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

DOI Web Site 被引用文献22件
  • S Cheng
    Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA 94501.
  • C Fockler
    Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA 94501.
  • W M Barnes
    Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA 94501.
  • R Higuchi
    Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA 94501.

書誌事項

公開日
1994-06-07
DOI
  • 10.1073/pnas.91.12.5695
公開者
Proceedings of the National Academy of Sciences

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説明

<jats:p>We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant lambda 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.</jats:p>

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