Genotyping SSR length variants by isothermal DNA amplification

Lee, David; Ialicicco, M.; Akkinepalli, Harika; Morreale, Giacomo; Liu, Yan; Leung, Hei; Scippa, Gabriella Stefania; Greenland, Andy; Mackay, Ian

doi:10.1139/g2012-058

Cited by 2 publications

(1 citation statement)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We chose RPA among the isothermal methods, as it allows an amplification of DNA at temperatures lower than 42°C while most of the others required a higher reaction temperature that would have no or little effects on SR (41). For example, loop-mediated isothermal amplification (LAMP) has already been evaluated on microsatellites with a reaction temperature of 55°C without reducing the formation of stutter peaks compared to PCR (53). Similarly, the reduction of the annealing and elongation of PCR at 56°C had only produced a very slight reduction of the stutter peaks compared to the standard PCR (54).…”

Section: Discussionmentioning

confidence: 99%

Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

Daunay

Duval

Baudrin

et al. 2019

Nucleic Acids Research

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

Daunay

Duval

Baudrin

et al. 2019

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

Pitfalls in mononucleotide microsatellite repeats instability assessing (MSI) in the patients with B-cell lymphomas

Sychevskaya¹,

Risinskaya²,

Кравченко³

et al. 2021

Klin. lab. diagn.

View full text Add to dashboard Cite

Analysis of microsatellite instability (MSI) is a routine study in the diagnostics of solid malignancies. The standard for determining MSI is a pentaplex PCR panel of mononucleotide repeats: NR-21, NR-24, NR-27, BAT-25, BAT-26. The presence of MSI is established based on differences in the length of markers in the tumor tissue and in the control, but due to the quasimonomorphic nature of standard mononucleotide loci the use of a control sample is not necessary in the diagnosis of MSI-positive solid tumors. The significance of the MSI phenomenon in oncohematology has not been established. This paper presents the results of a study of MSI in B-cell lymphomas: follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBL). We have shown that aberrations of mononucleotide markers occur in these diseases, but the nature of the changes does not correspond to the classical MSI in solid neoplasms. This fact requires further study of the pathogenesis of such genetic disorders. Due to the possibility of ambiguous interpretation of the results of the MSI study for previously uncharacterized diseases, strict compliance with the methodology of parallel analysis of the tumor tissue and the control sample is mandatory.

show abstract

Genotyping SSR length variants by isothermal DNA amplification

Cited by 2 publications

References 19 publications

Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

Pitfalls in mononucleotide microsatellite repeats instability assessing (MSI) in the patients with B-cell lymphomas

Contact Info

Product

Resources

About