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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.
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
April 1996,
DNA research : an international journal for rapid publication of reports on genes and genomes,
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
January 2002,
Methods in molecular biology (Clifton, N.J.),
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
January 1997,
Methods in molecular biology (Clifton, N.J.),
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
January 2011,
Methods in molecular biology (Clifton, N.J.),
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
September 1993,
BioTechniques,
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
August 2020,
Data in brief,
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
February 1996,
Methods (San Diego, Calif.),
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
June 2005,
Applied and environmental microbiology,
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
November 1988,
Gene,
S Cheng, and
C Fockler, and
W M Barnes, and
R Higuchi
October 2004,
Clinical chemistry,
