Clinical Chemistry AACC Online Job Center
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 53: 150-152, 2007; 10.1373/clinchem.2006.081240
This Article
Right arrow Extract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herrmann, M. G.
Right arrow Articles by Voelkerding, K. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herrmann, M. G.
Right arrow Articles by Voelkerding, K. V.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2007;53:150-152.)
© 2007 American Association for Clinical Chemistry, Inc.

Letters to the Editor

Instrument Comparison for Heterozygote Scanning of Single and Double Heterozygotes: A Correction and Extension of Herrmann et al., Clin Chem 2006;52:494-503

Mark G. Herrmann1,a, Jacob D. Durtschi1, L. Kathryn Bromley1, Carl T. Wittwer1,2 and Karl V. Voelkerding1,2

1 Institute for Clinical and, Experimental Pathology, ARUP Laboratories, 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.


To the Editor:

We have discovered that the DNA sample used in our recent paper (1) as a control heterozygote at the sickle cell locus of ß-globin (17A->T, also known as HBB c.20A->T by HUGO nomenclature) contained an additional variant. Subsequent sequencing revealed a double heterozygote, HBB c.[9C->T; 20A->T]. The HBB c. 9C->T is a silent variant for the 3rd amino acid, histidine. In view of this additional sequence variation, we reevaluated the heteroduplex scanning capabilities of the instruments, as reported in the original Fig. 3, to ascertain their ability to distinguish melting curves of heteroduplexes caused by single and double heterozygotes from melting curves of homoduplexes. The c. 9C->T is a common variant with an allele frequency of 38%, as determined in review of clinical samples submitted for ß-globin sequencing (courtesy of Dr. Elaine Lyon, ARUP Laboratories).

The study was repeated as previously described (1), including both single and double heterozygotes. Eight instruments were evaluated for genotyping and heteroduplex scanning resolution (ABI’s 7000, Bio-Rad’s iCycler, Cepheid’s SmartCycler, Corbett’s Rotor-Gene 3000, Idaho Technology’s HR-1 and LightScanner, and Roche’s LightCycler 1.2 and LightCycler 2.0). The resulting normalized melting curves for genotyping and temperature-shifted curves of the 4 genotypes [wild-type, homozygous variant (c.20A->T), single heterozygote (c.20A->T), and double heterozygote (c.[9C->T; 20A->T])] are shown in Fig. 1 . The melting curves of the homozygous genotypes are similar to those reported earlier. Melting curves of heterozygous genotypes segregated according to the number of mismatches present, with the double heterozygote resulting in more low-temperature melting than the single heterozygote. After temperature shifting, the heterozygotes were readily distinguishable by curve shape(2)(3)(4).


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		Figure 1. Melting curves of 110-bp HBB amplicon, including c.20A->T (sickle cell) and c.9C->T (silent variant).

Each genotype was melted and displayed in triplicate on 8 different instruments. Melting curves for the homozygous wild-type are shown in green, the homozygous mutant (c.20A->T) in red, the single heterozygous mutant (c.[20A->T]) in black, and the double heterozygote mutant (c.[9C->T; 20A->T] in blue. (A), normalized melting curves for genotyping; (B), temperature-shifted curves for heterozygote scanning.

The repeat analysis confirms our earlier results and further shows the capability of DNA melting analysis and its dependency on instrument resolution. Although all melting curves from heterozygotes are distinguishable, some resolve from the homozygotes with greater clarity than others after temperature correction (Fig. 1BUp ), with the fundamental shape of each genotype differing from instrument to instrument. As seen by comparing the single heterozygous sample to the wild-type, the high-temperature regions merge on some instruments but are clearly distinct on other instruments. Heterozygous and homozygous samples segregate either by subtle changes in the melting curve slope or by the formation of multiple distinct melting features separated by inflection points, as was also seen in melting curves from the double heterozygote, for which some instruments resolved 2 melting regions and others 3. The continuing development of instruments with finer temperature control and fluorescence acquisition will lead to increased detail derived from heteroduplex and homoduplex contributions to the overall melting curve, providing even greater ability to identify unique sequence variants.

Aspects of melting analysis are covered by issued and pending patents owned by the University of Utah and licensed to Idaho Technology. C.T.W. holds equity interest in Idaho Technology.


References

  1. Herrmann MG, Durtschi JD, Bromley LK, Wittwer CT, Voelkerding KV. Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem 2006;52:494-503.[Abstract/Free Full Text]
  2. Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 2004;50:1748-1754.[Abstract/Free Full Text]
  3. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, et al. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 2004;50:1156-1164.[Abstract/Free Full Text]
  4. Margraf RL, Mao R, Highsmith WE, Holtegaard LM, Wittwer CT. Mutation scanning of the RET protooncogene using high-resolution melting analysis. Clin Chem 2006;52:138-141.[Abstract/Free Full Text]



The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
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]

Home page
 J. Mol. Diagn.Home page
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]

Home page
 Nucleic Acids ResHome page
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]

Home page
 Clin. Chem.Home page
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 page
 Nucleic Acids ResHome page
A.-L. Chateigner-Boutin and I. Small
A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons
Nucleic Acids Res., September 27, 2007; 35(17): e114 - e114.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Extract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Herrmann, M. G.
Right arrow Articles by Voelkerding, K. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Herrmann, M. G.
Right arrow Articles by Voelkerding, K. V.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Automation and Analytical Techniques

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS