OrbitoAsia Diagnostics

Overview - What is Sanger sequencing?

Sanger sequencing, also known as chain-termination sequencing or dideoxy sequencing has been the powerhouse of DNA sequencing since its invention in the 1970s. The process is based on the detection of labelled chain-terminating nucleotides that are incorporated by a DNA polymerase during the replication of a template. The method has been extensively used to advance the field of functional and comparative genomics, evolutionary genetics and complex disease research. Notably, the dideoxy method was employed in sequencing the first human genome in 2002. Because of its suitability for routine validation of cloning experiments and PCR fragments, Sanger sequencing remains a popular technique in many laboratories across the world.

Applications - What are the advantages of Sanger sequencing?

Sanger DNA sequencing is widely used for research purposes like:

Workflow - Sanger sequencing methods & technologies

Dideoxy sequencing is based on synthesis of DNA strands that are complementary to a template DNA strand. The sequencing reaction uses normal deoxynucleoside triphosphates (dNTPs) and modified dideoxynucleoside triphosphates (ddNTPs) for strand elongation. The ddNTPs are chemically altered with a fluorescent label and with a chemical group that inhibits phosphodiester bond formation, causing DNA polymerase to stop DNA extension whenever a ddNTP is incorporated. The resulting DNA fragments are subjected to capillary electrophoresis, where the fragments flow through a gel-like matrix at different speeds according to their size. Each of the four modified ddNTPs carries a distinct fluorescent label. The emitted fluorescence signal from each excited fluorescent dye determines the identity of the nucleotide in the original DNA template

Sanger sequencing vs. next-generation sequencing

The DNA sequencing field did not stop evolving with the successful adaptation of Sanger sequencing. The establishment of next-generation sequencing (NGS) and third-generation sequencing technologies offered substantial benefits compared to the traditional dideoxy method. However, the chain-termination method remains widely used in the sequencing field, because it offers several distinct advantages. Specifically, Sanger sequencing is preferable over NGS for:
Nevertheless, next-generation sequencing is often considered to be superior to Sanger sequencing, especially for project objectives that require:

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:
  1. Predictive genomic testing in at-risk relatives (for example, for a familial BRCA1 variant conferring breast cancer risk).
  2. 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).

Clinical applications of Sanger sequencing

Advantages

Limitations

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.or more routine small-scale projects and NGS is applied to meet large-scale sequencing needs.