Gel-Based Nonradioactive Single-Strand Conformational Polymorphism and Mutation Detection: Limitations and Solutions

Gupta, Vibhuti; Arora, Reetakshi; Ranjan, Anand; Bairwa, Narendra K.; Malhotra, Dheeraj; Udhayasuriyan, P.T.; Saha, Ayan; Bamezai, R.

doi:10.1385/1-59259-840-4:247

Cited by 6 publications

(5 citation statements)

References 28 publications

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“…Screening of hotspots for mutation before sequencing could reduce this cost by excluding wild-type samples from further analysis. Techniques such as single-strand conformation polymorphism,7 temperature gradient gel electrophoresis,8 denaturing high-performance liquid chromatography9 and matrix-assisted laser desorption/ionisation-time of flight mass spectroscopy10 11 have been developed for the purposes of mutation detection. However, these techniques are complicated, expensive and tend to be available only in research institutions.…”

Section: Introductionmentioning

confidence: 99%

Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation detection in formalin-fixed paraffin-embedded tissue

et al. 2010

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Section: Introductionmentioning

confidence: 99%

Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation detection in formalin-fixed paraffin-embedded tissue

et al. 2010

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“…Sanger sequencing is usually regarded as the ‘gold standard’ for mutation detection but there are some doubts regarding its sensitivity. For any mutation detection method, the lower limit of detection of mutant alleles is an important consideration and a variety of more sensitive techniques are available (Gupta et al. 2005; Lee et al.…”

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confidence: 99%

“…Sanger sequencing is usually regarded as the 'gold standard' for mutation detection but there are some doubts regarding its sensitivity. For any mutation detection method, the lower limit of detection of mutant alleles is an important consideration and a variety of more sensitive techniques are available (Gupta et al 2005;Lee et al 2005;Janne et al 2006;van den Boom & Ehrich 2007;Koren-Michowitz et al 2008). Many of these are expensive, complicated and of unproven use when dealing with FFPE tissue.…”

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confidence: 99%

Comparative analysis of pyrosequencing and QMC‐PCR in conjunction with high resolution melting for KRAS/BRAF mutation detection

Ibrahem

Seth

O’Sullivan

et al. 2010

Int J Experimental Path

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Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing are the most versatile. We undertook a comparative analysis of these techniques. The methods are: To compare the limit of detection (LOD), mutations in KRAS (codon 12/13 hotspot) and BRAF (V600E hotspot) were tested. DNA mixtures containing mutant alleles at a frequency of around 25%/12.5%/6%/3%/ 1.5%/0.8% were analysed. To compare frequency of mutation detection, 22 DNA samples (nine high quality samples from cell lines, 13 low quality samples from formalin-fixed paraffin-embedded tissue) were tested for three hotspots in KRAS (codons 12/13, 61 and 146) and two hotspots in BRAF (V600E and exon 11). HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. Two of the samples tested had previously been called as wild type for KRAS by direct Sanger sequencing but were found to be mutant by both HRM and pyrosequencing. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing.

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“…TRAIL, BRCA2, and TP53 polymorphisms were detected by sequencing of the PCR products [31] and SSLP carried out [32] to detect IFNG CA-repeat length polymorphism. Casp8 -652 6N deletion polymorphism was genotyped by performing denaturing high performance liquid chromatography (DHPLC) [33].…”

Section: Cases and Controlsmentioning

confidence: 99%

Functional implication of TRAIL −716 C/T promoter polymorphism on its in vitro and in vivo expression and the susceptibility to sporadic breast tumor

Pal

Gochhait

Chattopadhyay

et al. 2010

Breast Cancer Res Treat

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Recently, TRAIL function has been elucidated beyond its known classical role of mediating cellular homeostasis and immune surveillance against transformed cells. Here, we show how CC genotype of -716 TRAIL promoter SNP rendered risk for sporadic breast cancer as compared to the CT and TT genotypes (P (recessive model) = 0.018, OR = 1.4, 95% CI = 1.1-1.9; P (allele model) = 0.010, OR = 1.3, 95% CI = 1.1-1.7). The in silico prediction of the introduction of core Sp1/Sp3-binding motif suggested the functional significance of the SNP variation. This functional implication was validated by luciferase assay in HeLa (P = 0.026), MCF-7 (P = 0.022), HepG2 (P = 0.024), and HT1080 (P = 0.030) cells and also by real-time expression studies on tumor tissues (P = 0.01), revealing the transcriptionally repressed status of -716 T when compared to -716 C allele. The SNP-SNP interactions reflected an enhanced protective effect of CT and TT genotypes with the protective genetic backgrounds of TP53-BRCA2 (P = 0.002, OR = 0.2, 95% CI = 0.1-0.6), IFNG (P = 0.0000002, OR = 0.3, 95% CI = 0.2-0.4), and common variant Casp8 (P = 0.0003, OR = 0.5, 95% CI = 0.3-0.7). Interestingly, a comparison with clinical parameters showed overrepresented CT and TT genotypes in progressing (P = 0.041) and ER/PR negative tumors (P = 0.024/0.006). This was explained by increased apoptotic index, calculated as a ratio of selected pro-apoptotic and anti-apoptotic gene expression profiles, in CC genotyped tumors, favoring either intrinsic (P = 0.008,0.018) or extrinsic (P = 0.025,0.217) pathway depending upon the ER/PR status. Our study reveals for the first time that a promoter SNP of TRAIL functionally modulates the gene and consequently its role in breast cancer pathogenesis, cautioning to consider the -716 TRAIL SNP status in patients undergoing TRAIL therapy.

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Gel-Based Nonradioactive Single-Strand Conformational Polymorphism and Mutation Detection: Limitations and Solutions

Cited by 6 publications

References 28 publications

Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation detection in formalin-fixed paraffin-embedded tissue

Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation detection in formalin-fixed paraffin-embedded tissue

Comparative analysis of pyrosequencing and QMC‐PCR in conjunction with high resolution melting for KRAS/BRAF mutation detection

Functional implication of TRAIL −716 C/T promoter polymorphism on its in vitro and in vivo expression and the susceptibility to sporadic breast tumor

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