Effective amplification of long targets from cloned inserts and human genomic DNA

Suzanne Cheng, Carita Fockler, Wayne M. Barnes, Russell Higuchi

Research output: Contribution to journalArticle

553 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)5695-5699
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number12
DOIs
StatePublished - Jun 7 1994

Keywords

  • PCR
  • cosolvents
  • genome mapping
  • thermostable 3'-to-5'-exonuclease

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