Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer.
Antoine Daunay,Alex Duval,Laura G. Baudrin,Olivier Buhard,Victor Renault,Jean-François Deleuze,Alexandre How-Kit +6 more
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It is shown that LT-RPA improves the limit of detection of MSI compared to PCR up to four times, notably for small deletions, and simplifies the identification of the mutant alleles.Abstract:
Microsatellites are polymorphic short tandem repeats of 1-6 nucleotides ubiquitously present in the genome that are extensively used in living organisms as genetic markers and in oncology to detect microsatellite instability (MSI). While the standard analysis method of microsatellites is based on PCR followed by capillary electrophoresis, it generates undesirable frameshift products known as 'stutter peaks' caused by the polymerase slippage that can greatly complicate the analysis and interpretation of the data. Here we present an easy multiplexable approach replacing PCR that is based on low temperature isothermal amplification using recombinase polymerase amplification (LT-RPA) that drastically reduces and sometimes completely abolishes the formation of stutter artifacts, thus greatly simplifying the calling of the alleles. Using HT17, a mononucleotide DNA repeat that was previously proposed as an optimal marker to detect MSI in tumor DNA, we showed that LT-RPA improves the limit of detection of MSI compared to PCR up to four times, notably for small deletions, and simplifies the identification of the mutant alleles. It was successfully applied to clinical colorectal cancer samples and enabled detection of MSI. This easy-to-handle, rapid and cost-effective approach may deeply improve the analysis of microsatellites in several biological and clinical applications.read more
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The clinical utility of microsatellite instability in colorectal cancer.
TL;DR: This review aims to assess the clinical utility of MSI as a biomarker in CRC and provides an overview of the available methods for evaluation of the analytical validity of MSI detection and elaborate the evidence on the clinical validity ofMSI in the management of CRC patients.
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Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies.
Ioannis Ladas,Fangyan Yu,Ka Wai Leong,Mariana Fitarelli-Kiehl,Chen Song,Ravina Ashtaputre,Matthew H. Kulke,Harvey J. Mamon,G. Mike Makrigiorgos +8 more
TL;DR: A nuclease-based approach that uses overlapping oligonucleotides to eliminate unaltered micro-satellites at the genomic DNA level, prior to PCR is described, improving detection sensitivity by 500–1000-fold relative to current HRM approaches.
Journal ArticleDOI
Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation.
Marc Ciosi,Sarah A. Cumming,Afroditi Chatzi,Eloise Larson,William Tottey,Vilija Lomeikaite,Graham Hamilton,Vanessa C. Wheeler,Ricardo Mouro Pinto,Seung Kwak,A. Jennifer Morton,Darren G. Monckton +11 more
TL;DR: In this paper, the authors applied MiSeq and PacBio sequencing to PCR products of the HTT CAG repeat in transgenic R6/2 mice carrying ∼55, ∼110, ∼255 and ∼470 CAGs and compared the repeat length distributions generated for different tissues at two ages.
Journal ArticleDOI
NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR.
Fangyan Yu,Ka Wai Leong,Alexander Makrigiorgos,Viktor A. Adalsteinsson,Ioannis Ladas,Kimmie Ng,Harvey J. Mamon,G. Mike Makrigiorgos +7 more
TL;DR: A new approach to detect microsatellite instability using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed- PCR with the breadth of information provided by NGS is presented.
Journal ArticleDOI
Sensitive detection of microsatellite instability in tissues and liquid biopsies: Recent developments and updates.
TL;DR: A review of the available microsatellite instability (MSI) detection methods and their detection limits is presented in this article, focusing on recently developed next-generation-sequencing based approaches and bioinformatics algorithms available for MSI analysis in various cancer types.
References
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Journal ArticleDOI
Microsatellites: simple sequences with complex evolution
TL;DR: Few genetic markers, if any, have found such widespread use as microsatellites, or simple/short tandem repeats, but features such as hypervariability and ubiquitous occurrence explain their usefulness, but these features also pose several questions.
Journal ArticleDOI
Microsatellite Instability in Colorectal Cancer
C. Richard Boland,Ajay Goel +1 more
TL;DR: Colorectal tumors with MSI have distinctive features, including a tendency to arise in the proximal colon, lymphocytic infiltrate, and a poorly differentiated, mucinous or signet ring appearance, and do not have the same response to chemotherapeutics.
Journal ArticleDOI
DNA Detection Using Recombination Proteins
TL;DR: RPA couples isothermal recombinase polymerase-driven primer targeting of template material with strand-displacement DNA synthesis and achieves exponential amplification with no need for pretreatment of sample DNA, thereby establishing an instrument-free DNA testing system.