# DBT JRF 2020 — Question 10

Exam: DBT JRF 2020 · Section: A · Topic: Molecular Biology · Difficulty: Easy

## Question

The most important step of an automated DNA sequencing reaction is

## Options

- **A.** ligation of DNA template
- **B.** cleavage of template DNA
- **C.** specific and systematic termination of the amplified DNA
- **D.** addition of calcium chloride

## Answer

Option C

## Explanation

Sanger (dideoxy chain termination) sequencing relies on the specific and systematic termination of DNA synthesis. During a sequencing reaction, four separate reactions contain all four normal dNTPs plus a small proportion of one ddNTP (dideoxyribonucleotide) each. ddNTPs lack the 3'-OH group required for the next phosphodiester bond formation. When a ddNTP is incorporated, chain elongation terminates at that position. Gel electrophoresis of fragments of different lengths (one reaction per base) gives the sequence. Modern automated sequencing uses fluorescently labelled ddNTPs in a single reaction read by a laser detector.

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