Identification of ryanodine receptor 1 single-nucleotide polymorphisms by high-resolution melting using the LightCycler 480 System.
High-resolution melting (HRM) allows single-nucleotide polymorphism (SNP) detection/typing using inexpensive generic heteroduplex-detecting double-stranded DNA (dsDNA) binding dyes. Until recently HRM has been a post-PCR process. With the LightCycler 480 System, however, the entire mutation screening process, including post-PCR analysis, can be performed using a single instrument. HRM assays were developed to allow screening of the ryanodine receptor gene (RYR1) for potential mutations causing malignant hyperthermia (MH) and/or central core disease (CCD) using the LightCycler 480 System. The assays were validated using engineered plasmids and/or genomic DNA samples that are either homozygous wild type or heterozygous for one of three SNPs that lead to the RyR1 amino acid substitutions T4826I, H4833Y, and/or R4861H. The HRM analyses were conducted using two different heteroduplex-detecting dsDNA binding dyes: LightCycler 480 HRM dye and LCGreen Plus. Heterozygous samples for each of the HRM assays were readily distinguished from homozygous samples with both dyes. By using engineered plasmids, it was shown that even homozygous sequence variations can be identified by using either small amplicons or the addition of exogenous DNA after PCR. Thus, the LightCycler 480 System provides a novel, integrated, real-time PCR/HRM platform that allows high throughput, inexpensive SNP detection, and genotyping based on high-resolution amplicon melting.
Grievink
Hilbert
H
Institute of Molecular Biosciences, Massey University, Palmerston North 11-222, New Zealand.
Stowell
Kathryn M
KM
D004247:DNA / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics*; D044366:Transition Temperature
D019837:Ryanodine Receptor Calcium Release Channel; D004247:DNA