Dataset Information

High resolution melting for mutation scanning of TP53 exons 5-8.

ABSTRACT:

Background

p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples.

Methods

We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status.

Results

One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons.

Conclusion

HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.

SUBMITTER: Krypuy M

PROVIDER: S-EPMC2025602 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Publications

High resolution melting for mutation scanning of TP53 exons 5-8.

Krypuy Michael M Ahmed Ahmed Ashour AA Etemadmoghadam Dariush D Hyland Sarah J SJ DeFazio Anna A Fox Stephen B SB Brenton James D JD Bowtell David D DD Dobrovic Alexander A

BMC cancer 20070831

<h4>Background</h4>p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples.<h4>Methods</h4>We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA f ...[more]

PMID: 17764544

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Project description:BackgroundTogether 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).ResultsHere, 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.ConclusionThe 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:BackgroundMutation scanning provides the simplest, lowest-cost method for identifying DNA variations on single PCR amplicons, and it may be performed before sequencing to avoid screening of noninformative wild-type samples. High-resolution melting (HRM) is the most commonly used method for mutation scanning. With PCR-HRM, however, mutations less abundant than approximately 3%-10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application.MethodsWe used serial dilution of cell lines containing the TP53 exon 8 mutation to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted coamplification at lower denaturation temperature (COLD)-PCR with an extra step for cross-hybridization, followed by preferential denaturation and amplification at optimized critical temperature (full-COLD-PCR), to further enrich low-level mutations before HRM with or without DMSO, and we used droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional PCR-HRM and full-COLD-PCR-HRM with and without DMSO were used for mutation scanning of TP53 exon 8 in cancer samples containing known mutations and myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined.ResultsThe detection sensitivity of PCR-HRM scanning increases 2- to 5-fold in the presence of DMSO, depending on mutation type and sequence context, and can typically detect mutation abundance of approximately 1%. When mutation enrichment is applied during amplification with full-COLD-PCR followed by HRM in the presence of DMSO, mutations with 0.2%-0.3% abundance in TP53 exon 8 can be detected.ConclusionsDMSO improves HRM mutation scanning sensitivity with saturating dyes. When full-COLD-PCR is used, followed by DMSO-HRM, the overall improvement is about 20-fold compared with conventional PCR-HRM.

Project description:BACKGROUND: TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor software, aimed at simultaneous detection of mutations in three homoeologous genes. RESULTS: We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. CONCLUSION: Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.

Dataset Information

High resolution melting for mutation scanning of TP53 exons 5-8.

Background

Methods

Results

Conclusion

Publications

High resolution melting for mutation scanning of TP53 exons 5-8.

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