EcoRI + PstI

3000 bp, 1300 bp, 790 bp

10

EcoRI + BamHI

4700 bp, 350 bp

11

BamHI + XhoI

2800 bp, 2200 bp

12

BamHI + PstI

2550 bp, 1275 bp, 1150 bp

13

PstI + XhoI

3600 bp, 1100 bp

14

XhoI

5050 bp

lane

Digest performed on PMA

Size of fragment obtained

1

BamHI

3600 bp

2

EcoRI +  XhoI

3600bp

3

EcoRI + PstI

2800 bp, 700 bp

4

EcoRI + BamHI

3000 bp, 400 bp

5

BamHI +  XhoI

3600 bp

6

BamHI + PstI

2400 bp, 1100 pb

7

PstI +  XhoI

3600 pb

 

FIGURE 5 Restriction endonuclease map for the digestion products presented in Table2. For pMA plasmid. The circular DNA has 3,600 bp (3.6 kb). With the EcoRI site arbitrarily placed at 12 o’clock position, the locations of the other mapped restriction endonuclease sites are marked.

FIGURE 6 Restriction endonuclease map for the digestion products presented in (Table3). For pMB plasmid. The circular DNA has 5,050 bp (5.05kb). With the EcoRI site arbitrarily placed at 12 o’clock position, the locations of the other mapped restriction endonuclease sites are marked.

The process of making restriction maps for circular DNA plasmid is, in general, the same as making a restriction maps for a liner DNA, except that each time that the restriction enzyme cleave produces a fragment. In other words, three fragments are formed when three sites are cut by the endonuclease enzyme, and so on. It is also important to mention that Each restriction enzyme cut at a specific site.

With the data obtained for pMA (Table 2),the deduction of a restriction digest map for the pMA plasmid is as follows.

       The source DNA plasmid is a 3.6 kilobase-pair (kb) produced with a single digest of BamHI, which suggests the length of the plasmid is 3.6 kilobase-pair since only one fragment has been obtained when the plasmid treated with single digest endonuclease.

        Digestion with (EcoRI + XhoI), (PstI + XhoI) or (BamHI + XhoI) produces a single restriction fragment, that suggests that there is no restriction site for XhoI, in other words, XhoI doesn’t cut pMA plasmid since there is no restriction site for XhoI.

       However, when double digest performed with (EcoRI + PstI), (EcoRI + BamHI) and (BamHI + PstI) produced two fragments. which suggests that EcoRI, PstI, BamHI each one of them has only one restriction site on the plasmid.

       The results of the EcoRI and PstI double digestion produced two fragments 2.8-kb, 700-bp. for the double digestion of EcoRI + BamHI produced 3-kb, 400 bp. for BamHI + PstI two fragments 2.4-kb, 1.1-kb produced. Based on that

 In general, the double digest of each of (EcoRI + PstI), (EcoRI + BamHI) and (BamHI + PstI) separately produced length, which roughly resonates with a length of the pMA plasmid.

 

With the data obtained for pMB (Table 3),the deduction of a restriction digest map for the pMB plasmid is as follows.

       The first single digestion (Fig 3/Table 3, lane 14) with XhoI produced one fragment, which is about 5.05-kb. The suggest the length of plasmid pMB is 5.05-kb.

       Digestion with XhoI and EcoRI (Fig 3/Table 3, lane 8), XhoI cleaves 5.05-kb EcoRI into two fragments 3.1-kb and 1.9-kb.

      Also, BamHI + XhoI (Fig 3/Table 3, lane 11), the double digestion of BamHI + XhoI, produced two fragments. That indicates XhoI, BamHI, and EcoRI cut at one site, and each one separately produces one single fragment.

      However, for the PstI restriction enzyme, when double digest performed with EcoRI (Fig 3/Table 3, lane 9), three fragments have been produced 3-kb, 1.3kb, and 790 bp. Moreover, when double digested with PstI + BamHI (Fig 3/Table 3, lane 12),three fragments also have been generated with different 2.55-kb 1.275-kb and 1.15-kb. Based on that, the date obtained for the gel electrophoresis image (Fig3) suggests that pstI cuts in two sites on the pMB plasmid. pstI has two recognized restriction sites inside the plasmid.

