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.
Highlights
The mechanisms of repeat instability remain tedious to assay in part because of the low yields during PCR amplification of expanded TNRs5
We adapted protocols and show the applicability of this method in four molecular biology techniques commonly used when working with expanded CAG repeats: PCR amplification, sequencing, molecular cloning, and small-pool PCR (SP-PCR)
We first wanted to determine whether expanded CAG repeats of varying lengths could be amplified using dUTP instead of dTTP in the PCR and that Uracil N-Glycosylase (Ung) did not interfere with the amplification
Summary
The mechanisms of repeat instability remain tedious to assay in part because of the low yields during PCR amplification of expanded TNRs5. These precautions reduce, but do not eliminate, the risk of contamination This is important in the context of small-pool PCR (SP-PCR), the gold standard assay to measure repeat instability[10,11]. The products are separated on agarose gels and transferred onto a membrane for Southern blotting This assay is exquisitely sensitive and many no-DNA controls must be included to ensure that there is no carry-over contamination. A common solution is to pre-treat the samples with a bacterial uracil N-glycosylase, Ung, that degrades any contaminating DNA containing uracil and to replace dTTP with dUTP in all PCRs15. It was unclear whether this method is suitable for molecular applications www.nature.com/scientificreports/. We adapted protocols and show the applicability of this method in four molecular biology techniques commonly used when working with expanded CAG repeats: PCR amplification, sequencing, molecular cloning, and SP-PCR
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