DNA sequencing

DNA sequencing involves the determination of the nucleotides that make up an actual strand of DNA. The DNA is digested into fragments and then subjected to gel electrophoresis to see the strands separated out to allow observations to be made.

Sanger method

The Sanger method involves separating the DNA to make it single stranded, and allowing it to form complementary strands according to base pairing rules. However, there is also going to be dideoxynucleotides (ddA, ddC,ddT & ddG). These are molecules synonymous to ordinary nucleotides except they lack a OH’ group so when they are added to the DNA strand, no more nucleotides can be added on afterwards because they lack the OH’ group that ordinarily allows more bases to be added. The dideoxynucleotides will be added in place of their ordinary nucleotides for example, ddA will be added instead of A and as a result replication of the DNA stops and we get DNA fragments of different lengths. Depending on what dideoxynucleoide was added and where the fragment was cut we can determine where the base is in the sequence. For example, if we add ddA and our strand is 5 bases long we know the 5th position has an A, because that is where the ddA must have been added instead of A to stop the replication.

Automated direct DNA sequencing

This is the most common method of DNA sequencing used today, which involves machines automating the process making the process more efficient. The dideoxynucleotides are labelled with different colour fluorescent dyes and are added to the DNA sample. The differences in the bases are observed through different colours they produce.

See also