TY - JOUR
T1 - Impact of mutation type and amplicon characteristics on genetic diversity measures generated using a high-resolution melting diversity assay
AU - Cousins, Matthew M.
AU - Donnell, Deborah
AU - Eshleman, Susan H.
N1 - Funding Information:
Supported by the HIV Prevention Trials Network, sponsored by the National Institute of Allergy and Infectious Diseases ; the National Institute on Drug Abuse ; the National Institute of Mental Health ; and grants from the Office of AIDS Research of the NIH and US Department of Health and Human Services ( U01AI068613 and UM1AI068613 to S.H.E.) and National Institute of Allergy and Infectious Diseases ( 1R01-AI095068 to S.H.E.).
PY - 2013
Y1 - 2013
N2 - We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change <3-bp insertion<9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology.
AB - We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change <3-bp insertion<9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology.
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U2 - 10.1016/j.jmoldx.2012.08.008
DO - 10.1016/j.jmoldx.2012.08.008
M3 - Article
C2 - 23178437
AN - SCOPUS:84875440825
SN - 1525-1578
VL - 15
SP - 130
EP - 137
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 1
ER -