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Molecular Pathology

DNA sequencing

Sanger DNA Sequencing


Author: Rodney E. Shackelford, D.O., Ph.D., Tulane University (see Reviewers page)
Revised: 12 February 2011, last major update February 2011
Copyright: (c) 2008-2011, PathologyOutlines.com, Inc.

Background
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● This method of DNA sequencing was developed by Frederick Sanger in 1977 (Proc Natl Acad Sci USA 1977;74:5463)
● It is also known as the chain termination method

Procedure
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● DNA polymerase is used to extend a DNA strand until a chain elongation-terminating radiolabeled or fluorescently labeled dideoxynucleotide triphosphate is incorporated
● The DNA samples are divided into four separate sequencing reactions, each containing deoxynucleotides (dATP, dGTP, dCTP and dTTP), DNA polymerase, a single-stranded DNA primer, and low concentrations of labeled, chain-terminating dideoxynucleotide (ddATP, ddGTP, ddCTP and ddTTP)
● The dideoxynucleotides lack the 3-OH deoxyribose sugar, blocking further phosphodiester bond formation and subsequent chain elongation
● Low dideoxynucleotide concentrations allow the formation of DNA fragments of varying length
● Although DNA is most commonly labeled at the 3 end, it can also be labeled at the 5 end
● Electrophoretic/autoradiographic analysis involves sequential reading of four lanes (A, T, G, and C)

● The shotgun sequencing and direct approaches are commonly used
● In shotgun sequencing (random priming), genomic DNA is randomly fragmented into 2-3 kb fragments, inserted into a vector, and replicated via bacterial culture, leading to many different and overlapping genomic regions that can be sequenced and aligned to give the larger genomic sequence
● Although the redundancy of this approach increases sequencing costs (because the same genomic areas may be sequenced 6-10 times), it has been used successfully in many applications, such as sequencing the Haemophilus influenzae genome
● In the direct approach (primer walking), a section of DNA to be sequenced (usually ~ 40 kb) is placed next to a known sequence, with the primer lying within the known sequence
● The DNA polymerase then extends the sequencing reaction into the unsequenced DNA
● Once the sequence is obtained, a new primer is synthesized complementary to the distal portion of the newly sequenced DNA and the process is repeated until the entire genomic region is sequenced
● The advantage if this process is that fewer genomic regions are sequenced multiple times; however each new round of sequencing requires a new primer to be synthesized, which increases the cost

● Several different radiolabels have been employed in the Sanger method, including 32P, 35S and more recently 33P. 32P emits high-energy b-particles, giving a strong penetrating signal, resulting in diffuse autoradiographic bands, lowering the number of bands that may be read. 35S emits lower energy b-particles, allowing more accurate and longer sequencing to be read via autoradiography. 33P has intermediate properties between 32P and 35S

● Many different non-radioactive labels have been employed in the Sanger method, including fluorescent dyes 5-carboxyfluorescein, 7-nitrobenzo-2-oxa-1-diazole, tetramethylrhodamine and Texas Red
● Non-radioactive dyes must: 1) have absorption and emission maxima in the visible spectrum; 2) emit at wavelengths that allow spectroscopic separation; 3) emit at a single wavelength; and 4) not affect the electrophoretic mobility of the DNA fragments
● The advantage of using four different labels vs. one radioisotope is that the entire sequencing reaction may be run in one reaction and analyzed in one gel lane or other single separation mechanism
● Commonly employed DNA polymerases include DNA polymerase I Klenow fragment, AMV reverse transcriptase, Taq DNA polymerase and T7 DNA polymerase
● Originally, Taq polymerase had a strong preference for incorporating ddNTPs over dNTPs, but this problem was solved using a Taq polymerase with a single amino acid substitution, which resulted in the dNTP and ddNTP incorporation rate becoming more equal

● Advantages of chain-termination method: readily adaptable to commercial kits; use far fewer toxic reagents than the Maxam-Gilbert method
● Disadvantages of chain-termination method: non-specific primer binding, less accurate read-out, formation of DNA secondary structures which alter sequencing fidelity
● After three decades of improvement, the Sanger based sequencing method has achieved read-lengths of ~1,000 bp with capillary electrophoresis and per-base sequencing accuracy as high as 99.999%
● Current costs to sequence one kB of DNA is $0.50

End of Molecular Pathology > DNA sequencing > Sanger DNA Sequencing


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