Abstract
Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and the same amplicon, although with limited discrimination power. By targeting the three C>T SNPs rs527559815, rs547832288, and rs16906252, located in the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene within a distance of 45 bp, we investigated whether the discrimination power can be increased by coupling HRM analysis with pyrosequencing (PSQ). After optimizing polymerase chain reaction (PCR) conditions, PCR products subjected to HRM analysis could directly be used for PSQ. By analyzing oligodeoxynucleotide controls, representing the 36 theoretically possible variant combinations for diploid human cells (8 triple-homozygous, 12 double-homozygous, 12 double-heterozygous and 4 triple-heterozygous combinations), 34 out of the 36 variant combinations could be genotyped unambiguously by combined analysis of HRM and PSQ data, compared to 22 variant combinations by HRM analysis and 16 variant combinations by PSQ. Our approach was successfully applied to genotype stable cell lines of different origin, primary human tumor cell lines from glioma patients, and breast tissue samples.
Highlights
Single nucleotide polymorphisms (SNPs) are the most abundant type of genetic variations in the human genome [1]
For investigation of the potential of high resolution melting (HRM) analysis coupled with PSQ to genotype multiple SNPs in one and the same amplicon, we selected the SNPs rs527559815, rs547832288, and rs16906252 due to the following reasons
The three SNPs are located in a differentially methylated region (DMR) of the methylguanine-DNA methyltransferase (MGMT) promoter, with rs527559815 being upstream of exon 1, rs547832288 located in exon 1, and rs16906252 occurring in an intra-promoter enhancer element [31]
Summary
Single nucleotide polymorphisms (SNPs) are the most abundant type of genetic variations in the human genome [1]. A detailed map of genetic variations constructed by the 1000 Genomes Project hints at more than 80 million SNPs [2]. By applying high-throughput platforms such as high-density SNP microarrays and generation sequencing (NGS). Technologies, genome-wide association studies (GWAS) have linked thousands of SNPs to multifactorial diseases [3], including rheumatoid arthritis [4], schizophrenia [5], and cancer [6,7]. More than 90% of disease-associated SNPs are located in non-coding regions, including promoter regions and enhancers. The suitability of regulatory SNPs as potential diagnostic, prognostic and/or predictive biomarker has been investigated in numerous studies [6,8,11]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
