1. The following is a DNA sequence that we want to amplify using PCR 5’CTCGAGGTG

Question

1. The following is a DNA sequence that we want to amplify using PCR

5’CTCGAGGTGAATATGAAAG---Gene of-- CATTTGGCGCGTAATCGATA3’

3’GAGCTCCACTT ATACT TTC---interest-- GTAAACCGCGCATTAGCTAT5’

(a) (2 points) If this DNA sequence needs to be taken through PCR, what are the reaction components that will be required?

(b) (4 points) Which set of primers from the options below, which you would use for PCR reaction in part (a) and Why? Set 1: 5’TACACTTATACTTTC3’ and 3’GTAAACCGCGCATTAG5’

Set 2: 5’CTCGAGGTGAATAT3’ and 3’CCGCGCATTAGCTAT5’

Set 3: 5’GAGTTACACTTATAC3’ and 3’TGGCGAGTAATCGATA5'

(c) (4 points) You decide to determine the complete nucleotide sequence of the gene by DNA sequencing using fluorescent nucleotides. Which of the below nucleotides(s) (1/2/3) are used in DNA sequencing reaction and in what type of sequencing? Show all that apply. 2 and 3 are used in DNA sequencing reaction.Which of the below nucleotides(s) (1/2/3) would you fluorescently tag for DNA sequencing and why

Explanation / Answer

a) Reaction components user are , Template , Taq-polymerase, Buffer, DNTPs, Primers forward and reverse.

ACCORDING TO CHEGG MINIMUM GUIDELINES I HAVE TO ANSWER FIRST FULL QUESTION BUT AS A COURTESY I WILL ANSWER FEW MORE

2) Set 2: 5’CTCGAGGTGAATAT3’ and 3’CCGCGCATTAGCTAT5’

3) In sanger sequences we would use 2 & 3 ( mixture of normal dNTPs and ddNTPs)

4) we would tag dideoxynucleotide chain terminator nucleotides (3) with four different flourescent labels so that it gives us the information about the terminal nucleotide added. Corresponding colour will represent representative terminal nucleotide in a random chain termination.

Hope it helps... Regards

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