Systematic studies on parameters influencing the performance of the polymerase chain reaction

To obtain a detailed understanding of the various factors influencing the polymerase chain reaction, we optimized parameters such as ion concentration, pH, primer sequence and concentration, hybridization stringency, cycle numbers, etc. Using 3 different plasmids (2 HIV2 clones and pUC18) and severa...

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Bibliographic Details
Published inJournal of clinical chemistry and clinical biochemistry Vol. 28; no. 1; p. 5
Main Authors Linz, U, Delling, U, Rübsamen-Waigmann, H
Format Journal Article
LanguageEnglish
Published Germany 01.01.1990
Subjects
Base Sequence
DNA Probes
DNA, Viral - genetics
DNA, Viral - metabolism
DNA-Directed DNA Polymerase - genetics
Gene Amplification
HIV - genetics
Humans
Hydrogen-Ion Concentration
Magnesium - metabolism
Molecular Sequence Data
Nucleic Acid Hybridization
Peptide Chain Elongation, Translational
Plasmids
Polymerase Chain Reaction
Templates, Genetic
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Summary:To obtain a detailed understanding of the various factors influencing the polymerase chain reaction, we optimized parameters such as ion concentration, pH, primer sequence and concentration, hybridization stringency, cycle numbers, etc. Using 3 different plasmids (2 HIV2 clones and pUC18) and several genomic DNA samples from HIV-positive patients as templates, together with 6 sets of primers, the following optimal conditions were found: the DNA should be linearized, the primer concentration should be 0.1-0.2 mumol/l, and the magnesium ion concentration should be less than 2 mmol/l. The pH of the reaction mixture should be 8.5-9.0. Twenty five cycles are sufficient. For fragments greater than 10(3) bases the elongation time should be 5 min. The elongation temperature is not critical and can vary between 50 and 70 degrees C. The hybridization temperature can be used to control the specificity of the polymerase chain reaction and, finally, mismatches at the 3' end of the primer can totally inhibit the amplification.
ISSN:0340-076X