Pre-boiling high GC content, mixed primers with 3′ complementation allows the successful PCR amplification of Pseudomonas aeruginosa DNA

Primer design is a major factor in the successful amplification of DNA by the polymerase chain reaction (PCR). Primers that have complementary 3' ends can fail to amplify the desired target because of mispriming, especially when the primer length is short and the GC content high. We encountered...

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Bibliographic Details
Published in:Nucleic acids research 1992-03, Vol.20 (5), p.1155-1155
Main Authors: Kureishi, Amar, Bryan, Larry E.
Format: Article
Language:English
Subjects:
Base Composition - genetics
Base Sequence
Biological and medical sciences
Diverse techniques
DNA, Bacterial - genetics
Fundamental and applied biological sciences. Psychology
Molecular and cellular biology
Molecular Sequence Data
Oligodeoxyribonucleotides - genetics
Polymerase Chain Reaction - methods
Pseudomonas aeruginosa
Pseudomonas aeruginosa - genetics
Temperature
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Summary:Primer design is a major factor in the successful amplification of DNA by the polymerase chain reaction (PCR). Primers that have complementary 3' ends can fail to amplify the desired target because of mispriming, especially when the primer length is short and the GC content high. We encountered this problem in attempting to use PCR to amplify and clone the Pseudomonas aeruginosa gyrA gene of clinical strain Y4492. We designed consensus primers based on areas of homology between the known gyrA genes of other organisms. The 2 areas selected corresponded to E. coli gyrA amino acids 39-46, and 454-460. From these regions, we designed mixed primers.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/20.5.1155