DNA sequencing

DNA sequencing (gene sequencing) The process of elucidating the nucleotide sequence of a DNA fragment. Two techniques are used. The Maxam–Gilbert method (named after Allan Maxam and Walter Gilbert) involves cleaving the DNA with a restriction enzyme and labelling each of the resulting smaller fragments with ³²P-phosphate at one end. The fragments are subjected to four different sets of reactions, each set specifically cleaving DNA at a particular base or bases. The cleaved fragments are separated by electrophoresis according to their chain length and identified by autoradiography. The base (nucleotide) sequence is deduced from the position of bands in each of the four lanes in the gel. The Sanger method (named after Frederick Sanger), also called the dideoxy method, involves synthesizing a new DNA strand using as template single-stranded DNA from the gene being sequenced. Synthesis of the new strand can be stopped at any of the four bases by adding the corresponding dideoxy (dd) derivative of the deoxyribonucleoside phosphates; for example, by adding ddATP the synthesis terminates at an adenosine; by adding ddGTP it terminates at a guanosine, etc. As in the first method, the fragments, which comprise radiolabelled nucleotides, are finally subjected to electrophoresis and autoradiography. A big advantage of the Sanger method is that it can easily be adapted to sequencing RNA, by making single-stranded DNA from the RNA template using the enzyme reverse transcriptase. This enables, for example, sequencing of ribosomal RNA for use in molecular systematics. Furthermore, by using fluorescent dyes as labels instead of radioisotopes, the Sanger method has been fully automated. After separation of the fragments, the products of all four reactions are detected by fluorescence spectroscopy and analysed by computer, which gives a printout of the base sequence. DNA sequencing is now employed on a major scale, for example in determining the nucleotide sequence of entire genomes (see Human Genome Project).