UCB BIo 1AL Part 2 August - III. DNA Sequencing th...

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LAB GMB I: Mitosis, Meiosis and DNA sequencing Page # 141 H H H O Base P O O O H H H CH 2 O 2 H H H H O P O H H H O O 2 H H O P O O P O O OH O O O O P O O O P O O P O O O O H H H O P O O O H H H 2 O 2 H H H H H H H O 2 H H O O P O O O P O O P O O O O O P O O O H H H O P O O O H H H 2 O 2 H H H H O O P O O O P O O P O O O O H O P O H H H O O 2 H H O P O O P O O O O H H H O Base P O O O H H H Base 2 O 2 H H H H O O P O O O P O O P O O O O Note: Free 3' OH group attacks the alpha 5' phosphate. X Time: additional polymerization. Time: NO additional polymerization. H Note: No free 3' OH group to attack the alpha 5' phosphate; polymerization stops. 4B) 5’ phosphate end 3’ OH end 5’ phosphate end 3’ OH end III. DNA Sequencing (Campbell, 7 th ed. pages. 397, 8 th ed. p. 410) Knowing the nucleotide sequence of a stretch of DNA can be valuable. Genetic codons can be used to determine the amino acid sequence of the protein encoded by the DNA. One can look for similarities with other genes for clues about the function of the DNA of interest. Most DNA sequencing is done using a chain- termination method. Two tools enable us to sequence DNA using this method: 1) the ability to replicate DNA in vitro using exogenous DNA as the template and 2) the ability to electrophoretically separate the resulting DNA fragments even though they differ in length by only 1 nucleotide. You must understand the structure and polymerization of nucleoside triphosphates (nucleotides). You should read Campbell, (7th ed. pages 299- 305, 8 th ed. 311-317, to learn about cellular DNA replication. During cellular replication the two strands of DNA separate at the origin of replication and primase adds ribonucleotides (r) complementary to the template strand (rA/dT, rC/dG, rG/dC, rU/dA). The addition of the RNA primer creates a 3' OH group. This is required because DNA polymerase requires a free 3' OH group to attach an incoming nucleotide. The incoming nucleotide is complementary to the base on the template. In summary, DNA polymerase synthesizes DNA in a 5' to 3' direction using a template to determine which nucleotide is added to the free 3' OH at the growing end. Figures 4A and 4B illustrate DNA polymerization. Note that only the growing strand is illustrated. Notice that in Figure 4A the incoming nucleotide attaches to the free 3' OH group. If one were to add a nucleotide that lacked a 3 ' OH group then the addition of that nucleotide would prevent polymerization as shown in Figure 4B. Deoxyribonucleotides lack a hydroxyl group at the 2' OH relative to a ribonucleotide. Dideoxy ribonucleotides, lack hydroxyl groups at both the 2' and 3' positions. This is clearly seen on the dinucleotide shown in Figure 4B. Now let’s focus on a sequencing reaction and how to determine the sequence of DNA using dideoxynucleotides to terminate synthesis. A typical sequencing reaction uses about 0.5 µ g of a given piece of single stranded DNA (ss DNA). Note that double stranded DNA (ds DNA) can be denatured to yield ss DNA. Realize that 0.5 µ g of ss DNA represents a large population of identical DNA strands.
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UCB BIo 1AL Part 2 August - III. DNA Sequencing th...

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