Primed synthesis methods of sequencing DNA and RNA

Abstract

Primed synthesis sequencing requires a partially single-stranded molecule in which a specific polynucleotide primer with a free 3' hydroxyl terminus is base paired to a single-stranded template. The template and primer strands can be DNA or RNA in any combination. A polymerase, either DNA polymerase for DNA/DNA or RNA/DNA primer-template combinations or reverse transcriptase for a DNA/RNA primer-template molecule, is used to copy the template by chain extension. In the chain termination method of sequencing (see Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463; 1977), nucleotide-specific chain terminators (dideoxy ATP(ddA), ddC, ddG, and ddT) are used to promote a nucleotide specific termination in the chain extension, so that some of the chains terminate at every position at which that nucleotide occurs in the sequence. By using each of the nucleotide specific-chain terminators in separate chain extension reactions and on the same primed template, the sequence of the template can be determined. This method is simple, rapid, and accurate and has been used to determine the sequences of whole DNA viruses (phi X174 and G4), plasmid cloned DNA, eukaryote mRNAs, and RNA viral genomes. These primed synthesis methods of sequencing are now being developed into methods for nucleotide mutagenesis.