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Sanger Sequencing Notes

Medicine Notes > Biochemistry Notes

This is an extract of our Sanger Sequencing document, which we sell as part of our Biochemistry Notes collection written by the top tier of Oxford University students.

The following is a more accessble plain text extract of the PDF sample above, taken from our Biochemistry Notes. Due to the challenges of extracting text from PDFs, it will have odd formatting:

Sanger sequencing Sanger sequencing is a method of sequencing the order of bases in DNA. It relies on the principle of controlled termination of replication. This is achieved by carrying out DNA replication in an experiment where a small amount one of the bases is radiolabelled, and altered such that when it is incorporated, it will terminate replication. Most of this base added will be normal, however some will be radiolabelled and if the experiment is done with enough molecules, DNA sequences of many different lengths will be synthesised, all of which will have a final radiolabelled base of a specific type, adenine, guanine, thymine or cytosine. This experiment is repeated four times, once for each base. The analogs used are the 2',3' dideoxy analogs, for example, 2'3,3'-dideoxyribosenucleoside triphosphate. Electrophoresis and autoradiography are then carried out on these strands, enabling researchers to see which positions bases occupy by their radiolabelled appearance at the end of chains of certain lengths. The sequence of labelled terminal bases will reveal the normal DNA sequence of bases. Alternatively, for DNA lengths of up to 500 bases, a fluorescence method may be used. Here each base is substituted by a fluorescently-tagged analog, so that when the DNA polymerase makes complement copies of the starting DNA strand, the copied sequences will have a colour sequence which can be measured using wavelengths. The colour sequence will correspond to the base sequence and the advantage of this method is that it can be carried out using one DNA mixture as the bases are labelled different colours; in the Sanger method one experiment must be carried out for each base as each radiolabelled base will darken the radiograph regardless of which base analog it is.

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