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Sanger Sequencing - A Brief History and Current Applications

Feb 10, 2022 11:03:00 AM / by Champions Oncology

3D illustration of a method of DNA sequencing

The first description of the “Sanger sequencing” method was published more than 40 years ago, and soon became the cornerstone method in DNA sequencing[1]. This method is also called the “chain termination method” and is a highly accurate method that can produce long reads and can be used for DNA or RNA sequence analysis. Here we describe the basics of the Sanger sequencing method and highlight its current applications.


The Method

The Sanger sequencing method is comprised of three main steps. The first step uses the DNA sequence of interest as a template for a specialized PCR reaction called chain termination PCR that includes normal nucleotides, or deoxynucleoside triphosphates (dNTPs), for DNA strand elongation by DNA polymerase mixed with either radioactively or fluorescently labeled modified dideoxyribonucleotide triphosphates (ddNTPs) that do not have 3’-OH groups and thus cannot form bonds with additional dNTPs. This PCR reaction generations billions of labeled oligonucleotides that are terminated at random lengths by these labeled ddNTPs. The first described method of manual Sanger sequencing required four different PCR reactions, with each reaction using radioactively labeled ddATP, ddTTP, ddCTP or ddGTP, where A = adenosine, T = thymidine, C = cytidine and G = guanosine. The more commonly used automated Sanger sequencing method mixes all ddNTPs into a single PCR reaction with different fluorescent labels on each ddNTP type in a method called dye-terminator sequencing[1].


Following the PCR reaction, chain-terminated oligonucleotides are denatured and separated by size using gel electrophoresis that pulls negatively charged DNA fragments toward the positively charged terminus of the gel. Since DNA fragments have the same charger per unit mass, smaller fragments move more quickly, and fragments are separated from smallest to largest on the gel and should cover all possible nucleotide positions for the sequence of interest. The manual method requires the four different reactions to be run in four separate lanes to know which nucleotide corresponds to each position. The final step of reading the DNA sequence for the manual method involves reading the relative positions of each ddNTP in the gel to determine the sequence. Automated methods use a machine that carries out single capillary electrophoresis and a laser that excites the fluorescent dyes at the terminus of each fragment. Each of the four types of ddNTPs emits light at a different wavelength, and high-throughput DNA analyzers detect these different wavelengths and record the sequence in a chromatogram.

DNA Nucleotides


Modern Methods and More

More recently, microfluidic Sanger sequencing methods have integrated the PCR and capillary electrophoresis steps into a single nanoscale chip. This method uses considerably less materials and labor than conventional methods, and the reads of the overlapping DNA fragments are assembled into a full-length sequence[2].

Automated sequencing methods can be run in high-throughput formats and had been the mainstay of sequencing technology since the human genome project but read lengths have been limited to several hundred base pairs because of technical limitations in resolving large DNA fragments. Nonetheless, Sanger sequencing is still considered the most accurate sequencing method compared with next-generation sequencing (NGS) methods and is essential to validating NGS data. Sanger sequencing is still critical to research that requires precise sequencing, including human leukocyte antigen (HLA) typing for bone marrow matching, molecular profiling of mutations in tumors for basic cancer research or personalized oncology treatments, as well as confirmation of genome editing in CRISPR applications. Sanger sequencing is a remarkable method that has stood the test of time and continues to evolve.


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[1] Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes JC, Hutchison CA, Slocombe PM, Smith M. Nucleotide sequence of bacteriophage ΦX174 DNA. Nature. 1977;265(5596):687–695.

[2] Smith LM, Fung S, Hunkapiller MW, Hunkapiller TJ, Hood LE. The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucleic Acids Res. 1985;13(7):2399-2412. doi:10.1093/nar/13.7.2399.


Tags: NGS