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

• Published in: Nucleic acids research Vol. 20; no. 5; p. 1155
• Main Authors: Kureishi, Amar, Bryan, Larry E.
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11.03.1992
• 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; Pseudomonadales; Amplification; Polymerase chain reaction; Heat treatment; DNA; Primer; Bacteria; Pseudomonadaceae; Pseudomonas aeruginosa; Method
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/20.5.1155

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.