Mechanism of in Vitro Expansion of Long DNA Regeats: Effect of Temperature, Repeat Kength, Repeat Sequence, and DNA polymerases

摘要

Studies of sequence repeat expansions fro duplexes consisting of DNA repeat sequences greater than three bases are currently lacking. These studies are needed in order to gain a better understanding of DNA expansions in general and as a first step in understanding expansions of longer sequence repeats that have been implicated in human diseases. We have undertaken an in vitro study of tetranucleotide, hexanucleotide, and octanucleitide repeat expansions from short DNA duplexes using Taq DNA polymerase. Expansions of hexanucleotide repeats were also studied with the klenow fragment of DNA polymerase I and with T4 DNA polymerase. Studies with Taq DNA polymerase show that expanions occur more readily as the lengthh of the repeat sequence decreases but are generally more efficiennt at reaction temperatures with Taq DNA polymerase is proposed that does not invoke strand slippage or DNA structure. Studies at 37 deg C with Lkenow pol I and T4 DNA polymerase indicate that the template-switching and/or strand-displacement activities of the polymerases used can play a major role in the apparent in vitro expansions of short repetitive DNA duplexes.