Kilo-base Range Isothermal DNA Amplification

Kong, Huimin

Abstract

The ability to amplify and subsequent detection a target sequence with a limited length may satisfy the need for diagnosing some of the diseases. Nevertheless, much biomedical research, such as cloning genes and analysis of genetics variation, often demands selective amplification of large DNA fragments in the kilo basepairs range. Currently Polymerase Chain Reaction (PCR) is the only method used by researchers to amplify large DNA fragments. Although PCR is able to amplify a target up to 10-20 kb, a relatively high error rate due to the lack of proof-reading activity of Taq polymerase and the requirement of thermo-cycling may limit the use of PCR. The research proposed provides an alternative method to exponentially amplify medium range and long range DNA fragment at a constant temperature. A new isothermal DNA amplification method has been recently developed. This amplification technology, Helicase-Dependent Amplification (HDA), is based on the unwinding activity of a DNA helicase to separate two DNA strands generating single-stranded templates for primer hybridization and subsequent extension by a DNA polymerase. This amplification method is more closer to nature's choice for the replication of genomic DNA. Thus, it may be a better way to amplify DNA. However, the current UvrD-based HDA system can only amplify target sequences less than one kb, probably due to low processivity of the UvrD helicase. The proposed research aims to increase the length of target that can be amplified by testing highly processive helicases in HDA reactions. The proposed product ultimately to be developed will be a helicase-based nucleic acid amplification kit capable of doing kilo-base range amplification at one temperature with high fidelity. Thus, amplification of a long DNA fragment could merely involve mixing a DNA sample with the HDA reagent mix and incubation in a water bath for one hour. ? ?