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J. Biol. Chem., Vol. 280, Issue 32, 28952-28958, August 12, 2005

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Characterization of a Thermostable UvrD Helicase and Its Participation in Helicase-dependent Amplification^*

Lixin An, Wen Tang, Tamara A. Ranalli, Hyun-Jin Kim, Jamie Wytiaz, and Huimin Kong¶

From the New England Biolabs and BioHelix, Beverly, Massachusetts 01915

Helicase-dependent amplification (HDA) is an isothermal in vitro DNA amplification method based upon the coordinated actions of helicases to separate double-stranded DNA and DNA polymerases to synthesize DNA. Previously, a mesophilic form of HDA (mHDA) utilizing the Escherichia coli UvrD helicase, DNA polymerase I Klenow fragment, two accessory proteins, MutL and single-stranded DNA-binding protein (SSB), was developed (1). In an effort to improve the specificity and performance of HDA, we have cloned and purified a thermostable UvrD helicase (Tte-UvrD) and the mutL homolog (Tte-MutL) from Thermoanaerobacter tengcongensis. Characterization of the Tte-UvrD helicase shows that it is stable and active from 45 to 65 °C. We have found that the Tte-UvrD helicase unwinds blunt-ended DNA duplexes as well as substrates possessing 3'- or 5'-ssDNA tails. Tte-UvrDwasusedtodevelopathermophilichelicase-dependent amplification (tHDA) system to selectively amplify target sequences at 60–65 °C. The tHDA system is more efficient than mHDA, displaying heightened amplification sensitivity without the need for the MutL and SSB accessory proteins. Amplification independent of MutL corresponds with studies demonstrating that maximal Tte-UvrD helicase activity does not require the mutL homolog. The tHDA system allows for rapid amplification and detection of targets present in genomic DNA. The expeditious nature and simplistic design of the tHDA platform makes the technology ideal for use in diagnostic applications requiring rapid identification of organisms at the point-of-need.

Received for publication, March 21, 2005 , and in revised form, June 10, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 978-998-7285; Fax: 978-921-1350; E-mail: kong{at}biohelix.com.

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