Determination of gene structure by intron trapping using polymerase chain reaction: application to the human plasma...

We have devised a method to determine gene structure that utilizes the known gene structure of homologous proteins and the polymerase chain reaction (PCR). Because homologous proteins have evolved from a common ancestral gene, it is possible to design primers corresponding to the adjacent exon sequences in the protein of interest. These primers could then be used in a PCR using normal genomic DNA as template. The resultant PCR product is then subcloned and the nuceotide sequence of the insert is determined. This information provides the exon-intron junction sequences and the partial sequences of the intron. We have applied this method to trap introns A, H, and J of human plasma prekallikrein gene and determined their exon-intron junction sequences. The primers were designed based on the known cDNA sequence of human plasma prekallikrein and gene structures of rat plasma prekallikrein and human coagulation factor XI (which is 50% identical in the primary sequence). The intron-exon junctions are at identical sites to those of the rat plasma prekallikrein gene. The intron sequences thus obtained will be useful in designing primers for exon trapping and sequence analysis of plasma prekallikrein gene from patients with defective plasma prekallikrein. This technique will also allow the determination of the entire gene structure.