9. Gene sequencing: Sanger sequencing, next-generation sequencing (NGS) · Genetics · MU Sofia

9. Gene sequencing: Sanger sequencing, next-generation sequencing (NGS).

Explains how gene sequencing determines the order of nucleotides, starting with the Sanger dideoxy chain-termination method and its principle, steps, features and uses. It then covers next-generation sequencing, including massively parallel sequencing, the main platforms and applications such as whole-genome and exome sequencing. A comparison table sets the two methods side by side, and the note closes with their clinical relevance, including confirmation of variants and cancer-driven targeted therapy.

Inside: 6 sections · 5 labelled pictures · 1 table · 14 flashcards · 4 min

Topic 9 of 65 in the MU Sofia syllabus for Genetics (Medicine, Year 3). The free AceMed note covers it in 6 sections, about 432 words (4 min of reading), with 14 flashcards.

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.