In vitro amplification of DNA fragments greater than 10 kb

The synthesis of a 10.9-kb DNA fragment from a bacteriophage lambda template was used in the search for conditions to extend the range for the polymerase chain reaction (PCR). Using the same primer sequences and conditions (denaturation at 94 degrees C, 1 min; annealing at 57 degrees C, 1 min; polymerization at 70 degrees C, 20 to 30 min) as published by W. Rychlik, W. J. Spencer, and R. E. Rhoads [(1990) Nucleic Acids Res. 18, 6409-6412], unsatisfactory results were obtained with AmpliTaq and native Taq polymerase (poor reproducibility, low product yield, nonspecific products), whereas Tub polymerase completely failed to amplify this fragment. Only after changes in the following parameters were reliable results obtained but only with Tub polymerase: A two-step PCR procedure with primer annealing and extension at 65 degrees C followed by DNA denaturation at 94 degrees C for 1.5 min was performed. The DNA fragment desired was specifically amplified when the enzyme concentration was reduced to 0.4 U/50 microliters and extension times as low as 4 to 12 min with an optimum at 8 min were used. A prolongation to 20 min or more resulted in an accumulation of unspecific products with a concomitant reduction in the yield of the fragment. Under the conditions described above it was also possible to amplify a DNA fragment even significantly longer (15.6 kb).