High-Resolution Melting Analysis as a Developed Method for Genotyping the PD Susceptibility Loci in LRRK2 Gene.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.
Enzhu
E
Jiang
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.|Department of Forensic Medicine, Baotou Medical College, Baotou, P.R. China.
Fengrui
F
Li
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.
Chenchen
C
Jing
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.
Pei
P
Li
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.
Honggang
H
Cui
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.
Baojie
B
Wang
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.
Mei
M
Ding
School of Forensic Medicine, China Medical University, Shenyang, P.R. China.
Hao
H
Pang
D056426:Genetic Loci; D020022:Genetic Predisposition to Disease; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation; D010300:Parkinson Disease / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*
C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases
HRM; LRRK2; Parkinson's disease; SNPs; genotyping; susceptibility locus