That been said, however, when double digests performed for pstI + XhoI  (Fig 3/Table 3, lane 13) only produced two fragments, 3.6-kb and 1.1-kb.

 

 

The similarities and differences between the two plasmids.

 

After the double digest and based on the data, the following similarities and differences between pMA and pMB plasmid have been observed:

First, the length, based on the restriction maps obtained from the gel electrophoresis (Table 3, Fig 3) pMA, is shorter than pMB (Fig 5) since pMA is about 3.6-kb whilst pMB is about 5.05-kb. That resonates with () that pMB derived from pMA.

Second the restriction site,

As shown and indicate in the restriction map above (Fig 5), XhoI doesn’t cut pMA plasmid, which indicates there is no restriction site for XhoI in pMA plasmid. However, with pMB, XhoI produce a single fragment during endonuclease (Fig 6/ Table 3)

For the PstI enzyme, based on the result that has been obtained, PstI has one site when digest pMA plasmid and produced only one fragment, which indicates that it has only one restriction site on the pMA plasmid. However, when pMB double digested PstI with EcoRI or BamHI, it produced three fragments, and since  EcoRI and BamHI cut only once based on the data ( fig 4,5 table 4,5) that indicates and suggest that PstI has two restriction sites in pMB plasmid.

3. DISCUSSION

For some restriction endonuclease mapping experiments, the sum of the fragments of some multiple digestions is less than the total length of the starting DNA because the fortuitous locations of some sites produce fragments of the same size. Under these conditions, two different fragments

with the same length that migrate to the same location in a gel after electrophoresis

often stain more heavily than a band with only one kind of fragment.

This difference in staining intensity gives an indication that

coincidental fragments have been produced by restriction endonuclease

digestion.

The resolution of fragments for restriction endonuclease mapping can

be enhanced by labeling the pieces of DNA, usually at the 5’ends, with a

radioactive compound or fluorescent dye and determining their lengths

after electrophoretic separation with autoradiography or fluorography,

respectively. A standard 5’-end-labeling procedure entails dephosphorylation

of the 5′ends of a linear DNA molecule with calf intestine alkaline

 

Reference

Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten. 2010

Molecular biotechnology : principles and applications of recombinant DNA /

. 4th ed.

Geertsma, E. R., and B. Poolman.

2007. High-throughput cloning and

expression in resistant bacteria. Nat.

Methods 4:705–707.

Radnedge, L., and H. Richards. 1999.

The development of plasmid vectors.

Methods Microbiol. 29:51–95.

Garfin, D. E., 1995. Electrophoretic

methods, p. 53–109. In J. A. Glasel and

M. P. Deutscher (ed.), Introduction to

Biophysical Methods for Protein and

Nucleic Acid Research. Academic Press,

Inc., San Diego, CA.

Pingoud, A., M. Fuxreiter, V.

Pingoudand W. Wende. 2005. Type II

restriction endonucleases: structure

and mechanism. Cell. Mol. Life Sci.

62:685-707.

 
 

Identification of Unknown Bacteria with DNA Sequencing

INTRODUCTION

 Bacteria are microscopic, single-celled organisms that can be found in all kinds of environment, that includes inside and outside of other organisms. They are the most diverse and successful group out of all prokaryotic organisms. The goal of this lab is to identify the unknown bacteria by traditional cellular and biochemical tests and by DNA sequence-based methods of species identification.

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 The first step of identifying the unknown bacteria is to determine the shape, size, color, and other characteristics of it. These characteristics help us determine if the colonies are bacteria, yeast, or mold. Bacteria will appear flatter and liquidity compared to yeast which will generally be puffier. Mold will look fuzzy and has a volcano-like elevated center. Bacterial colonies have multiple shapes such as circular, rhizoid, irregular, or filamentous. The colonies can also differentiate with different edges such as entire, undulate. Lobate, filamentous, or curl. Bacterial cells can vary in shape. For example, round (cocci), rod-like (bacilli), or helical (spirochete).

