9. You decide to perform dideoxy sequencing on a DNA template. You add the appro

Question

9. You decide to perform dideoxy sequencing on a DNA template. You add the appropriate 32P-labelled primer, DNA polymerase, DNA template, buffer, dNTP mix and a small amount of one of the four ddNTPs to 4 reaction tubes. After running PCR, you load cach reaction in a separate lane and separate the products by gel electrophoresis. - ve ddATP ddTTP ddCTP ddGTP + ve Lane 1 e 2 Lane 3 Le 4 e 5 A. What is the sequence of the template strand? Write the sequence from 5' to 3' B. If you accidently add 10 fold more ddGTP to reaction in lane 4, what effect would it have on banding pattern in lane 4? C. In lane 5, draw what you would expect to see if you prepared a reaction using nucleotide mix containing dATP, dTTP, dCTP and dGTP, but no deoxvnuclcotide

Explanation / Answer

A. sequence of template strand from 5' to 3' is 5'CAGATTAC'

B. If you add 10 fold more ddDTP to reaction in lane 4 then possibley it makes shorter fragment and makes smear at the bottom of gel.

C if we prepare reaction without deoxynucleotide then there is no band will appear on gel because there is no template for synthesis in reaction.

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