DNASequencing Methods

The most detailed physical maps are based on direct DNA sequence information. The first methods for quickly sequencing DNA were developed between 1975 and 1977. Frederick Sanger and his colleagues created the dideoxy sequencing method based on the elongation of DNA; Allan

Maxam and Walter Gilbert developed a second method based on the chemical degradation of DNA. The Sanger method quickly became the standard procedure for sequencing any purified fragment of DNA.

The Sanger, or dideoxy, method of DNA sequencing is based on the process of replication. The fragment to be sequenced is used as a template to make a series of new DNA molecules. In the process, replication is sometimes (but not always) terminated when a specific base is encountered, producing DNA strands of different length, each of which ends in the same base.

The method relies on the use of a special substrate for DNA synthesis. Normally, DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs), which have an OH group on the 3'-carbon atom (I Figure 19.5a). In DNA synthesis, two phosphate groups on the 5'-carbon atom of a dNTP are removed, and a phosphodiester bond is formed between the remaining 5'-phosphate group of the dNTP and the 3'-OH group of the last nucleotide on the growing DNA chain (see p. 000 in Chapter 12). In the Sanger method, a special nucleotide, called a dideoxyri-bonucleoside triphosphate (ddNTP; i Figure 19.5b), is used as substrate. The ddNTPs are identical with dNTPs, except that they lack a 3'-OH group. Like dNTPs, ddNTPs possess three phosphate groups on their 5' ends, and so they are incorporated into a growing DNA chain. When a ddNTP has been incorporated into a DNA chain, however, no more nucleotides can be added, because there is

Was this article helpful?

0 0

Post a comment