Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application toMLH1germline mutations in Lynch syndrome

Abstract

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called ''quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM),'' which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)1multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC1MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

High-resolution DNA melting analysis: advancements and limitations

“…Therefore, an additional technique such as multiplex ligation-dependent probe amplification (MLPA) or quantitative PCR (qPCR) is needed to assess gene dosage. In this issue, Rouleau et al (2009) combine qPCR for detection of large rearrangements with high resolution melting to detect small variants. In 90 min on a single instrument, they determine both exon-by-exon gene dosage and scan the mismatch repair gene MLH1 for variants.…”

“…However, the optimal number of melting domains for HRMA remains controversial. For example, Tindall et al (2009) argue strongly for small products and against multiple domain melting, while half of the targets used by Rouleau et al (2009) have more than one domain and most of the PCR products used by Dobrowolski et al (2009) have two or more domains, all in this issue. Controversy and competition may be good signs for this fledgling utilitarian method.…”

High-Resolution Melting Analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine

“…A combination of qPCR and HRMA has been proposed as a low-cost and rapid method to test for D. pinea when the number of samples is very large (Rouleau et al 2009). Such a combination may also be the best way to detect D. pinea DNA, ensuring both specificity and accuracy.…”

Microsatellite Instability Detection by High-Resolution Melting Analysis

“…HRM analysis is similar to DHPLC in that both methods identify heteroduplexes by their lower thermal stability, and HRM has been shown to be comparable to DHPLC in terms of its analytical sensitivity and specificity (19 ). Major drawbacks of the DHPLC method are chemical waste, high maintenance cost, the need for post-PCR manipulations, and low throughput (37 ). Unlike DHPLC, HRM analysis scans through a range of temperatures rather than depending on a specific temperature that requires intensive optimization for each run.…”

“…HRM analysis with saturating DNA dyes as a scanning tool for heteroduplex identification has apparent advantages, including assay design simplicity, no post-PCR manipulation, accuracy, versatility, speed of analysis, and cost efficiency (19,37 ). If necessary, more mononucleotide MSI markers can be easily incorporated into HRM analysis.…”