Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis

Vorkas, Panagiotis A.; Christopoulos, Konstantinos; Kroupis, Christos; Lianidou, Evi

doi:10.1016/j.clinbiochem.2009.08.024

Cited by 13 publications

(15 citation statements)

References 24 publications

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“…It has been previously shown that this could be an important issue in HRMA. 24,25 We also tested the amount of template used (100 ng, 50 ng, and 25 ng) in PCR reactions, using two wild-type FFPE samples presenting high and one presenting low variation. The samples that presented high variation showed a reduction of variation with template amount of 25 ng (see Supplemental Figure S2, C and D at http://jmd.amjpathol.org).…”

Section: Resultsmentioning

confidence: 99%

“…However, FFPE samples present difficulties in PCR amplification and a reduction of diagnostic specificity when analyzed by HRMA. 24,25 Herein, we report the development and validation of a highly sensitive and reliable HRMA mutation scanning methodology, using small amplicons, for hotspot regions of the PIK3CA gene. These mutations present a high prevalence in breast, as well as in other cancer types, and have an important clinical significance.…”

Section: Discussionmentioning

confidence: 99%

“…20 Moreover, small amplicons display a higher analytical sensitivity compared with larger amplicons. 23,24 This is a major advantage when scanning for somatic mutations, where the tumor tissue is contaminated with adjacent normal tissue.…”

Section: The Introduction Of High-resolution Melting Analysis (Hrma) mentioning

confidence: 99%

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PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method

Vorkas

Poumpouridou

Agelaki

et al. 2010

The Journal of Molecular Diagnostics

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Somatic mutations in the PIK3CA gene have been discovered in many human cancers , and their presence correlates to therapy response. Three "hotspot" mutations within the PIK3CA gene are localized in exons 9 and 20. High-resolution melting analysis (HRMA) is a highly sensitive, robust, rapid, and cost-effective mutation analysis technique. We developed a novel methodology for the detection of hotspot mutations in exons 9 and 20 of the PIK3CA gene that is based on a combination of PCR and HRMA. The PIK3CA HRMA assay was evaluated by performing repeatability, sensitivity , and comparison with DNA sequencing studies and was further validated in 129 formalin-fixed paraffin-embedded breast tissue samples: 99 tumors, 20 noncancerous , and 10 fibroadenomas. The developed methodology was further applied in a selected group of 75 breast cancer patients who underwent Trastuzumab treatment. In sensitivity studies , the assay presented a capability to detect as low as 1% of mutated dsDNA in the presence of wtDNA for both exons. In the 99 tumor samples (validation group) , 12/99 (12.1%) exon 9 mutations and 20/99 (20.2%) exon 20 mutations were found. No mutations were found in noncancerous tissues. In fibroadenomas , we report one PIK3CA mutation for the first time. In the selected group , 30/75 (40%) samples were detected as mutants. The PIK3CA HRMA assay is highly sensitive , reliable , cost-effective , and easy-to-perform, and therefore can be used as a screening test in a highthroughput pharmacodiagnostic setting. PIK3CA mutations appear to have a clinical significance.12 Recent studies have shown that PIK3CA mutations can independently hamper the therapeutic response to anti-EGFR biological therapies (panitumumab or cetuximab) in metastatic colorectal cancer 13 and demonstrate resistance to dietary restriction therapies. 14 Moreover, several efforts are underway nowadays to target the PI3K pathway with therapeutic inhibitors.Additionally, keeping in mind that not all patients with HER2-overexpressing metastatic breast cancer respond to Trastuzumab (Herceptin), activated PI3K signaling has been proposed to predict Trastuzumab resistance. 15,16 Loss of the PTEN (Phosphatase and Tensin Homolog) protein has been suggested as a key factor for the development of resistance to this drug. 15,16 However, PTEN loss alone, 16,17 and in combination with phosphorylated AKT expression, 17 proved inadequate to predict response to therapy. Conversely, combining PTEN loss and gain-of-function mutations of the PIK3CA gene, resistance to Trastuzumab was successfully predicted.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method

Vorkas

Poumpouridou

Agelaki

et al. 2010

The Journal of Molecular Diagnostics

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“…In plants, such an approach has been applied to detect SSRs, which was used for genotype identification and authenticity testing (Jaakola et al, 2010;Ganopoulos et al, 2011;Distefano et al, 2012;Madesis et al, 2012). Moreover, it can also be used for mutation scanning (Vorkas et al, 2010). The application of HRM to discriminate SSR genotypes in domestic animals has not been reported.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the melting process is faster, data analysis can be performed automatically in a few minutes, and the sensitivity is very high relative to other methods (Vossen et al, 2009). HRM has been widely used to detect single nucleotide polymorphisms (SNPs) and to scan mutations (Vorkas et al, 2010;Trujillo et al, 2012). Recently, more and more studies have focused on the genotyping of microsatellites by HRM.…”

Section: Introductionmentioning

confidence: 99%

Limitation of high-resolution melting curve analysis for genotyping simple sequence repeats in sheep

Yang¹,

Yuan²,

Guo³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Variation in microsatellite or simple sequence repeat (SSR) loci has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to genotype. Non gel-based systems are therefore important to increase simplicity and improve turn-around time without compromising assay sensitivity and accuracy. In this report, we assessed the latest of the non-gelbased methods, high-resolution melting (HRM) curve analysis. HRM is a technique that monitors exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double-stranded DNA. The measurement immediately follows polymerase chain reaction in a one-step, closed-tube method. Four SSR loci of different complexity in sheep, namely MAF209, MCM140, CB226, and SRCRSP5, were assessed using the LightScanners System with LC Greens PLUS DNA binding dye. In order to improve the accuracy of genotyping, we applied internal oligo nucleotide calibrators while performing HRM. DNA polymorphisms were previously identified using capillary electrophoresis analysis (CE). The result showed that CE detected more genotypes than HRM in the same loci regardless of the level of polymorphism at the SSR loci. We demonstrate current limitations of the HRM method for the analysis of SSR loci.

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Ultrafast, low-power, PCB manufacturable, continuous-flow microdevice for DNA amplification

et al. 2019

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The design and fabrication of a continuous flow μPCR device with very short amplification time and low power consumption is presented. Commercially available, 4-layer printed circuit board (PCB) substrates are employed, with inhouse designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network, where DNA amplification takes place. The 1.9 m-long microchannel in combination with desirably high flow velocities (for fast amplification) challenged the robustness of the sealing, that was overcome with the development of a novel bonding method rendering the microdevice robust even at extreme pressure drops (12 bars). The proposed fabrication methods are PCB compatible, allowing for mass and reliable production of the μPCR device in the established PCB industry. The μPCR chip was successfully validated during the amplification of two different DNA fragments (and with different target DNA copies) corresponding to the exon 20 of the BRCA1 gene, and to the plasmid pBR322, a commonly used cloning vector in E. coli. Successful DNA amplification was demonstrated at total reaction

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Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis

Cited by 13 publications

References 24 publications

PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method

PIK3CA Hotspot Mutation Scanning by a Novel and Highly Sensitive High-Resolution Small Amplicon Melting Analysis Method

Limitation of high-resolution melting curve analysis for genotyping simple sequence repeats in sheep

Ultrafast, low-power, PCB manufacturable, continuous-flow microdevice for DNA amplification

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