 The next step of identifying the unknown bacteria is to determine if enzyme catalase is present in the bacteria or not. Catalase is an enzyme that is produced by microorganisms that live in oxygenated environments that neutralize the bactericidal effects of H2O2. The catalase will break down the H2O2 into oxygen and Water. In order to find out if the bacteria can produce catalase enzyme, a small sample of the bacteria will be mixed with 3% hydrogen peroxide and observed for bubbles.

 An oxidase test will also help identify the unknown bacteria. The oxidase test is used to reveal whether the bacteria can produce cytochrome c oxidase, an enzyme for the bacterial electron transport chain. Which means bacteria that test positive for the oxidase test are aerobic, which means it can use oxygen a terminal electron acceptor in respiration. Bacteria that test negative either cannot use oxygen as an electron acceptor or utilize a different cytochrome to transfer electrons to oxygen. For this test, we will be observing for color change on the oxidase slide.

 Then a Mannitol Salt Agar (MSA) test is performed. This test is used to test for salt tolerant. Because MSA contains a high concentration of sodium chloride, only the salt-tolerant species can grow on it. Non-salt tolerance species cannot survive to reproduce on this medium. And the fermenting species would change the color of the medium from red to yellow or orange.

 Next, we determined whether the bacterial sample was Gram-Positive or Gram-Negative. Gram-Positive bacteria have cell walls composed of thick layers of peptidoglycan while Gram-Negative have cell walls with a thin layer of peptidoglycan. With that, we started culturing our bacteria on three different mediums: EMB-Lactase, PEA, and vancomycin. Gram-Positive bacteria will grow on PEA medium only. While the Gram-Negative bacteria will grow on EMB-Lactose and vancomycin medium. The potassium hydroxide test (KOH string test) is another test that we did to determine if the bacterial sample was Gram-Positive or Gram-Negative. The KOH will dissolve the thin layer of peptidoglycan of the cell walls on the Gram-Negative bacteria, but it has no effect on the Gram-positive cell walls. The KOH will dissolve the cell walls and the content of the bacteria including the DNA will be released. Because of the DNA the solution will become viscous and it will stick to the toothpick creating a “string”.

 To perform the last test, which is gel electrophoresis, we used PCR to amplified DNA from our unknown bacteria. The 16S Ribosome gene will be isolated and amplified to provide us with the template for the DNA sequence used to help us identify the unknown bacteria. Once the DNA is sequenced it was edited in Chromas and ran through BLAST, a database that has millions of gene sequences, to be aligned with the best possible match allowing us to identify the unknown bacteria.

MATERIALS AND METHOD

Making a Live Culture

 To make the live culture, we scraped a colony of the unknown bacteria and transfer it to a PCR tube containing 295 μL of sterile water. Then we vortex the tube for 2-3 seconds to uniformly distribute the cells in the water (Holbrook and Leicht, 2019).

Setting up PCR Tubes

 Once the live culture has been created, we set up the PCR tube. We made two PCR tube; one with bacterial DNA and the other for control which contains no DNA and both containing 25 μL of 2X PCR Master Mix. We added 5 μL of live bacterial culture and 20 μL of 16S rRNA Primer Mix to the tube with bacterial DNA. In our control tube, we added 5 μL of sterile water and 20 μL of 16S rRNA Primer Mix. Then both tubes were briefly centrifuged in the microcentrifuge. Once mixed, the DNA was amplified by the following procedure (Holbrook and Leicht, 2019):

1X: 94oC, 3 min

30X: 94oC, 30 sec; 50oC, 30 sec; 72oC, 45 sec

1X: 72oC, 5 min

Purification of PCR Products

 After the DNA amplification is done, the content in the tube with bacterial DNA was transfer to a new 1.5 mL microcentrifuge tube. Next 250 μL of Buffer BB was added to the PCR sample. Then the PCR sample was transferred to a spin column in a collection tube and centrifuge for 30 seconds at room temperature in the Eppendorf5430. Next, the flow through was discarded and placed back into the collection tube. 200 μL Buffer WB was added to the spin column and centrifuge for 30 seconds in the Eppendorf5430. The flow through was discarded again. Then the process of adding WB Buffer, centrifuge, and discarding was repeated. Then the spin column was transferred to a 1.5 mL microcentrifuge tube. Next, 25 μL Buffer EB was added to the center of the membrane of the spin column and left to stand for 1 minute. After 1 minute, it is centrifuge for 30 seconds in the Eppendorf5430. Then the spin column was discarded and the 1.5 collection tube that contains the DNA will be used for gel electrophoresis and sequencing (Holbrook and Leicht, 2019).

