Some Properties of Site-Specific Nickase BspD6I and the Possibility of Its Use in Hybridization Analysis of DNA

L. A. Zheleznaya¹, T. A. Perevyazova¹, E. N. Zheleznyakova², and N. I. Matvienko^2*

¹Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia; fax: (0967) 790-553

²Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia; fax: (095) 924-0493; E-mail: nikmatv@vega.protres.ru

^* To whom correspondence should be addressed.

Received November 5, 2001; Revision received November 30, 2001

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A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand. The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55°C). The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target. Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule. The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon). The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19. Unfortunately, nickase BspD6I does not cleave DNA in the RNA-DNA duplexes and therefore cannot be used for detection of RNA targets.

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KEY WORDS: site-specific nickases, hybridization of nucleic acids, restriction endonucleases, molecular beacons

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