[The improvement of a direct DNA sequencing method by devising the primer design: detection of lipoprotein lipase gene aberrations]

We developed an improved method of direct DNA sequencing which makes it possible to detect heterozygous mutations in an exon and the splicing consensus regions (acceptor and donor) in introns even in the cases where there is limited information available regarding the base sequences of the introns flanking the exon. Human lipoprotein lipase (LPL) gene was utilized to develop this method, since the reported intron base sequences of the LPL gene are limited to 40 bases. We constructed PCR primers with extra 9 bases for the LPL gene amplification and 20-mers from 5' end of the PCR primers were used as sequencing primers in order to obtain the necessary distance from the 3' end of the sequencing primer to 5' end of the splicing acceptor consensus sequence for the accurate determination of the sequence of the consensus region. Furthermore, the attachment of the 9 bases made it possible to optimize the Tm value of the sequencing primers by adjusting their G + C/A + T ratio. The direct sequencing method using the sequencing primer with an appropriate Tm value (i.e., 48 degrees C-58 degrees C in this study) was effective in reducing nonspecific bands on the sequence ladder pattern, and allowed the determination of the base sequences of both the sense and antisense strands of the splicing consensus sequences and exon. As a result, this improved direct sequencing method was capable of detecting heterozygous as well as homozygous LPL gene mutations.