Untitled

Abstract

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 degrees 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.

Nicking endonucleases

“…We have used the ability of nicking endonucleases to cut only one DNA strand in elaboration of a new method of DNA target detection in a reaction proceeding at con stant temperature (55°C) [41,42]. A molecular beacon and a nickase (Nt.BspD6I isolated by us was used in this work) are involved in the reaction.…”

Crystallization and preliminary crystallographic analysis of the site-specific DNA nickase Nb.BspD6I

“…The plasmid pRARE/sscL1IM contains the methylase M.SscL1I gene inserted into plasmid pRARE. The M.SscL1I has been shown to protect host DNA against hydrolysis by the nickase (Zheleznaya et al, 2002). The pRARE (Novagen) contains genes of tRNA that rarely occur in E. coli.…”

“…Four naturally occurring enzymes have so far been identi®ed in bacterial sources (Abdurashitov et al, 1996;Morgan et al, 2000;Zheleznaya et al, 2002;Dedkov et al, 2001). One of them, the nickase Nb.BspD6I (N.BspD6I in old nomenclature), was found and characterized by Zheleznaya et al (2002). All the enzymes recognize the DNA sequence 5 H -GAGTC-3 H /5 H -GACTC-3 H and cleave only the top strand 4 bp downstream of the recognition site.…”

“…Diagnostics assays based on the nickases have already been designed for the detection of DNA targets using molecular beacons (Zheleznaya et al, 2002) and for isothermal reactions for the ampli®cation of oligonucleotides (Van Ness et al, 2003). New methods and technologies may be designed to exploit the nickases and some DNA nanotechnology and DNA computing problems may also be solved by the application of nickases (Zhang et al, 2002;Wang et al, 2001).…”

“…Thus, the nickases are both site-speci®c and strand-speci®c endonucleases. Four naturally occurring enzymes have so far been iden-ti®ed in bacterial sources (Abdurashitov et al, 1996;Morgan et al, 2000;Zheleznaya et al, 2002;Dedkov et al, 2001). One of them, the nickase Nb.BspD6I (N.BspD6I in old nomenclature), was found and characterized by Zheleznaya et al (2002).…”