| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Technical Briefs |
1 Institute for Clinical and Experimental Pathology, ARUP, Salt Lake City, UT; 2 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT;
aaddress correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108; fax 801-584-5114, e-mail mark.herrmann{at}aruplab.com
Abstract
Background: Additional instruments have become available since instruments for DNA melting analysis of PCR products for genotyping and mutation scanning were compared. We assessed the performance of these new instruments for genotyping and scanning for mutations.
Methods: A 110-bp fragment of the β-globin gene including the sickle cell anemia locus (HBB c. 20A>T) was amplified by PCR in the presence of LCGreen Plus or SYBR Green I. Amplicons of 4 different genotypes [wild-type, homozygous, and heterozygous HBB c. 20A>T and double-heterozygote HBB c. (9C>T; 20A>T)] were melted on 7 different instruments [Applied Biosystems 7300, Corbett Life Sciences Rotor-Gene 6500HRM, Eppendorf Mastercycler RealPlex4S, Idaho Technology LightScanner (384 well), Roche LightCycler 480 (96 and 384 well) and Stratagene Mx3005p] at a rate of 0.61 °C/s or when this was not possible, at 0.50 °C steps. We evaluated the ability of each instrument to genotype by melting temperature (Tm) and to scan for heterozygotes by curve shape.
Results: The ability of most instruments to accurately genotype single-base changes by amplicon melting was limited by spatial temperature variation across the plate (SD of Tm = 0.020 to 0.264 °C). Other variables such as data density, signal-to-noise ratio, and melting rate also affected heterozygote scanning.
Conclusions: Different instruments vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole amplicon melting analysis. Instruments specifically designed for high-resolution melting, however, displayed the least variation, suggesting better genotyping accuracy and scanning sensitivity and specificity.
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  L. S. Kristensen and L. L. Hansen PCR-Based Methods for Detecting Single-Locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment Clin. Chem., August 1, 2009; 55(8): 1471 - 1483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhou, R. J. Errigo, H. Lu, M. A. Poritz, M. T. Seipp, and C. T. Wittwer Snapback Primer Genotyping with Saturating DNA Dye and Melting Analysis Clin. Chem., October 1, 2008; 54(10): 1648 - 1656. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-P. Audrezet, A. Dabricot, C. Le Marechal, and C. Ferec Validation of High-Resolution DNA Melting Analysis for Mutation Scanning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene J. Mol. Diagn., September 1, 2008; 10(5): 424 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. N. Gundry, S. F. Dobrowolski, Y. R. Martin, T. C. Robbins, L. M. Nay, N. Boyd, T. Coyne, M. D. Wall, C. T. Wittwer, and D. H.-F. Teng Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons Nucleic Acids Res., June 1, 2008; 36(10): 3401 - 3408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. De Leeneer, I. Coene, B. Poppe, A. De Paepe, and K. Claes Rapid and Sensitive Detection of BRCA1/2 Mutations in a Diagnostic Setting: Comparison of Two High-Resolution Melting Platforms Clin. Chem., June 1, 2008; 54(6): 982 - 989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Kristensen and A. Dobrovic Direct Genotyping of Single Nucleotide Polymorphisms in Methyl Metabolism Genes Using Probe-Free High-Resolution Melting Analysis Cancer Epidemiol. Biomarkers Prev., May 1, 2008; 17(5): 1240 - 1247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Margraf, R. Mao, and C. T. Wittwer Rapid Diagnosis of MEN2B Using Unlabeled Probe Melting Analysis and the LightCycler 480 Instrument J. Mol. Diagn., March 1, 2008; 10(2): 123 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Seipp, D. Pattison, J. D. Durtschi, M. Jama, K. V. Voelkerding, and C. T. Wittwer Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting Clin. Chem., January 1, 2008; 54(1): 108 - 115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Montgomery, C. T. Wittwer, J. O. Kent, and L. Zhou Scanning the Cystic Fibrosis Transmembrane Conductance Regulator Gene Using High-Resolution DNA Melting Analysis Clin. Chem., November 1, 2007; 53(11): 1891 - 1898. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
