Please use this identifier to cite or link to this item: https://doi.org/10.1371/journal.pone.0180984
Title: Improved high sensitivity screen for huntington disease using a one-step triplet-primed PCR and melting curve assay
Authors: Zhao M. 
Cheah F.S.H.
Chen M. 
Lee C.G. 
Law H.-Y. 
Chong S.S. 
Keywords: allele
Article
capillary electrophoresis
controlled study
correlational study
fluorescence
genotype
health care cost
high resolution melting analysis
human
human cell
Huntington chorea
measurement accuracy
oral biopsy
plasmid
real time polymerase chain reaction
screening test
segregation analysis
sensitivity and specificity
genetics
Huntington chorea
polymerase chain reaction
procedures
trinucleotide repeat
Alleles
Electrophoresis, Capillary
Genotype
Huntington Disease
Plasmids
Polymerase Chain Reaction
Trinucleotide Repeat Expansion
Issue Date: 2017
Publisher: Public Library of Science
Citation: Zhao M., Cheah F.S.H., Chen M., Lee C.G., Law H.-Y., Chong S.S. (2017). Improved high sensitivity screen for huntington disease using a one-step triplet-primed PCR and melting curve assay. PLoS ONE 12 (7) : e0180984. ScholarBank@NUS Repository. https://doi.org/10.1371/journal.pone.0180984
Abstract: Molecular diagnosis of Huntington disease (HD) is currently performed by fluorescent repeat-flanking or triplet-primed PCR (TP-PCR) with capillary electrophoresis (CE). However, CE requires multiple post-PCR steps and may result in high cost in high-throughput settings. We previously described a cost-effective single-step molecular screening strategy employing the use of melting curve analysis (MCA). However, because it relies on repeat-flanking PCR, its efficiency in detecting expansion mutations decreases with increasing size of the repeat, which could lead to false-negative results. To address this pitfall, we have developed an improved screening assay coupling TP-PCR, which has been shown in CE-based assays to detect all expanded alleles regardless of size, with MCA in a rapid one-step assay. A companion protocol for rapid size confirmation of expansion-positive samples is also described. The assay was optimized on 30 genotype-known DNAs, and two plasmids pHTT(CAG) 26 and pHTT(CAG) 33 were used to establish the threshold temperatures (TTs) distinguishing normal from expansion-positive samples. In contrast to repeat-flanking PCR MCA, TP-PCR MCA displayed much higher sensitivity for detecting large expansions. All 30 DNAs generated distinct melt peak T m s which correlated well with each sample’s larger allele. Normal samples were clearly distinguished from affected samples. The companion sizing protocol accurately sized even the largest expanded allele of ~180 CAGs. Blinded analysis of 69 clinical samples enriched for HD demonstrated 100% assay sensitivity and specificity in sample segregation. The assay targets the HTT CAG repeat specifically, tolerates a wide range of input DNA, and works well using DNA from saliva and buccal swab in addition to blood. Therefore, rapid, accurate, reliable, and high-throughput detection/exclusion of HD can be achieved using this one-step screening assay, at less than half the cost of fluorescent PCR with CE. © 2017 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Source Title: PLoS ONE
URI: https://scholarbank.nus.edu.sg/handle/10635/165788
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0180984
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