Sanger Sequencing
Related: PCR · Broad-Range 16S PCR · Whole-Genome Sequencing · Sequencing Technologies
1. Principle
Sanger (chain-termination) sequencing determines the nucleotide sequence of a PCR amplicon using dideoxynucleotides and capillary electrophoresis — still the workhorse for single-gene identification and confirmatory sequencing.
2. Step-by-Step Procedure (conceptual)
- Amplify target (PCR / Broad-Range 16S PCR).
- Purify amplicon; set up cycle sequencing with forward/reverse primers.
- Capillary electrophoresis → chromatogram.
- Base-call; inspect quality; BLAST / reference align; report ID or mutation.
3. Interpretation
- Clean single consensus → reliable ID/mutation call.
- Mixed bases → mixed infection, heterozygosity (eukaryotes), or poor template.
- Pitfalls: Primer-binding mutations; low resolution for species complexes; not for genomes.
4. Clinical Use Cases
- 16S/ITS identification from culture-negative specimens or unusual isolates.
- Confirming resistance SNPs / toxin alleles.
- Validating NGS findings at critical positions.
5. Comparison with Other Methods
| Method | Scale | Best use |
|---|---|---|
| Sanger | One amplicon | Confirm / ID |
| Whole-Genome Sequencing | Genome | Outbreaks, resistome |
| qPCR | Targeted detect | Speed, not full sequence |
6. Mnemonic / Visual Aid
Stop bases paint the chromatogram — ddNTPs terminate with color.
Active Recall
- Why run both forward and reverse reads?
- When is Sanger preferred over WGS?
- What does a mixed chromatogram peak suggest in a 16S assay?