Question

9. A fragment of DNA has been isolated from bat that could possibly code for the binding region of coronavirus on respiratory cells. Based on the Sanger Sequencing Method, what is the DNA sequence of this fragment? (Shortest fragments running the fastest are at the TOP of this gel) [ A 15] Determined DNA Sequence from this gel: If a deletion mutation was introduced where there are 3 guanines in a row GGG --> GG, then what would be the result of this mutation using the expressed protein? What is this type of mutation? What is the new DNA sequence? What is the new amino acid sequence? G CAT

Answer 1

9. In Sanger sequencing, radiolabeled dideoxynucleotides are added separately (one type in one tube) to tubes containing dNTPs, primers, DNA polymerase, template and buffer. Amplification will generate different fragment where there is premature termination of amplification by DNA polymerase on encountering the ddNTPs, giving a signal. The ddNTPs cannot be added as DNA polymerase requires a 3’ OH group for adding nucleotides to growing strand which is absent in ddNTPs. The fragments are run on the gel. The shortest fragment will run fastest, while largest fragments will be seen on top of the gel. The sequence is read in 5’ to 3’ direction from top to bottom. However, in the gel shown, the run shown is reversed, with shortest fragments on the top.

A. DNA Sequence from gel is read as

5’ ATG CTA TCG GGA CCA GTA G 3’

mRNA: 5’ AUG CUA UCG GGA CCA GUA G 3’

In mRNA, uracil replaces thymine.

Amino acid sequence:

N- Methionine-Leucine-Serine-Glycine-Proline-Valine- ……-C

N= N terminus

C= C terminus

B. The deletion of G from the GGG will lead to frameshift nonsense mutation that leads to formation of a premature stop codon. The new protein formed will be non-functional.

The new DNA sequence is

5’ ATG CTA TCG GAC CAG TAG 3’

mRNA: 5’ AUG CUA UCG GAC CAG UAG 3’

New Amino acid sequence:

N- Methionine-Leucine-Serine-Aspartic acid-Glutamine-Stop-C

Final truncated new sequence:

N- Methionine-Leucine-Serine-Aspartic acid-Glutamine-C

Thus, there is a shift in open reading frame after second codon. This is a frameshift point mutation. This frameshift mutation is also a nonsense mutation as it results in formation of stop codon UAG. This leads to premature termination of the sequence, forming a non-functional protein.