19. Molecular genetic methods: types of DNA sequencing.

I. Introduction

• Definition: Gene sequencing is the process of determining the exact order of nucleotides (A, T, G, C) in a DNA fragment or whole genome.
• Importance:
• Understanding genetic variation.
• Identifying disease-causing mutations.
• Guiding targeted therapy.
• Forensic and ancestry studies.
• Two main categories:
• First-generation sequencing → Sanger method (gold standard for decades).
• Next-generation sequencing (NGS) → high-throughput, parallel sequencing technologies.

II. Sanger Sequencing (Dideoxy Chain-Termination Method)

Principle

• Uses dideoxynucleotides (ddNTPs), which lack a 3′-OH group.
• Incorporation of ddNTP stops DNA synthesis at that base.
• Produces DNA fragments of varying lengths ending with labeled ddNTPs.

Steps

1. Template preparation: Single-stranded DNA of interest.
2. Reaction setup: DNA polymerase + primer + 4 normal dNTPs + small amount of labeled ddNTPs (fluorescently tagged).
3. Chain termination: Whenever ddNTP is incorporated, elongation stops.
4. Fragment separation: Capillary electrophoresis separates DNA fragments by size.
5. Detection: Fluorescent labels read by laser → sequence determined by fragment order.

Features

• Sequence read length: ~500–1000 bp.
• Accuracy: very high (>99.9%).
• Throughput: low (one gene at a time).
• Cost: higher per base compared to NGS.

Applications

• Confirming specific mutations identified by other methods.
• Small gene or exon sequencing (e.g., CFTR, BRCA1/2 gene testing).
• Used in early Human Genome Project.