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Sanger sequencing was the first method of DNA sequencing, developed by Fred Sanger in 1975. It was the method used for the ground-breaking Human Genome Project, completed in 2003. Other names for this techniques are ‘chain-termination sequencing’ or ‘dideoxy sequencing’.

How does Sanger sequencing work?

  • Patient DNA is used as a template in a polymerase chain reaction (PCR).
  • A mix of normal bases (dNTPs) and chain terminating bases (ddNTPs) is used in the PCR reaction. When a chain terminating base is randomly incorporated into a growing DNA chain, it cannot grow any further. This means that DNA fragments of different lengths are generated. Each fragment ends in a chain-terminating base.
  • The DNA fragments are then separated by size using capillary electrophoresis.
  • Each of the four chain terminating bases (A/T/C/G) has a different fluorescent label. A laser is used to excite these fluorescently labelled bases at the end of each fragment.
  • Shorter fragments come first in the sequence followed by increasingly longer fragments.
  • The fluorescence of the base that terminated each length of fragment is recorded, and a chromatograph is generated showing which base is present at which position along the DNA fragment.
  • The chromatogram is compared to a reference file to identify any variants.

Clinical applications of Sanger sequencing

Sanger sequencing remains the most accurate form of DNA sequencing. It is still widely used in clinical laboratories for the following applications:

  • Diagnostic sequencing of a single gene.
  • Testing for a specific familial sequence variant. This can include:
    • predictive genomic testing in at-risk relatives (for example, for a familial BRCA1 variant conferring breast cancer risk);
    • carrier testing for parents, where a child has an autosomal recessive condition (for example, cystic fibrosis);
    • prenatal testing for known familial variants; and
    • segregation analysis, to aid the interpretation of the pathogenicity of a variant (for example, by establishing if a variant being investigated is present in an affected sibling as well as in the proband).
  • To confirm variants that are identified by next-generation sequencing (NGS).
  • To fill gaps in NGS data.

Advantages and limitations of Sanger sequencing


  • Gold standard method for accurate detection of single nucleotide variants and small insertions/deletions.
  • More flexible for testing for a specific familial variant than NGS.
  • Cost effective where single samples need to be tested very urgently, so they cannot be batched up (for example, in prenatal testing, or parental carrier testing during a pregnancy).
  • Less reliant on computational tools than NGS.
  • In some cases, longer fragments (up to approximately 1000bp) can be sequenced than in short read NGS.


  • Limited throughput.
  • Not cost effective for sequencing many genes in parallel, or for sequencing the same region in many samples.
  • May not detect mosaicism.
  • Can require a larger amount of input DNA than NGS.

For a summary table comparing the advantages and disadvantages of the different approaches to gene sequencing (gene panel/WES/WGS), view our Knowledge Hub article, Different approaches to gene sequencing.

Key messages

  • Sanger sequencing is a fast and cost effective way of reading the sequence of small targeted regions of the genome.
  • Sanger sequencing is the gold standard method for accurate detection of single nucleotide variants and small insertions/deletions.
  • Sanger sequencing is widely used to test for known familial variants, for validation of results obtained through NGS and for some single gene sequencing.


For clincians

Tagged: Sequencing, Technologies

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  • Last reviewed: 08/06/2022
  • Next review due: 08/06/2024
  • Authors: Dr Amy Frost, Dr Julia van Campen
  • Reviewers: Dr Siobhan Simpson, Professor Kate Tatton-Brown