Effective amplification of long targets from cloned inserts and human genomic DNA.
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.
Cheng
S
S
Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA 94501.
Fockler
C
C
Barnes
W M
WM
Higuchi
R
R
D010582:Bacteriophage lambda; D002021:Buffers; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D005914:Globins / Q000235:genetics; D006801:Humans; D008970:Molecular Weight; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins; D012997:Solvents; D013045:Species Specificity; D013379:Substrate Specificity; D013698:Templates, Genetic
D002021:Buffers; D017931:DNA Primers; D011994:Recombinant Proteins; D012997:Solvents; D005914:Globins; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases