A. What is the sequence of the template strand? Be sure to label the 5’ and 3’ e

Question

A. What is the sequence of the template strand? Be sure to label the 5’ and 3’ ends.(5 pt)

B. Suppose that you accidentally added 10-fold more ddGTP to the reaction in lane 4. What effect would that have on the banding pattern in lane 4? (5 pt)

C. In lane 5, draw the band at the proper location whehere you would expect to see if you prepared a reaction using nucleotide mix containing only dATP, dTTP, dCTP, and dGTP. (5 pt)

D. In lane 6, draw the band at the proper location where you would expect to see if you prepared a reaction using a nucleotide mix containing only ddATP, dTTP, dCTP, and dGTP. (5 pt)

4. You decide to perform dideoxy sequencing on a PCR product. You add the appropriate 32P labeled primer, DNA polymerase, DNA template (the PCR product), buffer, dNTP mix, and a small amount of one of the four ddNTPs to four reaction tubes. You run the reactions in the thermal cycler, load each reaction into a separate lane of a polyacrylamide gel, and separate the products by gel electrophoresis. In the figure below, the lanes are labeled according to the ddNTP added. ddATP ddTTP ddCTP ddGTP C D Lane 1 9:08 PM

Explanation / Answer

A) In electrophoresis, smaller fragments runs faster towards anode, that will be 5' end when DNA has to be sequenced. Therefore, 5'- ACGGTCTCAATG -3'

B) Addition of dideoxynucleotides adds H to the 3' end of the growing strand rather then OH, so no further additon of nucleotide will take place.

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