Jiang Enzhu, Li Fengrui, Jing Chenchen, Li Pei, Cui Honggang, Wang Baojie, Ding Mei, Pang Hao
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.
Department of Forensic Medicine, Baotou Medical College, Baotou, P.R. China.
J Clin Lab Anal. 2015 Jul;29(4):299-304. doi: 10.1002/jcla.21769. Epub 2014 May 21.
Single-nucleotide polymorphisms (SNPs) have been reported as a highly relevant point for the mechanisms of Parkinson's disease (PD). The invention of saturating dye makes it possible to identify heteroduplex DNA without redistribution during melting, which allows using high-resolution melting (HRM) to detect SNPs. However, the HRM analysis for detection of those SNPs associated with PD was rarely applied.
Two SNPs, G2385R and R1628P, located in leucine-rich repeat kinase 2 (LRRK2) gene were individually and multiplexedly genotyped using HRM analysis. The sequence variant observed in unexpected HRM curves was confirmed by DNA sequencing.
HRM analysis identified successfully all genotypes both on R1628P and G2385R loci. The unexpected HRM curves appeared in R1628P amplicon generated from combinations of R1628P and rs11176013 loci. A multiplexed HRM assay that genotyped R1628P, rs11176013, and G2385R loci was efficiently established.
The present HRM assay is a reliable and rapid method for genotyping R1628P and G2385R loci in LRRK2 gene, and multiplex HRM analysis results in high throughput and has the potential to facilitate a wide range of genotyping studies on PD susceptibility genes.
单核苷酸多态性（SNP）已被报道为帕金森病（PD）发病机制的一个高度相关点。饱和染料的发明使得在熔解过程中无需重新分配即可鉴定异源双链DNA成为可能，这使得利用高分辨率熔解（HRM）检测SNP成为现实。然而，用于检测与PD相关的SNP的HRM分析很少被应用。
使用HRM分析对位于富含亮氨酸重复激酶2（LRRK2）基因中的两个SNP，即G2385R和R1628P进行单独和多重基因分型。通过DNA测序确认在意外的HRM曲线中观察到的序列变异。
HRM分析成功鉴定了R1628P和G2385R位点上的所有基因型。从R1628P和rs11176013位点组合产生的R1628P扩增子中出现了意外的HRM曲线。高效建立了对R1628P、rs11176013和G2385R位点进行基因分型的多重HRM检测方法。
本HRM检测方法是一种可靠且快速的对LRRK2基因中的R1628P和G2385R位点进行基因分型的方法，多重HRM分析具有高通量的特点，有潜力促进对PD易感基因进行广泛的基因分型研究。
