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Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis.

ABSTRACT: Mutation scanning using high-resolution melting curve analysis (HR-melt) is an effective and sensitive method to detect sequence variations. However, the presence of a common SNP within a mutation scanning amplicon may considerably complicate the interpretation of results and increase the number of samples flagged for sequencing by interfering with the clustering of samples according to melting profiles. A protocol describing simultaneous high-resolution gene scanning and genotyping has been reported. Here, we show that it can improve the sensitivity and the efficiency of large-scale case-control mutation screening. Two exons of ATM, both containing an SNP interfering with standard mutation scanning, were selected for screening of 1,356 subjects from an international breast cancer genetics study. Asymmetric PCR was performed in the presence of an SNP-specific unlabeled probe. Stratification of the samples according to their probe-target melting was aided by customized HR-melt software. This approach improved identification of rare known and unknown variants, while dramatically reducing the sequencing effort. It even allowed genotyping of tandem SNPs using a single probe. Hence, HR-melt is a rapid, efficient, and cost-effective tool that can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes.

SUBMITTER: Nguyen-Dumont T

PROVIDER: S-EPMC3478947 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis.

Nguyen-Dumont Tú T Calvez-Kelm Florence Le FL Forey Nathalie N McKay-Chopin Sandrine S Garritano Sonia S Gioia-Patricola Lydie L De Silva Deepika D Weigel Ron R Sangrajrang Suleeporn S Lesueur Fabienne F Tavtigian Sean V SV

Human mutation 20090601 6

Mutation scanning using high-resolution melting curve analysis (HR-melt) is an effective and sensitive method to detect sequence variations. However, the presence of a common SNP within a mutation scanning amplicon may considerably complicate the interpretation of results and increase the number of samples flagged for sequencing by interfering with the clustering of samples according to melting profiles. A protocol describing simultaneous high-resolution gene scanning and genotyping has been rep ...[more]

PMID: 19347964

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Project description:With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.

Project description:BACKGROUND: Together single nucleotide substitutions and small insertion/deletion variants are the most common form of sequence variation in the human gene pool.High-resolution SNP profile and/or haplotype analyses enable the identification of modest-risk susceptibility genes to common diseases, genes that may modulate responses to pharmaceutical agents, and SNPs that can affect either their expression or function. In addition, sensitive techniques for germline or somatic mutation detection are important tools for characterizing sequence variations in genes responsible for tumor predisposition. Cost-effective methods are highly desirable. Many of the recently developed high-throughput technologies are geared toward industrial scale genetic studies and arguably do not provide useful solutions for small laboratory investigator-initiated projects. Recently, the use of new fluorescent dyes allowed the high-resolution analysis of DNA melting curves (HRM). RESULTS: Here, we compared the capacity of HRM, applicable to both genotyping and mutation scanning, to detect genetic variations in the tumor suppressor gene TP53 with that of mutation screening by full resequencing. We also assessed the performance of a variety of available HRM-based genotyping assays by genotyping 30 TP53 SNPs. We describe a series of solutions to handle the difficulties that may arise in large-scale application of HRM to mutation screening and genotyping at the TP53 locus. In particular, we developed specific HRM assays that render possible genotyping of 2 or more, sometimes closely spaced, polymorphisms within the same amplicon. We also show that simultaneous genotyping of 2 SNPs from 2 different amplicons using a multiplex PCR reaction is feasible; the data can be analyzed in a single HRM run, potentially improving the efficiency of HRM genotyping workflows. CONCLUSION: The HRM technique showed high sensitivity and specificity (1.0, and 0.8, respectively, for amplicons of <400 bp) for mutation screening and provided useful genotyping assays as assessed by comparing the results with those obtained with Sanger sequencing. Thus, HRM is particularly suitable for either performing mutation scanning of a large number of samples, even in the situation where the amplicon(s) of interest harbor a common variant that may disturb the analysis, or in a context where gathering common SNP genotypes is of interest.

Project description:Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5.Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP.Intra-assay (n=5) precision of Tms for all SNPs was ?0.19%, while inter-assay (n=20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/?L.The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

Dataset Information

Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis.

Publications

Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis.

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