Question

3. Now, you repeat the procedure of question 2, but you add a complete dNTP mix (containing ATP, CTP, dGTP, and dTTP), and ddGTP. In this way, when the DNA polymerase incorporates a nucleotide carrying guanine into the growing DNA strand, it will use sometimes dGTP and sometimes ddGTP. When ddGTP is used, the DNApol will no longer add nucleotides to the DNA strand. For ala3, write below three the possible molecules produced. For b1-b3, consider that you used ddATP instead of ddGTP. (15 points) al.dNTPs+ddGTP e gate pole-cathode e 5'-CG Lane A Lane B 3'-GCATCGTTACTCGC-5' a2.dNTPs+ddGTP 5'-CG 31-GCATCGTTACTCGC-5 a3.dNTPs+ddGTP 5'-CG 3'-GCATCGTTACTCGC-5' bi.dNTPs+ddATP 5'-CG 3'-GCATCGTTACTCGC-5' b2.dNTPs+ddATP 5'-CG 3'-GCATCGTTACTCGC-5 b3.dNTPs+ddATP 5'-CG 3'-GCATCGTTACTCGC-5' 5) The gel below shows the results from the DNA sequencing of a DNA template sample. What is the sequence of the DNA sample (template) (label the 5' end of your sequence)? (30 points)

Answer 1

a1)  

5'-CGTAG-3'

a2)

5'-CGTAGCAATG-3'

a3)

5'-CGTAGCAATGAG-3'

Reason:

ddGTP doesn't contain any -OH group at 3' end. Hence further elongation of chain cannot occur, and the chain will terminate abruptly when G from ddGTP is added to the chain. The above three possibilities include the addition of ddGTP at 3 potential sites where its addition is possible. There are 3 C present in 3' strand, so making 3 possible sites for addition of dGTP and ddGTP. Wherever ddGTP is added in any of these 3 sites, the three possiblilites of 5' strand will arise. All three are listed above.

b1)

5'- CGTA -3'

b2)

5' - CGTAGCA - 3'

b3)

5' - CGTAGCAA - 3'

Reason:

ddATP also doesn't contain any -OH group at 3' end. Hence further elongation of chain cannot occur, and the chain will terminate abruptly when A from ddATP is added to the chain. So the same previous explanation is also followed here.

5)

The sequence of DNA sample will be :

5' - ATCAGAGCTATAGCAGTGC - 3'

Reason :

Since the gel denotes the addition of ddNTP's, so it means that the template strand will be containing the complementary nucleotides corresponding to the site where ddATP,ddGTP, ddTTP and ddCTP is added. Hence the resultant sequence will be complementary too.

Secondly, since the +ve electrode is at the bottom, so we need to read the sequence in reverse. The smallest fragment will be at the bottom due to highest mobility (charge to mass ratio) and the last nucleotide will be at the top. Hence we start reading the sequence from bottom and then write the complementary sequence to get the exact sequence of DNA template.

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