Catalase Test

 To conduct the catalase test, we obtained a microscope slide and place it in an empty Petri dish. Then the unknown bacterial were collected and smeared onto the microscope slide. We added one drop of 3% hydrogen peroxide onto the smeared bacterial and watched for bubble formation (Holbrook and Leicht, 2019).

Oxidase Test

 First, we obtained a dry oxidase slide with four test areas for an oxidase-positive control, an oxidase negative control, and for two unknown bacteria. We collected each bacterial sample to spread onto the corresponding areas of the oxidase slide, then leaving the oxidase slide to incubate at room temperature for at least 20 seconds. After 20 seconds, an oxidase-positive bacterium should exhibit a color change (Holbrook and Leicht, 2019).

MSA Test

 For the MSA test first, we added the unknown bacteria to a tube containing sterile water. Then, using a micropipette we added 50-75 μL of the liquid bacterial culture and 5-10 sterile glass beads to MSA containing Petri dish. The Petri dish was shaken up and removed once the liquid bacterial culture is evenly distributed. They were incubated for 2 days at 37oC before we could observe the growth on the dish (Holbrook and Leicht, 2019).

Plating on Test Media

 A liquid culture containing unknown bacterium and 300 μL of sterile water was added to EMB-lactose, PEA, and vancomycin containing agar. The 5-10 sterile beads were also added and remove once the liquid culture is distributed evenly. The Petri dishes were incubated for 1-2 days at 37oC before we could observe the growth on the dish (Holbrook and Leicht, 2019).

 

 

KOH String Test

 50 μL of 3% KOH were added to a microscope slide with the unknown bacteria. The solution was stirred for about 60 seconds. Then using a sterile toothpick, we test to see if the solution has a “string” appearance (Holbrook and Leicht, 2019).

Preparing the Gel for Analysis of Purified PCR Product

 To prepare the 40ml of 1.5% agarose solution we added 0.6g of agarose and 40ml of 1X TBE buffer into a glass flask. Then we microwave the solution for 45-60 seconds until the solution boiled. While the gel is cooling, we set up the gel tray. After the flask has cooled to the point where it is warm but not hot, ethidium bromide was added to the flask. Then we gently swirled the flask to mix the solution. Once mixed we poured the liquid into the gel tray and leave it to set completely. When the gel has solidified, we removed the comb and placed the tray with the gel onto the electrophoresis chamber and filled the chamber with 1X TBE buffer until the gel completely covered with buffer (Holbrook and Leicht, 2019).

Loading and Running the Gel

 First, we added 6 μL of purified 16S rRNA PCR products and 4 μL Loading Dye into the microcentrifuge tube with the bacterial sample. As for the control microcentrifuge tube, we added 12 μL of the control PCR reaction and 4 μL of the Loading Dye. Then both tubes were spin for about 10 seconds in the mini centrifuge. Next, using the P20 micropipette we load 10 μL of bacterial DNA into wells 2, 5, and 7 (three different groups shared the gel, so each well had a different bacterial DNA), 10 μL of the controls were added to wells 3, 6, and 8, 10 μL of DNA Size Stand were added to well 4, and leaving well 1 empty. The lid was placed onto the electrophoresis apparatus and was set at a high voltage. Then we ran the gel until the blue dye moved about ¾ of the length of the gel. Once the gel had finished running, it was removed and photographed with the Fotodyne UV illuminator and camera (Holbrook and Leicht, 2019).

