Please use this identifier to cite or link to this item:
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
capillary electrophoresis
controlled study
correlational study
health care cost
high resolution melting analysis
human cell
Huntington chorea
measurement accuracy
oral biopsy
real time polymerase chain reaction
screening test
segregation analysis
sensitivity and specificity
Huntington chorea
polymerase chain reaction
trinucleotide repeat
Electrophoresis, Capillary
Huntington Disease
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.
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
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0180984
Appears in Collections:Staff Publications

Show full item record
Files in This Item:
File Description SizeFormatAccess SettingsVersion 
10_1371_journal_pone_0180984.pdf13.17 MBAdobe PDF




checked on Apr 18, 2021

Page view(s)

checked on Apr 16, 2021


checked on Apr 16, 2021

Google ScholarTM



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.