Cardiff University | Prifysgol Caerdydd ORCA
Online Research @ Cardiff 
WelshClear Cookie - decide language by browser settings

Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

Dion, Vincent and Aeschbach, Lorène 2017. Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications. Scientific Reports 7 , 18026. 10.1038/s41598-017-18168-2

[img]
Preview
PDF - Published Version
Available under License Creative Commons Attribution No Derivatives.

Download (2MB) | Preview

Abstract

Expanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR (SP-PCR), such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We performed SP-PCR on myotonic-dystrophy-derived samples containing an expansion as large as 1000 repeats, demonstrating the applicability to clinically-relevant material. Thus, we expect the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders.

Item Type: Article
Date Type: Published Online
Status: Published
Schools: Medicine
ISSN: 2045-2322
Date of First Compliant Deposit: 1 April 2019
Date of Acceptance: 6 December 2017
Last Modified: 01 Apr 2019 15:47
URI: http://orca-mwe.cf.ac.uk/id/eprint/120287

Citation Data

Cited 3 times in Scopus. View in Scopus. Powered By Scopus® Data

Actions (repository staff only)

Edit Item Edit Item

Downloads

Downloads per month over past year

View more statistics