DNA Sequencing Reaction

 To create the dilution, we mixed together 2 μL of purified PCR product and 6 μL of sterile water. Then to get to the concentration of 3-10 ng of PCR DNA, we used 4 μL of the dilution and 6 μL of Big Dye mix. The tube was mixed and placed into the thermocycler. The following program was used (Holbrook and Leicht, 2019):

1X: 96oC, 1 min

30X: 96oC, 10 sec; 50oC, 5 sec; 60oC, 2 min

Hold: 4oC

Chromas and BLAST Database

 Once the DNA is sequenced, we used Chromas to analyze it. The poorly read ends were trimmed out, and the “N” basses are changed to whatever base had a peek at that location. Once edited, the sequence is copy and pastes into the BLAST database to determine the species of the bacteria (Holbrook and Leicht, 2019).

RESULTS

Cell Morphology

 When observing our petri dish containing the unknown bacteria, it was slightly raised with a yellow tint color throughout the colonies. All the colonies were circular with a smooth and glistening surface. When observing our live culture under a microscope, we observed the bacteria shape to be rod-like.

Catalase/ Oxidase Tests

Figure 1: Results of Catalase and Oxidase Tests.

When we performed the catalase test with our unknown bacteria, the hydrogen peroxide reacted strongly with the bacteria. There was an immediate formation of bubbles which was created by the conversion of H2O2 to O2 and H2O. As for the Oxidase test, the bacteria reacted strongly on the oxidase slide. When the bacteria were smeared onto the slide it turned blue indicating that the bacteria possess cytochrome c oxidase.

MSA Test

Test

Results

MSA Test

No growth

Figure 2: Results of the MSA test.

 The growth of the unknown bacteria was observed when it was placed on MSA. No growth was observed which means the unknown bacteria is a non-salt tolerant species.

 

 

Growth on Different Medium

Growth on Selective Medium

Forms a String KOH

Vancomycin

EMB-lactose

PEA

Amount of growth

+

N/A

Colony color

Yellow

N/A

N/A

N/A

Gram-positive or

Gram-Negative

Gram-Positive

Gram- Negative 

Gram-Negative

Figure 3: Classification of Unknown Bacteria as Gram-Positive or Gram-Negative.

Figure 4: Plate growth from 3 media. Vancomycin (Left), EMB-lactose, and PEA (right).

 The unknown bacteria were observed for growth on here different media; Vancomycin, EMB-lactose, and PEA. Only Vancomycin showed growth of a yellow color colony which indicates that it is a Gram-Negative Bacteria. Since there was no growth in EMB-lactose media, it indicated that the unknown bacteria are Gram-Positive. There was also no growth shown on the PEA media which means that the unknown bacteria are Gram-Negative. We also conducted a string test to help us determine if our bacteria are Gram-Positive or Gram-Negative. The string test indicated that our unknown bacteria is Gram-Negative.

Gel Electrophoresis

Figured 5: Gel electrophoresis of unknown bacterial DNA

1  B2  C3  SS B5 C6  B7 C8 88

 

 

 

 Figure 5 is the results of the gel electrophoresis of the unknown bacteria.

We can see all three lanes that have bacterial DNA. The band in lane B2 is half as bright compared to the size standard band. As for band in lane B5 and B7, it is as bright as the size standard band. The negative control lane (C3, C6, and C8) did not have any band which means there was no contamination.

Chromas and BLAST Database

Figure 6: The edited chromatogram from sequenced DNA.

Figure 7: Top 5 Match from the BLAST database for unknown bacteria

Figure 8: Best nucleotide alignment of Unknown Bacteria DNA sequence and the closest match species.

With the sequenced DNA, we were able to edit it and submit it to the BLAST database. With this, we found out that our bacteria stand was Aeromonas shown in figure 7.

Discussion

 The unknown bacterial DNA sequence of 16S rRNA gene matched up with the sequence of Aeromonas. Aeromonas is rod-shaped bacteria. Their colonies are smooth, convex, rounded and are tan/buff-colored. It is also Gram-Positive, Oxidase positive, and Catalase Positive. Aeromonas can exist in both aerobic and anaerobic environment. Aeromonas is also salt tolerance but up to a certain concentration.

