Maximizing sensitivity and specificity of PCR by preamplification heating

We have found that assembling the reaction mixture at a temperature greater than the annealing temperature improved both product yield and specificity of PCR. When reactions were maintained at 70 degree C in a dry heating block during addition of denatured samples to aliquotted reagent master mix, a...

Full description

Saved in:
Bibliographic Details
Published inNucleic acids research Vol. 19; no. 13; p. 3749
Main Authors D'AQUILA, R. T, BECHTEL, L. J, VIDELER, J. A, ERON, J. J, GORCZYCA, P, KAPLAN, J. C
Format Journal Article
LanguageEnglish
Published Oxford Oxford University Press 01.01.1991
Subjects
Biological and medical sciences
Biotechnology
DNA
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
In vitro gene amplification. Pcr technique
Methods. Procedures. Technologies
Polymerase chain reaction
Sensitivity
Specificity
Temperature
Process improvement
Method
Optimization
Online AccessGet full text

Cover

More Information
Summary:We have found that assembling the reaction mixture at a temperature greater than the annealing temperature improved both product yield and specificity of PCR. When reactions were maintained at 70 degree C in a dry heating block during addition of denatured samples to aliquotted reagent master mix, a reproducible increase in product yield was observed compared to duplicates maintained at room temperature. Greater specificity was also seen with heating in a gel electrophoretic analysis. In addition, improved sensitivity and specificity was seen with pre-amplification heating using templates of double stranded plasmid DNA that had not been previously denatured, both with and without the addition of tetramethylammonium chloride to the reaction master mix.
Bibliography:ObjectType-Article-2
SourceType-Scholarly Journals-1
ObjectType-Feature-1
content type line 23
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/19.13.3749