PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.
A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.
Barnes
W M
WM
Department of Biochemistry and Molecular Biophysics 8231, Washington University School of Medicine, St. Louis, MO 63110.
D010582:Bacteriophage lambda / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry; D005092:Exonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D010316:Particle Size; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D005092:Exonucleases