 The characteristic of Aeromonas is consistent with our finding. We observed the same morphology to what was described of Aeromonas. The colonies were smooth, with a glistening surface. As the for the bacteria cell itself, it is a rod-like shape. Our catalase test and oxidase test shows that Aeromonas can produce enzyme catalase and possesses cytochrome c oxidase. Aeromonas is a Gram-Negative bacterium.  It is consistent with the other test we have done, for example, growth on the Vancomycin, growth on the PEA, and the string test. With the similarities in the physiology and morphology, we can conclude that the DNA sequence was accurate, and the unknown bacteria is Aeromonas.

 The tests that were not consistent with our finding is the EMB-lactase test. There was no growth on the EMB-lactose medium. A reason why could have occurred is that while making our live culture, we did not put enough of the cells into the solution. Another inconsistent is the MSA test. There was no growth on our MSA medium which indicates that the bacteria are non-salt tolerance. Aeromonas is a salt-tolerance bacterium but only to about 6%. MSA contains about 7.5% – 10% concentration of salt. This could have been a reason why we had no growth in our MSA medium.

 Overall, most of our results were consistent with the sequencing and the BLAST results. We conclude that the unknown bacteria was Aeromonas, a gastrointestinal pathogen that can 

cause food poisoning and diarrhea.

References

“BLAST: Basic Local Alignment Search Tool.” National Center for Biotechnology Information, U.S. National Library of Medicine, https://blast.ncbi.nlm.nih.gov/Blast.cgi.

“Chromas: Technelysium Pty Ltd.” Chromas | Technelysium Pty Ltd, https://technelysium.com.au/wp/chromas/.

Holbrook, Mark A., Brenda G. Leicht. Diversity of Form and Function Biology 1412 Lab Manual. Eight edition, Department Of Biology at the University of Iowa, 2019

Igbinosa, Isoken H, et al. “Emerging Aeromonas Species Infections and Their Significance in Public Health.” TheScientificWorldJournal, The Scientific World Journal, 2012, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373137/.

Sadava, Hillis, Heller, and Berenbaum. Life: The Science of Biology. Tenth edition, Sinauer Associates, 2014.

 

Methods for Extracting DNA from the cells of Green Split Peas and Chicken Livers

Abstract:
In this experiment, I extracted DNA from the cells of Green Split Peas and Chicken Livers. I used different variables for each. First, I did the experiment with all materials being cold. The second time I did the experiment with materials at room temperature. My objective was to see which method would extract more DNA. The results were that the materials being colder extracted more DNA than the room temperature materials. In either case, I was able to extract more from the Green Split Peas than the Chicken Livers both times.
Introduction:
DNA is short for deoxyribonucleic acid. Nucleic acid, which is the genetic material determining the makeup of all living cells and many viruses. It consists of two long strands of nucleotides linked together in a structure resembling a ladder twisted into a spiral. The rungs of the ladder are made up of nucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G). DNA can replicate itself. DNA also serves as a template for synthesis of RNA in the presence of RNA polymerase. (APA, Dictionary. com)

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DNA is located in the chromosomes in of the human body. It is like blueprints or instructions for hair color, eye color, height, pretty much everything. DNA can be used to identify criminals with unbelievable exactness when genetic evidence exists. Similarly, DNA can be used to clear suspects and clear persons wrongly accused or convicted of crimes. In all, DNA is ever-increasingly crucial to ensuring precision and fairness in the criminal justice system. (The United States Department of Justice) DNA is used in a few ways to solve crimes. Comparing a suspects DNA to DNA found at the scene, or it can be put in the database to try to find the offender’s match. DNA can also be used to identify victims.
Materials and Methods:

Chicken Livers
Green Split Peas
91% Rubbing Alcohol
Meat Tenderizer
Salt
Blender
4 small glass Pyrex bowels
Small strainer
Wooden Chopstick
Measuring cup
Liquid soap

