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


     

Clinical Chemistry 47: 1956-1961, 2001;
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental Data
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 (28)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by von Ahsen, N.
Right arrow Articles by Schütz, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by von Ahsen, N.
Right arrow Articles by Schütz, E.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2001;47:1956-1961.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg2+, Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas

Nicolas von Ahsen1a, Carl T. Wittwer2 and Ekkehard Schütz1,3

1 Department of Clinical Chemistry, Georg-August University, Robert Koch Strasse 40, 37075 Goettingen, Germany.

2 Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132.

3 Chronic Illness Research Foundation, San Francisco, CA 94107.

aAuthor for correspondence. Fax 49-551-39-12504; e-mail nahsen{at}gwdg.de

Background: Many techniques in molecular biology depend on the oligonucleotide melting temperature (Tm), and several formulas have been developed to estimate Tm. Nearest-neighbor (N-N) models provide the highest accuracy for Tm prediction, but it is not clear how to adjust these models for the effects of reagents commonly used in PCR, such as Mg2+, deoxynucleotide triphosphates (dNTPs), and dimethyl sulfoxide (DMSO).

Methods: The experimental Tms of 475 matched or mismatched target/probe duplexes were obtained in our laboratories or were compiled from the literature based on studies using the same real-time PCR platform. This data set was used to evaluate the contributions of [Mg2+], [dNTPs], and [DMSO] in N-N calculations. In addition, best-fit coefficients for common empirical formulas based on GC content, length, and the equivalent sodium ion concentration of cations [Na+eq] were obtained by multiple regression.

Results: When we used [Na+eq] = [Monovalent cations] + 120() (the concentrations in this formula are mmol/L) to correct {Delta}S0 and a DMSO term of 0.75 °C (%DMSO), the SE of the N-N Tm estimate was 1.76 °C for perfectly matched duplexes (n = 217). Alternatively, the empirical formula Tm (°C) = 77.1 °C + 11.7 x log[Na+eq] + 0.41(%GC) - 528/bp - 0.75 °C(%DMSO) gave a slightly higher SE of 1.87 °C. When all duplexes (matched and mismatched; n = 475) were included in N-N calculations, the SE was 2.06 °C.

Conclusions: This robust model, accounting for the effects of Mg2+, DMSO, and dNTPs on oligonucleotide Tm in PCR, gives reliable Tm predictions using thermodynamic N-N calculations or empirical formulas.




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


Home page
 Clin. Chem.Home page
G. Pont-Kingdon, R. L. Margraf, K. Sumner, A. Millson, E. Lyon, and E. Schutz
Design and Application of Noncontinuously Binding Probes Used for Haplotyping and Genotyping
Clin. Chem., June 1, 2008; 54(6): 990 - 999.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Y.-F. Chen, R.-C. Chen, L.-Y. Tseng, E. Lin, Y.-K. Chan, and R.-H. Pan
NTMG (N-terminal Truncated Mutants Generator for cDNA): an automatic multiplex PCR assays design for generating various N-terminal truncated cDNA mutants
Nucleic Acids Res., July 13, 2007; 35(suppl_2): W66 - W70.
[Abstract] [Full Text] [PDF]

Home page
 BioinformaticsHome page
T. Koressaar and M. Remm
Enhancements and modifications of primer design program Primer3
Bioinformatics, May 15, 2007; 23(10): 1289 - 1291.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
Z.-J. Tan and S.-J. Chen
RNA Helix Stability in Mixed Na+/Mg2+ Solution
Biophys. J., May 15, 2007; 92(10): 3615 - 3632.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. D. Levin, D. Fiala, M. F. Samala, J. D. Kahn, and R. J. Peterson
Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers
Nucleic Acids Res., November 6, 2006; 34(20): e142 - e142.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
B. S. Robinson, P. T. Monis, and P. J. Dobson
Rapid, Sensitive, and Discriminating Identification of Naegleria spp. by Real-Time PCR and Melting-Curve Analysis
Appl. Envir. Microbiol., September 1, 2006; 72(9): 5857 - 5863.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
E. Schutz, M. Scharfenstein, and B. Brenig
Genotyping of Ovine Prion Protein Gene (PRNP) Variants by PCR with Melting Curve Analysis,
Clin. Chem., July 1, 2006; 52(7): 1426 - 1429.
[Abstract] [Full Text] [PDF]

Home page
 BioinformaticsHome page
S. Chavali, A. Mahajan, R. Tabassum, S. Maiti, and D. Bharadwaj
Oligonucleotide properties determination and primer designing: a critical examination of predictions
Bioinformatics, October 15, 2005; 21(20): 3918 - 3925.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
L. Zhou, A. N. Myers, J. G. Vandersteen, L. Wang, and C. T. Wittwer
Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye
Clin. Chem., August 1, 2004; 50(8): 1328 - 1335.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
M. Liew, R. Pryor, R. Palais, C. Meadows, M. Erali, E. Lyon, and C. Wittwer
Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons
Clin. Chem., July 1, 2004; 50(7): 1156 - 1164.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
X. Wang and B. Seed
A PCR primer bank for quantitative gene expression analysis
Nucleic Acids Res., December 15, 2003; 31(24): e154 - e154.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
B. Muller, K. Zopf, J. Bachofer, and W. Steimer
Optimized Strategy for Rapid Cytochrome P450 2D6 Genotyping by Real-Time Long PCR
Clin. Chem., October 1, 2003; 49(10): 1624 - 1631.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. Maheshri and D. V. Schaffer
Computational and experimental analysis of DNA shuffling
PNAS, March 18, 2003; 100(6): 3071 - 3076.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
C. N. Gundry, J. G. Vandersteen, G. H. Reed, R. J. Pryor, J. Chen, and C. T. Wittwer
Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes
Clin. Chem., March 1, 2003; 49(3): 396 - 406.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2001 by the American Association for Clinical Chemistry.