1. You are in a lab that is studying the process of DNA replication. You are particularly interested to know what exactly happens at the replication fork. To answer this question, you have set up a series of DNA replication reactions in test tubes using physiological buffers and conditions. However, the student that you have hired to help you has accidentally set up every reaction incorrectly. For each scenario below, indicate if DNA replication will be affected by the mistake and explain why or why not.
A. No DNA Polymerase was added
b) rNTPs were added instead of dNTPs
c) No primase was added
d) Only dNTPs were added (no rNTPs)
2. The nucleic acid from various viruses was extracted and the base compositions determined. What type of nucleic acid (RNA or DNA) and single-stranded or double-stranded occurs in each virus?
Virus 1) 35% A, 35% T, 15% G, 15%C
Virus 2) 35% A, 15% T, 25% G, 25% C
Virus 3) 35% A, 30% U, 30% G, 5% C
Virus 4) 20% A, 20% U, 30% G, 30% C
3. Rank the following four double stranded DNA molecules in terms of their Tm's:
GTGCAC GTGCGCAC GTACTA GTAGTA
CACGTG CACGCGTG CAGCAT CATCAT
3. For the Cot curve shown below label the highly repetitive DNA, moderately repetitive DNA, and the unique DNA fractions.
4. A duplex of DNA is found to have [T] = 29%.
A. What can be said about the relative proportions of remaining bases in the duplex?
B. What is the melting temperature (Tm) of the DNA?
5. The DNA from the bacteriophage øX174 is single-stranded. Would you expect the DNA base composition to follow Chargaff's rules? Why?
6. A single-stranded DNA molecules has the following sequence:
5' GCATCATCATTTAAACCCGGG 3'
A. What chemical groups protrude from each end of this DNA chain?
B. Give the complementary DNA base sequence to include its polarity. Draw an arrow in the direction that replication would occur for polymerization of the complementary strand.
C. What is the %GC of this molecule?
7. What is the function of each of the following in DNA replication?
A. 3'-5'-exonuclease activity of a DNA polymerase
B. 5'-3'-exonuclease activity of DNA polymerase I in E. coli.
D. Single stranded binding proteins (SSBPs)
1a -- DNA polymerase is the main enzyme responsible for DNA replication. Therefore, if it's not in the tube, there will be no replication.
1b -- DNA replication requires dNTPs, not rNTPs; therefore, there will be no DNA replication. rNTPs are needed for RNA synthesis.
1c -- Primase is required to "prime" the synthesis of DNA on the leading strand (and on the Okazaki fragments). Therefore, without primase, no DNA synthesis will occur. Remember, primase actually makes a short RNA fragment first, which later gets replaced by DNA.
1d -- Without rNTPs, primase cannot do its job of priming synthesis of the short primers. Therefore, even though there are dNTPs present, the RNA primers don't get made, and therefore, no DNA synthesis can occur.
3 -- The higher the GC content, the higher the melting temperature. Why is this? Because a GC base pair is bonded together by 3 hydrogen bonds, while an AT base pair is bonded together by only two hydrogen bonds. In addition, the longer strands should have a higher melting point as well, for much the same reason. Longer strands are held together by more hydrogen bonds than shorter strands. Therefore, the 8bp fragment will have the highest Tm. Not only is it the longest fragment, but it has ...
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