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DNA Concentration Can Specify DNA Melting Point in a High-Resolution Melting Analysis Master Mix

Ng, Jacklyn W.S., Holt, Deborah C., Andersson, Patiyan and Giffard, Philip M. (2014). DNA Concentration Can Specify DNA Melting Point in a High-Resolution Melting Analysis Master Mix. Clinical Chemistry,60(2):414-416.

Document type: Journal Article
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IRMA ID cmartelxPUB169
Title DNA Concentration Can Specify DNA Melting Point in a High-Resolution Melting Analysis Master Mix
Author Ng, Jacklyn W.S.
Holt, Deborah C.
Andersson, Patiyan
Giffard, Philip M.
Journal Name Clinical Chemistry
Publication Date 2014
Volume Number 60
Issue Number 2
ISSN 0009-9147   (check CDU catalogue  open catalogue search in new window)
Scopus ID 2-s2.0-84893457532
Start Page 414
End Page 416
Total Pages 3
Place of Publication United States of America
Publisher American Association for Clinical Chemistry, Inc.
HERDC Category C1 - Journal Article (DIISR)
Abstract Extract
High-resolution melting analysis (HRMA)1 can be used to discriminate sequence variants of PCR-amplified DNA fragments (1). We attempted to develop an HRMA-based procedure to discriminate multilocus sequence typing (MLST) (2) alleles from a divergent lineage of Staphylococcus aureus (3) using a Corbett Rotorgene 6000. In the case of the 573-bp glycerol uptake facilitator (glpF) fragment, there were 4 known alleles in our culture collection. Allele discrimination performed with the nonsaturating dye SYBR Green was not reliable. We therefore tested the saturating dye LCGreenPlus (Biofire Diagnostics) (4). Initial experiments revealed very poor allele discrimination due to lack of correlation between the melting temperature (Tm) and allele identity/allele percentage of guanine plus cytosine (%G+C) content. Here we report an investigation of the basis for this poor performance.

Our initial results with LCGreenPlus suggested that the Tm was primarily a function of the PCR yield rather than the allele sequence. This was tested by subjecting a glpF allele 321 sample to PCR in LCGreenPlus master mix, making a dilution series of the reaction products in the same master mix, and then subjecting the dilutions to HRMA. As predicted, the Tm values in both melting domains were inversely correlated with DNA concentration (Fig. 1 …
DOI http://dx.doi.org/10.1373/clinchem.2013.215582   (check subscription with CDU E-Gateway service for CDU Staff and Students  check subscription with CDU E-Gateway in new window)
Description for Link Link to publisher's version
URL http://www.clinchem.org/content/60/2/414
 
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