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EXCESSIVE THERMAL CYCLING CONVERTS AMPLIFIED PCR PRODUCTS TO RANDOM-LENGTH HIGHER MOLECULAR WEIGHT FRAGMENTS
Citation:
Bell, D. AND D.M. DeMarini. EXCESSIVE THERMAL CYCLING CONVERTS AMPLIFIED PCR PRODUCTS TO RANDOM-LENGTH HIGHER MOLECULAR WEIGHT FRAGMENTS. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-91/301 (NTIS PB92124577).
Description:
Poorly optimized thermal cycling conditions are known to produce artifacts during the polymerase chain reaction (PCR). e report here on a problem encountered while developing a PCR protocol that may occur frequently in other laboratories, but may 20 unrecognized by many investigators. CR reactions were cycled for 20, 26, 32, 38, or 44 cycles, the products of the reactions were electrophoresed, and the resulting gel was probed by means of performing a Southern blot. he results suggested that after approximately 30 cycles, most of the PCR primers are converted into PCR product, and that 3'-OH ends of the PCR product start to anneal to the genomic template DNA. hese are then extended during continued cycling, resulting in large molecular weight products that are not the desired product for DNA sequencing. e recommend that the total number of cycles in PCR be minimized to avoid the buildup of such nonspecific products. he results presented here support this recommendation and also demonstrate that excessive cycling can lead to a dramatic loss of specific PCR product.