![]() The fragments are run through a single long glass capillary filled with a gel polymer, where they migrate according to their length. The elongation reaction is repeated for 30–40 cycles.įollowing sequencing clean-up, the newly synthesized DNA fragments are separated by electrophoresis. Occasionally, one of the four chain-terminating ddNTPs will be inserted by chance, stopping elongation of the DNA strand. An adenine base (A) is paired with every thymine (T) on the template and a cytosine (C) with every guanine (G) and vice versa. In the extension step, the DNA polymerase extends the primer from its 3´ hydroxyl group to synthesize a new strand. A subsequent annealing step allows for hybridization of the oligonucleotide primer close to the sequence of interest. The sequence of the template DNA strand can thus be derived by analysis (Figure 1).ĭuring PCR and cycle sequencing, the DNA is first denatured (the double-stranded DNA template becomes single-stranded DNA). The chain-terminated fragments are detected by their fluorescent labels, with each color identifying one of the terminating ddNTPs. ![]() The extension products are then separated by electrophoresis, resolved to single-nucleotide differences in size. By mixing ddNTPs that have been labeled with a different color for each base, unlabeled dNTPs, and template DNA in a polymerase-driven reaction, strands of each possible length are produced when the ddNTPs are randomly incorporated and terminate the chain. These analogs, called dideoxyribonucleotides (ddNTPs), are missing the 3´ hydroxyl group that is required for 5’ to 3’ extension of a DNA polynucleotide chain. (There must be an area of known sequence close to the target DNA.)W In order to determine the sequence, Sanger sequencing makes use of chemical analogs of the four nucleotides in DNA. Sanger sequencing targets a specific region of template DNA using an oligonucleotide sequencing primer, which binds to the DNA adjacent to the region of interest.
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