Use of site-specific nicking endonucleases to create single-stranded regions and applications thereof

Abstract

The present invention relates to the use of site-specific nucleic acid nicking enzymes to create single-stranded regions in duplex nucleic acids. Such single-stranded regions can take the form of gaps interior to the duplex, or terminal single-stranded regions. Single-stranded termini can be crafted to allow linkage of various elements via base-pairing with elements containing a complementary single-stranded region. This joining is useful, for example, in an ordered, oriented assembly of DNA modules to create cloning or expression vectors. This joining is also useful in attaching detection probes and purifying DNA molecules containing the single-stranded region. Gaps are useful in similar applications, including attaching detection or purification probes.

1 - 21 . (cancelled) 
 Add new claims  22 - 29  as-filed in the Parent Application Ser. No. 09/738,444 on Apr. 19, 2002 in Applicants Response A.    
     
     
         22 . A nucleic acid molecule having at least one recognition site and an associated nicking site for a site specific single strand nicking endonuclease, the nucleic acid further comprising a target single strand region in double-stranded DNA.  
     
     
         23 . A nucleic acid molecule according to  claim 22 , having a first and a second single strand nicking site wherein the first nicking site is orientated in tandem with respect to the second nicking site.  
     
     
         24 . A nucleic acid molecule according to  claim 22 , having a first and optionally a second single strand nicking site such that the number of nucleotides between the first and the second nicking site or the first nicking site and a terminus of the nucleic acid molecule determines the length of a single-stranded region in the target single stranded region created.  
     
     
         25 . A nucleic acid molecule according to  claim 22  having a first single-stranded target region, the first region forming a stable pairing with a second single stranded target region of DNA having a complementary sequence.  
     
     
         26 . A nucleic acid molecule according to  claim 24 , wherein the single-stranded region has a length in the range of 12 to 18 nucleotides.  
     
     
         28 . A nucleic acid molecule according to claim  27 , wherein the single-stranded region has a sequence selected from any of SEQ ID NO:16 through SEQ ID NO:32.  
     
     
         29 . A nucleic acid molecule according to claim  27 , wherein the single-stranded region has a sequence selected from any of SEQ ID NO:35 through SEQ ID NO:49.