Sanger Sequencing: Introduction, Workflow, and Applications

Introduction to Sanger Sequencing

Sanger sequencing, also known as the "chain termination method," was developed by the English biochemist Frederick Sanger and his colleagues in 1977. This method is designed for determining the sequence of nucleotide bases in a piece of DNA. Sanger sequencing with 99.99% base accuracy is considered the "gold standard" for validating DNA sequences, including those already sequenced through next-generation sequencing. Sanger sequencing was used in the Human Genome Project to determine the sequences of relatively small fragments of human DNA (900 base pairs or less). These fragments were used to assemble larger DNA fragments and, eventually, entire chromosomes.

Sanger Sequencing Workflow

There are three main steps to Sanger sequencing which are the following:

DNA Sequence for Chain Termination PCR

The DNA sequence of interest is used as a template for a special type of PCR called chain-termination PCR. Chain-termination PCR works just like standard PCR, but with one major difference: the addition of modified nucleotides called dideoxyribonucleotides. In the extension step of standard PCR, DNA polymerase adds dNTPs to a growing DNA strand by catalyzing the formation of a phosphodiester bond between the free 3'-OH group of the last nucleotide and the 5'-phosphate of the next.

Size Separation by Gel Electrophoresis

In the second step, the chain-terminated oligonucleotides are separated by size via gel electrophoresis. In gel electrophoresis, DNA samples are loaded into one end of a gel matrix, and an electric current is applied; DNA is negatively charged, so the oligonucleotides will be pulled toward the positive electrode on the opposite side of the gel. Because all DNA fragments have the same charge per unit of mass, the speed at which the oligonucleotides move will be determined only by size. The smaller a fragment is, the less friction it will experience as it moves through the gel, and the faster it will move. As result, the oligonucleotides will be arranged from smallest to largest, reading the gel from bottom to top.

Gel Analysis and Determination of DNA Sequence

The last step simply involves reading the gel to determine the sequence of the input DNA. Because DNA polymerase only synthesizes DNA in the 5' to 3' direction starting at a provided primer, each terminal ddNTP will correspond to a specific nucleotide in the original sequence (e.g., the shortest fragment must terminate at the first nucleotide from the 5' end, the second-shortest fragment must terminate at the second nucleotide from the 5' end, etc.) Therefore, by reading the gel bands from smallest to largest, we can determine the 5' to 3' sequence of the original DNA strand.

Applications of Sanger Sequencing

Sanger DNA sequencing is widely used for research purposes like (1) targeting smaller genomic regions in a larger number of samples, (2) sequencing of variable regions, (3) validating results from next-generation sequencing (NGS) studies, (4) verifying plasmid sequences, inserts, mutations, (5) HLA typing, (6) genotyping of microsatellite markers, and (6) identifying single disease-causing genetic variants.

References:

1. Crossley BM, Bai J, Glaser A, et al. Guidelines for Sanger sequencing and molecular assay monitoring. Journal of Veterinary Diagnostic Investigation. 2020 Nov;32(6).
2. Mardis ER. DNA sequencing technologies: 2006–2016. Nature protocols. 2017 Feb;12(2).