Heterozygote PCR product melting curve prediction

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Publication Type pre-print
School or College School of Medicine
Department Pathology
Creator Dwight, Zachary Lawrence
Other Author Palais, Robert; Kent, Jana; Wittwer, Carl T.
Title Heterozygote PCR product melting curve prediction
Date 2014-01-01
Description Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple domains are calculated by recursive nearest-neighbor thermodynamics. However, the melting curve of an amplicon containing a heterozygous single nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heteroduplexes. To better predict the shape of composite heterozygote melting curves, 52 experimental curves were compared to brute force in silico predictions varying two parameters simultaneously: the relative contribution of heteroduplex products and an ionic scaling factor for mismatched tetrads. Heteroduplex products contributed 25.7 +/- 6.7% to the composite melting curve, varying from 23-28% for different SNV classes. The effect of ions on mismatch tetrads scaled to 76-96% of normal (depending on SNV class) and averaged 88 +/-16.4%. Based on uMelt (www.dna.utah.edu/umelt/umelt.html) with an expanded nearest neighbor thermodynamic set that UU IR Author Manuscript UU IR Author Manuscript University of Utah Institutional Repository Author Manuscript HETEROZYGOTE PCR PRODUCT MELTING CURVE PREDICTION 2 includes mismatched base pairs, uMelt HETS calculates helicity as a function of temperature for homoduplex and heteroduplex products, as well as the composite curve expected from heterozygotes. It is an interactive web tool for efficient genotyping design, heterozygote melting curve prediction, and quality control of melting curve experiments. The application was developed in Actionscript and can be found online at http://www.dna.utah.edu/hets/.
Type Text
Publisher Wiley-Blackwell
First Page 1
Last Page 14
Language eng
Bibliographic Citation Dwight, Z. L., Palais, R., Kent, J., & Wittwer, C. T. (2014). Heterozygote PCR product melting curve prediction. Human Mutation, 1-14.
Rights Management (c) Wiley-Blackwell ; This is the pre-peer reviewed version of the following article: Dwight, Z. L., Palais, R., Kent, J., & Wittwer, C. T. (2014). Heterozygote PCR product melting curve prediction. Human Mutation, which has been published in final form at doi: 10.1002/humu.22494 ; The definitive version is available at www3.interscience.wiley.com
Format Medium application/pdf
Format Extent 3,847,712 bytes
Identifier uspace,18396
ARK ark:/87278/s6vx3rj0
Setname ir_uspace
Date Created 2014-03-11
Date Modified 2021-05-06
ID 711704
Reference URL https://collections.lib.utah.edu/ark:/87278/s6vx3rj0