[Shadow bands in PCR amplification of trinucleotide repeats and their elimination]

To explore a method for eliminating the shadow bands in PCR amplification of trinucleotide repeats.

CTG trinucleotide repeat sequence in the 3'-untranslated region of the human myotonin protein kinase gene was used as templates DNA in PCR amplification, and the effects of Taq DNA polymerase alone and its mixture with Pwo DNA polymerase on occurrence of the shadow bands' were compared. The PCR products containing(CTG)5, both from Taq DNA polymerase and from Taq+Pwo polymerase amplification, were cloned into pBluecript KS and sequenced on ALF express TM DNA sequencer respectively.

The PCR of DNA sequence containing CTG repeats frequently produced a main band (usually darker) and a shadow band (lighter) that differed from the main band by 3 base pairs when Taq DNA polymerase was used alone. However, when the mixture of Taq DNA polymerase and Pwo DNA polymerase was used, the shadow bands usually disappeared. In the 4 clones containing PCR products amplificated by Taq DNA polymerase, one clone contained only 4 CTG repeats. However, this kind of decrease of repeat copy was not observed in the 5 clones containing PCR products amplified by using mixture of Taq+Pwo DNA polymerase.

The results of this experiment demonstrate that shadow bands occur during the PCR. Pwo DNA polymerase in known to possess not only DNA polymerase activity, but also 3'-5' exonuclease activity, or proofreading ability. The mixture of Taq DNA polymerase and Pwo DNA polymerase has higher processivity than Taq DNA polymerase itself. This explain the effect on eliminating or reducing the occurrence of shadow bands in polymerase chain reaction.