LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection.

Abstract

Microsatellites are short tandem repeats of one to six nucleotides that are highly polymorphic and extensively used as genetic markers in numerous biomedical applications, including the detection of microsatellite instability (MSI) in cancer. The standard analytical method for microsatellite analysis relies on PCR amplification followed by capillary electrophoresis or, more recently, next-generation sequencing (NGS). However, their amplification during PCR generates undesirable frameshift products known as stutter peaks caused by polymerase slippage, complicating data analysis and interpretation, while very few alternative methods for microsatellite amplification have been developed to reduce the formation of these artifacts. In this context, the recently developed low-temperature recombinase polymerase amplification (LT-RPA) is an isothermal DNA amplification method at low temperature (32 °C) that drastically reduces and sometimes completely abolishes the formation of stutter peaks. LT-RPA greatly simplifies the genotyping of microsatellites and improves the detection of MSI in cancer. In this chapter, we describe in detail all the experimental steps necessary for the development of LT-RPA simplex and multiplex assays for microsatellite genotyping and MSI detection, including the design, optimization, and validation of the assays combined with capillary electrophoresis or NGS.

• Authors: Sophie I Jeanjean, Victor Renault, Antoine Daunay, Yimin Shen, Lise M Hardy, Jean-François Deleuze, Alexandre How-Kit
• Journal: Methods in molecular biology (Clifton, N.J.) (Methods Mol Biol) Vol. 2621 Pg. 91-109 ( 2023) ISSN: 1940-6029 [Electronic] United States
• PMID: 37041442 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Chemical References

• Recombinases
• DNA
• Nucleotidyltransferases

Topics

• Humans
• Microsatellite Instability
• Recombinases (genetics)
• Genotype
• Microsatellite Repeats
• DNA (genetics)
• Nucleotidyltransferases
• Neoplasms (genetics)