For sequencing DNA by Sanger’s method, the chain elongation is terminated by:
(a) 1′, 4′ dideoxy nucleotides,
(b) 2, 3′ dideoxy nucleotides,
(c) 2, 4′ dideoxy nucleotides,
(d) 4, 3′ dideoxy nucleotides,

Which Dideoxy Nucleotides Are Used in Sanger’s DNA Sequencing Method?

Sanger sequencing, also known as the chain termination method, revolutionized molecular biology by providing a reliable way to determine DNA sequences. One of its key innovations is the use of dideoxynucleotides (ddNTPs) to halt DNA chain elongation.

Q: For sequencing DNA by Sanger’s method, the chain elongation is terminated by:
(a) 1′, 4′ dideoxy nucleotides
(b) 2, 3′ dideoxy nucleotides
(c) 2, 4′ dideoxy nucleotides
(d) 4, 3′ dideoxy nucleotides

Correct Answer:

(b) 2, 3′ dideoxy nucleotides

Explanation:

Normal DNA synthesis involves the addition of deoxynucleotides (dNTPs) to a growing DNA strand. These dNTPs have a 3′-OH (hydroxyl) group on the sugar, which is crucial for forming the next phosphodiester bond.

What Are Dideoxynucleotides (ddNTPs)?

• ddNTPs are modified nucleotides lacking both the 2′-OH and the 3′-OH on the ribose sugar.

• Without the 3′-OH group, the DNA polymerase cannot add any more nucleotides, thus terminating chain elongation.

🔍 Why the Other Options Are Incorrect:

• (a) 1′, 4′ dideoxy nucleotides – Not relevant to bonding in DNA synthesis.

• (c) 2, 4′ dideoxy nucleotides – The 4′ position does not participate in nucleotide addition.

• (d) 4, 3′ dideoxy nucleotides – The 4′ carbon is not involved in forming the phosphodiester bond, and removal at this site is irrelevant for chain termination.

🧬 Sanger Sequencing in Brief:

1. DNA template is mixed with primers, DNA polymerase, normal dNTPs, and fluorescently-labeled ddNTPs.

2. During synthesis, random incorporation of a ddNTP terminates the strand.

3. The result is a mixture of DNA fragments of varying lengths, each ending at a specific base.

4. These fragments are separated by capillary electrophoresis, and the sequence is read based on fluorescence.

Conclusion:

In Sanger’s DNA sequencing method, 2,3′-dideoxy nucleotides (ddNTPs) are used to terminate DNA chain elongation, allowing precise determination of nucleotide sequences. This principle remains foundational even in today’s advanced sequencing technologies.