Open AccessEffective amplification of long targets from cloned inserts and human genomic DNA.
Author(s) -
Suzanne Cheng,
Carita Fockler,
Wayne M. Barnes,
Russell Higuchi
Publication year - 1994
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.91.12.5695
Subject(s) - genomic dna , taq polymerase , primer dimer , microbiology and biotechnology , biology , thermus aquaticus , multiple displacement amplification , hot start pcr , inverse polymerase chain reaction , dna nanoball sequencing , polymerase chain reaction , primer (cosmetics) , recombinant dna , dna , dna polymerase , genomic library , multiplex polymerase chain reaction , genetics , polymerase , gene , chemistry , dna extraction , base sequence , organic chemistry
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.