Question

Given below is the diagram of Sanger's method of DNA sequencing. Based on this answer the following questions.

(a) Read and write the original DNA sequence from the autoradiogram below.

(b) Define the principle and steps of this technique.

Answer 1

(a) 3’ AGCTTCAGTC 3’ 

(b) Principle – When a ddNTP gets incorporated in the growing chain, the reaction stops due to non availability of 3’hydroxyl group.

Steps- Each test tube out of four carries single stranded DNA templates, dNTPs and DNA polymerase. Small amount of four ddNTPs are added separately into the four test tubes. For example in test tube containing ddATP, all chains will terminate at ddA but at different positions of T present in the template. The prematurely terminated fragments are resolved and read with agarose gel electrophoresis.