Effective Amplification of Long Targets from Cloned Inserts and Human Genomic DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-06, Vol.91 (12), p.5695-5699
• Main Authors: Cheng, Suzanne, Fockler, Carita, Barnes, Wayne M., Higuchi, Russell
• Format: Article
• Language: English
• Subjects: Annealing; Bacteriophage lambda; Bacteriophages; beta chain; Buffers; Deoxyribonucleic acid; DNA; DNA Primers - chemistry; DNA-Directed DNA Polymerase - metabolism; Exonucleases - metabolism; Gels; gene amplification; Genetics; genomes; Genomics; Globins - genetics; hemoglobin; Human genetics; Humans; man; mapping; Molecular Weight; Multigene family; Nucleotides; Osmolar Concentration; phage lambda; Polymerase chain reaction; Polymerase Chain Reaction - methods; Product category rules; Recombinant Proteins; Solvents; Species Specificity; Substrate Specificity; Templates, Genetic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.12.5695

We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the β-globin gene cluster from human genomic DNA and up to 42 kb from phage λ DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant λ plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to-5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.