Question

Briefly write the steps of Sanger’s chain termination method of DNA sequencing.

Answer 1

Procedure for Sanger's Chain Termination DNA Sequencing:
– Reaction Preparation: In four distinct tubes, the target DNA serves as a template. Each tube is also supplied with DNA polymerase, a primer, deoxynucleotide triphosphates (dNTPs), and a single type of dideoxynucleotide triphosphate (ddATP, ddGTP, ddCTP, or ddTTP) at a reduced concentration.
– DNA Elongation: DNA polymerase initiates primer extension. Incorporation of any ddNTP halts new strand synthesis prematurely. 
– Strand Termination: Elongation continues until a ddNTP is randomly incorporated into the nascent strand. Due to the absence of a 3’OH group in ddNTPs, further extension is impossible, resulting in a collection of fragments of varying lengths, each ending with a ddNTP. 
– Gel Separation: Gel electrophoresis is employed to separate these DNA fragments by size. Smaller fragments migrate further through the gel than larger ones. 
– Imaging: Radioactive labels or fluorescent dyes are used to detect the fragments within the gel. This generates a band pattern resembling a ladder, with the shortest fragment at the bottom, allowing for sequence determination from bottom to top.