Sanger sequencing

Sanger sequencing is a method of DNA sequencing first commercialized by Applied Biosystems, based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. Developed by Frederick Sanger and colleagues in 1977, it was the most widely used sequencing method for approximately 40 years. More recently, higher volume Sanger sequencing has been replaced by 'Next-Gen' sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use, for smaller-scale projects, and for validation of Next-Gen results. It still has the advantage over short-read sequencing technologies (like Illumina) that it can produce DNA sequence reads of > 500 nucleotides. Sanger sequencing is a method of DNA sequencing first commercialized by Applied Biosystems, based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. Developed by Frederick Sanger and colleagues in 1977, it was the most widely used sequencing method for approximately 40 years. More recently, higher volume Sanger sequencing has been replaced by 'Next-Gen' sequencing methods, especially for large-scale, automated genome analyses. However, the Sanger method remains in wide use, for smaller-scale projects, and for validation of Next-Gen results. It still has the advantage over short-read sequencing technologies (like Illumina) that it can produce DNA sequence reads of > 500 nucleotides. The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates (ddNTPs), the latter of which terminate DNA strand elongation. These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labelled for detection in automated sequencing machines. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), while the other added nucleotides are ordinary ones. The dideoxynucleotide concentration should be approximately 100-fold lower than that of the corresponding deoxynucleotide (e.g. 0.005mM ddTTP : 0.5mM dTTP) to allow enough fragments to be produced while still transcribing the complete sequence. Putting it in a more sensible order, four separate reactions are needed in this process to test all four ddNTPs. Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. In the original publication of 1977, the formation of base-paired loops of ssDNA was a cause of serious difficulty in resolving bands at some locations. This is frequently performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C). The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.