Put in a blender: 1/2 cup of split peas (with the chicken livers I used 3 medium sized livers. ), 1/8 teaspoon salt, 1 cup cold water
Blend on high for 15 seconds
Pour through a strainer into measuring cup.
Add 2 tablespoons liquid soap and swirl to mix.
Let the mixture sit for 5-10 minutes.
Pour the mixture into glass bowls, each about 1/3 full.
Add a pinch of meat tenderizer and swirl very carefully
Tilt bowls and pour in rubbing alcohol. (Until it forms a layer on top of mixture)

Results:
The first experiment I used very cold materials, with the peas and with the chicken livers. After blending the peas, I got a very light green colored water solution. After straining, there was a lot of shell like material at the bottom of the blender. I then added the 2 tablespoons of liquid soap to the mixture and allowed it to sit for about 10 minutes. I then transferred the “soup” into the Pyrex bowls, where I added a pinch of meat tenderizer and stirred very gently with the wooden chopstick. After stirring the meat tenderizer in, I then added the rubbing alcohol until it formed a layer on top of the mixture. Almost immediately, I was able to see the white colored stringy material in the peas. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top. I was able to transfer the DNA to another Pyrex bowl with alcohol in it to get a better look.

After blending the livers, I got a very reddish, beige colored water solution. After straining, there was liver material at the bottom of the blender. I then added the 2 tablespoons of liquid soap to the mixture and allowed it to sit for about 10 minutes. I then transferred the “soup” into the Pyrex bowls, where I added a pinch of meat tenderizer and stirred very gently with the wooden chopstick. After stirring the meat tenderizer in, I then added the rubbing alcohol until it formed a layer on top of the mixture. Almost immediately, I was able to see the white colored stringy material in the livers. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top I was able to transfer the DNA to another Pyrex bowl with alcohol in it to get a better look.
The second experiment I used almost room temperature materials with the peas and with the chicken livers. After blending the peas, I got a very light green colored water solution. After straining, there was a lot of shell like material at the bottom of the blender. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top. I then added the 2 tablespoons of liquid soap to the mixture and allowed it to sit for about 10 minutes. I then transferred the “soup” into the Pyrex bowls, where I added a pinch of meat tenderizer and stirred very gently with the wooden chopstick. After stirring the meat tenderizer in, I then added the rubbing alcohol until it formed a layer on top of the mixture. This time there was hardly any of the white stringy material I found in the first experiments, and barely anything to transfer.

After blending the livers, I again got a very reddish, beige colored water solution. After straining, there was a lot of material at the bottom of the blender. I then added the 2 tablespoons of liquid soap to the mixture and allowed it to sit for about 10 minutes. I then transferred the “soup” into the Pyrex bowls, where I added a pinch of meat tenderizer and stirred very gently with the wooden chopstick. After stirring the meat tenderizer in, I then added the rubbing alcohol until it formed a layer on top of the mixture. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top. This time there was hardly any of the white stringy material I found in the first experiments, and yet again, barely anything to transfer.
Discussion:
My results were surprising. These experiments also produced 3 layers: debris at the bottom, water in the middle, DNA floating at the top.
It really made a difference in the outcome just by using colder materials. Using ice-cold water and ice-cold alcohol will increase the yield of DNA. “Low temperatures protect the DNA by slowing down the activity of enzymes that could break it apart. The cold alcohol helps the DNA precipitate (solidify and appear) more quickly. Why would a cell contain enzymes that destroy DNA? These enzymes are present in the cell cytoplasm (not the nucleus) to destroy the DNA of viruses that may enter our cells and make us sick. A cell’s DNA is usually protected from such enzymes (called DNases) by the nuclear membrane, but adding detergent destroys that membrane. ” (gslc. genetics. utah. edu)
Out of the two, the warmer water experiments eliminated the most debris, but were not very successful at extracting as much DNA material.
References:
American Psychological Association: dna. (n. d. ).  Dictionary. com Unabridged. Retrieved April 12, 2015, from Dictionary. com website:http://dictionary. reference. com/browse/dna
Explorable : https://explorable. com/who-discovered-dna
Learn. Genetics, The Genetic Science Learning Center, Retrieved from: http://learn. genetics. utah. edu/content/labs/extraction/howto/faq/