High-resolution melting (HRM) analysis is an emerging technology to screen microsatellites for polymorphism. A potential issue surrounding this method is that amplicon sizes for HRM should typically be short (80–100 bp) for highest sensitivity to reveal polymorphism via the presence of two peaks in the curve of the derivative of fluorescence over temperature (dF/dT). In contrast, microsatellite amplicons are typically 100–400 bp. Therefore, we compared HRM analysis melting temperature range (ΔTm) and multiple dF/dT peaks for predicting microsatellite polymorphism. We assessed polymorphism at 27 microsatellite loci, with estimated lengths of 122–321 bp, in Providence Petrel (Pterodroma solandri). We validated HRM assessment using traditional capillary electrophoresis (CE). While 100 % of loci exhibiting multiple peaks in the dF/dT curve were confirmed as polymorphic by CE, 16 % improvement in sensitivity (83 vs. 67 %) was achieved by using ΔTm, and 25 % (92 vs. 67 %) by using ΔTm in addition to multiple dF/dT peaks. We suggest HRM melting temperature range as new predictor of polymorphism that can be used to rapidly assess microsatellites polymorphism.
Funding
Sea World Research and Rescue Foundation Inc
History
Publication title
Genes and Genomics
Volume
37
Issue
12
Pagination
977-983
ISSN
1976-9571
Department/School
School of Natural Sciences
Publisher
Genetics Society of Korea
Place of publication
Korea, Republic of
Rights statement
Copyright 2015 The Genetics Society of Korea and Springer-Science and Media