摘要
目的:应用基于适配器连接介导的等位基因特异性扩增法(Adapter Ligation-mediated Allele-specific Amplification,简称ALM-ASA)技术,检测与帕金森病(PD)发病相关的LRRK2基因中的4个SNP位点(6055G>A,7153G>A,4321C>T和2264C>T),探讨该法用于筛查帕金森病相关SNP位点的可行性,研究多个SNP位点同时检测的准确性和可靠性。方法:运用ALM-ASA法原理,改进使用多重PCR法代替单一预扩增法,使用4对引物在单管中预扩增含所待测SNP位点的四段片段,通过酶切、酶连和PCR特异性扩增检测判断SNP的类型。经PCR体外定点突变实验制备的相应位点的突变阳性片段,检验方法的准确性和可靠性。结果:采用该法成功测定了20名PD病人和20名健康中国人的LRRK2基因中最受关注的4个SNP位点的多态性。并随机对其中10分样本的检测结果以测序法检测进行验证,结果完全一致。结论:ALM-ASA法极大提高了PCR反应的特异性,是一种准确、可靠,且费用低廉的SNP筛查方法,可推广应用于临床和实验室进行与PD有关的单核苷酸多态性的筛查检测。
Objective： To apply a new method named as adapter-legation mediated allele-specific amplification （ALM-ASA in short） for multiplex single nucleotide polymorphism （SNP） genotyping of leucine rich repeat kinase 2 （LRRK2） in Parkinson＇s disease （PD） patients. Four SNPs loci 6055G〉A （G2019S）,7153G〉A （G2385R）,4321C〉T（R1441C）and 2264C〉T （P755L）in LRRK2 gene were selected as targets for investigation. Methods： The ALM-ASA method was applied to the genotyping with a slight modification, such as multiple DNA fragments containing SNPs of interest were preamplification instead of one fragment with all SNPs. By comparing with the positive samples artificially prepared from the PCR-based site-directed mutagenesis, the accuracy and reliability of the method was testified. Results： The four SNPs in the samples of 20 patients and 20 healthy were successfully typed. The results were in agreement with those by Sanger＇s DNA sequencing method. Conclusion： ALM-ASA is an accurate and efficient method for LRRK2 SNP typing with the advantages of simple set-up, low sample consuming, and lower running cost. It will be very powerful for screening the SNPs in the LRRK2 gene associated with PD in clinical research.
出处
《现代生物医学进展》
CAS
2009年第14期2716-2720,2734,共6页
Progress in Modern Biomedicine
基金
国家自然科学基金资助项目(No.30270368)
关键词
ALM-ASA法
LRRK2
单核苷酸多态
多重PCR
Adapter-ligation mediated allele-specific amplification
leucine rich repeat kinase 2
single nucleotide polymorphism
multiplex PCR
