Question

Q3 In the presence of a template DNA, a primer and deoxyribonucleotides, DNA polymerase synthesises DNA by adding complementary bases at the end of the primer/growing chain. The DNA template and the primer sequences are as follows DNA template 5`- ATGCGTGCGTACATACGCAATTCGAATGCGCGCTAACGGCCTTGCG-3` Primer 5`-CGCAAGGCCGTTAGCGC-3`

a. Deduce the sequence of the next five bases that will be added to the primer when all reagents needed for DNA synthesis are supplied in the reaction.

b. Generate the sequence of the newly synthesised shortest labelled fragment if labelled ddATP was also added in the reaction mixture?  

Answer 1

a. DNA template sequence is 5`- ATGCGTGCGTACATACGCAATTCGAATGCGCGCTAACGGCCTTGCG-3` Complementary Strand sequence is 3'- TACGCACGCATGTATGCGTTAAGCTTACGCGCGATTGCCGGAACGC-5'

Primer sequence is 5`-CGCAAGGCCGTTAGCGC-3`.

The sequence of the next five bases that will be added to the primer when all reagents needed for DNA synthesis are supplied in the reaction are the 5`-CGCAAGGCCGTTAGCGCGCATT-3`

3'-GCGTTCCGGCAATCGCGCGTAA-5'

In presence of dNTPs, primer and DNA template and DNA polymerase and reaction buffer the primer attached to the DNA template at annealing temprature.The DNA polymerase detect the template-primer complex and add the dNTPs at the 5'- end and DNA synthesis start at 5'------3' end.

B.In the reaction of DNA synthesis add ddATPs instead of dATPs and all other reagent(primer,DNA template, DNA polymerase and reaction buffer).Dideoxyadenine triphosphates (ddATPs) lack the 3’ OH group to which the next dNTP of the synthesized DNA chain is added. Lack  of 3’ OH group on ddATPs no more nucleotides can be added, and DNA polymerase stop DNA synthesis and form small fragments of DNA .(A= Labelled ddATP)

5'-TGCGCGCTAACGGCCTTGCG-3`

3'-ACGCGCGATTGCCGGAACGC-5' .