DNA Sequence Determination Using Dideoxy Analogs

Dideoxy chain termination
DNA
sequencing was developed by Sanger and colleagues (
1
,
2
) and is a simple and extremely accurate method of obtaining thousands of bases of sequence data per day. The procedure requires a single-stranded DNA template and a primer complementary to the 3′ end of the region of DNA to be sequenced. In the presence of the four nucleoside triphosphates (dNTPs), one of which is radioactively labeled, and the Klenow fragment of DNA polymerase, primed synthesis occurs across the region of interest. In the presence of competing dideoxynucleoside triphosphates (ddNTPs), specific termination occurs at each of the four different nucleotides. Chain termination occurs when a ddNTP is incorporated randomly at the 3′ end of the growing chain; the chain cannot be further extended because the ddNTP lacks the 3′ hydroxyl group. If this is carried out separately with each of the ddNTPs and the four sets of reaction products are fractionated on a polyacrylamide gel, which separates the synthesized single strands according to length, the DNA sequence can be deduced from the ascending order of the bands in the four different tracks. The principle of this method is summarized in Fig. 1 .Fig. 1.
Primed DNA synthesis is initiated by addition of the Klenow fragment of DNA polymerase I. In the example shown here, DNA has been cloned into the Sma site of M13mp8. DNA synthesis proceeds through the polylinker and into the insert. Only the sequence of the insert (TATT-) is shown. In the presence of the appropriate dideoxynucleotide (
see
Fig. 2 for the composition of A�, G�, C�, T�) chain termination occurs. These chains are separated according to length and the sequence read from the autoradiograph.