Question

Q1) List the limitations and challenges of dideoxy termination method of DNA sequencing?

Q2) In the context of mutation detection, describe the differences between first- and second-generation sequencing?

Q3) 6-year-old child presenting with sensorineural deafness, sequencing of MYO15A gene by Sanger method was performed and showed the following chromatogram:

A- Describe the principle of Sanger sequencing method?

B- Describe the DNA change: position, type of variant and genotype?

Answer 1

Sanger method is the method of DNA sequencing discovered by Frederick Sanger in 1977 which utilizes dideoxynucleotides in the growing chain of DNA. It is the chain termination method where small fragments of DNA are synthesized and the sequence is analaysed by utilizing these small fragments through PAGE. There are various limitations and challenges in dideoxy chain termination method such as:

1) It can sequence only small fragments ranging usually from 300-1000 bp.

2) Sequence quality degrades after 700-800 bp.

3) Sensitivity is very low.

4) It is not cost effective for high number of targets.

5) No. of base pairs that we can sequence is limited to the resolution of the gel.

6) Template DNA concentration needed is very high.

7) Lack of automation and manual involvement makes it tedious and erroneous.