8. Describe the possible outcome of a PCR experiment in which (a) one of the primers is inadvertently omitted from the reaction mixture; (b) one of the primers is complementary to several sites in the starting DNA sample; (c) there is a single- stranded break in the target DNA sequence, which is present in only one copy in the starting sample; (d) there is a double-stranded break in the target DNA sequence, which is present in only one copy in the starting sample. e
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- 36. Which is the sequence of steps used to amplify DNA with PCR during a single cycle? 1) Primers bind (anneal) to complementary sequences of the target DNA. 2) DNA is heated to a high temperature to cause denaturation (DNA is single-stranded). 3) DNA polymerase extends the 3’OH of primers to make copies of the target DNA. A) 1, 2 and 3 B) 3, 2, and 1 C) 2, 1, and 3 D) 1, 3, and 21. For each of the processes below, fill in blanks with one or more of the items in the following list: Ligase (L), Primase (P), enzyme with 5' to 3' activity (E), RNA-dependent polymerase (RNA-DP = polymerase which uses RNA template), DNA-dependent polymerase (DNA-DP=polymerase which uses DNA template). for PCR you need............. for normal mRNA synthesis you need........ for normal DNA synthesis you need........ for normal tRNA synthesis you need....... 2. (please explain) Suppose the DNA synthesis that occurs in PCR is less accurate than cellular DNA synthesis (as in, PCR has a higher error rate than normal DNA synthesis). This difference in error would most likely be because the polymerase used in PCR: a) has a low level of 5' to 3' exonuclease activity b)has a low level of 3' to 5' exonuclease activity c) has a low temp at which it denatures d) adds to the 5' end of the growing DNA chain e) does not use template16. Which of the following enzyme repairs the DNA backbone during molecular cloning (that is you are inserting the DNA fragment into a plasmid and the bases in the sticky ends are aligned but the backbone needs to be repaired). a) reverse transcriptase b) restriction enzymes c) DNA ligase d) polymerase
- Describe the possible outcome of a PCR experiment in which (a) there is a single-stranded break in the target DNA sequence, which is present in only one copy in the starting sample, and (b) there is a doublestranded break in the target DNA sequence, which is present in only one copy in the starting sample.5) PCR primers have a melting temperature (Tm) at which they become dissociated from the template DNA. This Tm is calculated by their length and their base composition. Suppose you have two PCR primers that are the exactly same length (each 20 nucleotides long). Primer A has a Tm of 60°C and Primer B has a Tm of 63°C. Based on what you know about DNA structure, hypothesize why there is a difference in Tm between the two primers?1. The polymerase chain reaction (PCR) is used by scientists to amplify DNA, particularly when the quantity of DNA is very small, mixed, or contaminated with other organisms. Explain (with words) the how PCR works using a diagram to help illustrate this (show a minimum of three cycles to illustrate your point). 2. How would one go about designing primers (I am interested in the concepts you would use to accomplish this task) that would amplify a specific sequence of the "16S rDNA," that would be found in the Clostridium family? Be sure you consider the importance of both "highly conserved" and "highly variable" regions during the amplification process. 3. How many molecules (target sequence copies) will be produced by 30 PCR cycles? Assume you start with only 1 copy of the target sequence (very unlikely)? Show your work! 4. Explain how gel electrophoresis works? Why are DNA samples that are to be separated by gel electrophoresis always loaded at the cathode end of the power source? The…
- 11. Which of the following enzymes remove supercoiling in replicating DNA ahead of the replication fork? a) Topoisomerases b) Primases c) Helicases d) DNA polymerases32. What is the basic principle of the original Sanger sequencing method? A) Single stranded DNA is replicated using a modified DNA polymerase enzyme (e.g., Klenow fragment) B) Individual sequencing reactions are terminated using ddNTPs, which lack both 2'- and 3'-OH groups. C) Requires [P]-radiolabeling of 5'-ends of DNA D) Requires detection of [P]-radiolabeled DNA by autoradiography. E) PAGE is used to separate DNA strands that terminate at an A, a G, a T, or a C.1. A DNA sequence 10 kb long contains one site at which it may be cut by the restriction enzyme Pstl. The same 10 kb long sequence also contains one site that can be cut by restriction enzyme Bg/II. Three separate fractions of this DNA were treated separately, as follows: 1) digestion by Pstl; 2) digestion by Bg/II; 3) digestion by both enzymes. The three treated fractions were then subjected to gel electrophoresis. The following table shows the lengths of the restriction fragments associated with each treated fraction. From the result, draw a restriction map that shows the Pst and the Bg/II cutting sites in the 10 kb DNA. Enzyme in mixture Pstl BgIII Pstl and BgIII Fragment Lengths (kb) 6.4 and 3.6 8.8 and 1.2 5.2, 3.6 and 1.2
- 2) The authors investigate if the switch from the polymerase to the exonuclease site involves releasing and re-binding of the DNA or if it utilizes an intramolecular rearrangement. To accomplish this, they use a primer-extension assay. How is a primer extension assay performed? In answering this question, be sure to mention a.) why heparin is used, b.) how they generate mismatched primers, and c.) how they visualize only the primer strand and not the template strand on their gel.90. The DNA template fragment shown was sequenced by the Sanger method. A sample of the DNA was reacted with DNA polymerase and each of the four dNTPs. One of the ddNTPs was added to each of the nucleotide mixtures as ddGTP ddATP ddTTP ddCTP labelled on the drawing. What is the sequence of the template strand? a) 5'ATCАTGСС3 b) 5' СCGTAСТАЗ c) 5'TAGTACGG 3' d) 5'GGCATGAT3' e) 5'СССАТGAT3'1. TRUE OR FALSE: a) Okazaki fragments are short DNA pieces that explain how the DNA polymerase can continue the synthesis of the new strand. b) Okazaki fragments are short DNA pieces that explain how the DNA polymerase can continue the synthesis of the new strand.