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 DMA 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 closer to nature's choice for the replication of genomic DNA. Thus, it may be a better way to amplify DNA. During the Phase I research, we demonstrated that, by using highly processive helicase RecBCD, we could amplify 1 kb DNA fragment and that it is feasible to increase the size of amplicon by coordinate activities of helicase and DNA polymerase. Therefore, we propose to further optimize the RecBCD-based HDA system and to improve coordination of HDA reaction by modulating the activity of helicase and DNA polymerase. The proposed product ultimately to be developed will be a RecBCD-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.
| Cao, Yun; Kim, Hyun-Jin; Li, Ying et al. (2013) Helicase-dependent amplification of nucleic acids. Curr Protoc Mol Biol 104:Unit 15.11 |
| An, Lixin; Tang, Wen; Ranalli, Tamara A et al. (2005) Characterization of a thermostable UvrD helicase and its participation in helicase-dependent amplification. J Biol Chem 280:28952-8 |
