Sanger Sequencing

Sanger sequencing (dideoxy chain termination method) is a first-generation DNA sequencing technique developed by Frederick Sanger in 1977. It remains the gold standard for confirming variants identified by other methods and for sequencing individual genes or small genomic regions with high accuracy.

• Chain termination principle: The reaction includes normal dNTPs and a small proportion of dideoxynucleotides (ddNTPs): ddATP, ddTTP, ddCTP, ddGTP. ddNTPs lack the 3'-OH group required for phosphodiester bond formation, so incorporation of a ddNTP terminates the growing DNA chain.
• Fluorescent labeling: Each of the four ddNTPs is labeled with a different fluorescent dye. Terminated fragments of varying lengths are separated by capillary electrophoresis and detected by a laser.
• Electropherogram (chromatogram): The output is a series of colored peaks representing the sequence. Each peak corresponds to a nucleotide at a specific position. Heterozygous variants appear as overlapping peaks at a single position.
• Read length: Typically 600–900 bp per reaction with high accuracy (99.99% per base).
• Bidirectional sequencing: Forward and reverse reads are generated to confirm variants on both strands.
• PCR-based: The target region is first amplified by PCR, then subjected to the sequencing reaction. One primer is used per reaction (unlike PCR, which uses a primer pair).

• Variant confirmation: Sanger sequencing is used to confirm clinically significant variants identified by NGS, CMA, or other screening methods
• Single gene testing: When a specific gene is suspected based on clinical presentation (e.g., sequencing CFTR for cystic fibrosis, FGFR3 for achondroplasia)
• Familial variant testing: Targeted sequencing of a known familial variant in at-risk relatives
• Cascade screening: Efficient for testing a single known variant across multiple family members
• Gap filling: When NGS has poor coverage in a specific region, Sanger can fill in the gaps
• Prenatal diagnosis: Rapid confirmation of a known familial variant in fetal DNA

• Low throughput: Sequences one fragment at a time; impractical for large genes or multiple genes
• Cost-inefficient for multi-gene testing: NGS panels are more cost-effective when multiple genes need analysis
• Cannot detect large deletions/duplications: A heterozygous whole-exon deletion will appear as a normal homozygous sequence (only the intact allele amplifies)
• Limited sensitivity for mosaicism: Generally requires ~15–20% variant allele fraction to be reliably detected on the chromatogram
• Cannot detect copy-neutral changes: Does not identify balanced rearrangements, methylation, or uniparental disomy
• Requires prior knowledge of target: Must know which gene and region to sequence