Mori A, Takagi A, Ikeda Y, Yamamoto A
Department of Etiology and Pathophysiology, National Cardiovascular Center Research Institute, Suita.
Rinsho Byori. 1996 Oct;44(10):983-90.
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.
我们开发了一种改进的直接DNA测序方法，即使在外显子侧翼内含子的碱基序列信息有限的情况下，也能够检测外显子以及内含子中的剪接共有区域（受体和供体）中的杂合突变。由于已报道的脂蛋白脂肪酶（LPL）基因的内含子碱基序列仅限于40个碱基，因此利用该基因来开发此方法。我们构建了带有额外9个碱基的PCR引物用于LPL基因扩增，并将PCR引物5'端的20聚体用作测序引物，以便从测序引物的3'端到剪接受体共有序列的5'端获得必要的距离，从而准确确定共有区域的序列。此外，添加这9个碱基使得通过调整其G + C/A + T比例来优化测序引物的Tm值成为可能。使用具有适当Tm值（即本研究中为48℃ - 58℃）的测序引物的直接测序方法有效地减少了序列梯带模式上的非特异性条带，并能够确定剪接共有序列和外显子的有义链和反义链的碱基序列。结果，这种改进的直接测序方法能够检测LPL基因的杂合和纯合突变。
