Capture PCR: Efficient amplification of DNA fragments adjacent to a known sequence in human and YAC DNA

Abstract

We have devised a procedure, termed capture PCR (CPCR), that permits the rapid isolation of DNA segments situated adjacent to a characterized nucleotide sequence. In this procedure, a DNA sample is restriction-digested and a linker, comprising two base-paired oligonucleotides, is added to the ends by ligation. Multiple extension reactions are performed using a biotinylated primer derived from the known sequence, permitting the subsequent isolation of extension products on a streptavidin-coated support. The enriched fragments are amplified exponentially using another specific oligonucleotide, hybridizing 3′ to the biotinylated primer in combination with one of the linker oligonucleotides, now functioning as a PCR primer. The convenience of CPCR is greatly enhanced by using a novel streptavidin-coated manifold, which is constructed so that it projects into each individual well of a microtiter plate. The procedure permits the simultaneous isolation of fragments from large numbers of DNA samples and minimizes the risk of contamination between reactions. We have applied this method to identify DNA sequences located downstream of known sequences in the human genome. The technique has also been used to identify end fragments of sequences cloned in a yeast artificial chromosome (YAC) vector. The reactions can be initiated directly from yeast colonies and provide access to DNA sequence information for these end fragments in a minimal number of steps. With the aid of the present technique, we have isolated over 100 end fragments from YACs derived from the human X chromosome. Isolated end sequences have been used to order YAC clones into a contig.