Sequence-independent, single-primer amplification (SISPA) of complex DNA populations

Abstract

Sequence-Independent, Single-Primer Amplification (SISPA) is a primer initiated technique that requires target sequence modification to achieve the non-selective logarithmic amplification of heterogeneous DNA populations. The method contrasts with the polymerase chain reaction (PCR), and its modified approaches, that have as their objective the amplification of unique or homologous sequences. SISPA requires the directional ligation of an asymmetric linker/primer oligonucleotide onto the target population of blunt ended DNA molecules. The common end sequence allows one strand of the double-stranded linker/primer to be used in repeated rounds of annealing, extension and denaturation in the presence of thermostable Taq DNA polymerase. The linker/primers contain restriction endonuclease sites to facilitate the molecular cloning of as little as 1 pg of starting material after amplification. SISPA is especially useful when the nucleotide sequence of the desired molecule is both unknown and present in limited amounts making its recovery by standard cloning procedures technically difficult. These conditions are present in the initial isolation and cloning of previously uncharacterized viral genomes. The application and quantitation of SISPA is described, together with its utility in the cloning and recovery of low abundance genetic sequences, as illustrated here with the hepatitis C virus.