Methods and compositions for seamless cloning of nucleic acid molecules

Abstract

The present invention is in the fields of biotechnology and molecular biology. More particularly, the present invention relates to cloning or subcloning one or more nucleic acid molecules comprising one or more type IIs restriction enzyme recognition sites. The present invention also embodies cloning such nucleic acid molecules using recombinational cloning methods such as those employing recombination sites and recombination proteins. The present invention also relates to nucleic acid molecules (including RNA and iRNA), as well as proteins, expressed from host cells produced using the methods of the present invention.

1 . A method of producing an RNA molecule for use as an interfering RNA comprising:
 (a) identifying one or more target nucleic acid sequences;   (b) preparing one or more nucleic acid molecules which   encode one or more interfering RNAs, wherein said interfering RNAs bind to said one or more target nucleic acid sequences;   (c) combining   (i) one or more first nucleic acid molecules   encoding one or more interfering RNAs flanked by one or more first type IIs restriction enzyme recognition sites;   (ii) one or more second nucleic acid molecules   comprising one or more selectable markers flanked by one or more second type IIs restriction enzyme recognition sites; and   (iii) one or more site-specific type IIs restriction enzymes; and   (d) incubating said combination under conditions sufficient   to join one or more of said nucleic acid molecules encoding one or more interfering RNAs and one or more of said second nucleic acid molecules, thereby producing one or more desired product nucleic acid molecules;   (e) inserting said one or more product nucleic acid molecules into a host cell; and   (f) expressing said one or more interfering RNAs in said host cell.   
     
     
         2 - 5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein said one or more selectable markers comprises at least one DNA segment encoding an element selected from the group consisting of an antibiotic resistance gene, a gene that encodes a fluorescent protein, an auxotrophic marker, a toxic gene and a phenotypic marker. 
     
     
         7 . The method of  claim 6 , wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a Zeocin resistance gene, a spectinomycin resistance gene and a kanamycin resistance gene. 
     
     
         8 . The method of  claim 6 , wherein said toxic gene is selected from the group consisting of a ccdB gene, a gene encoding a tus protein, a kicB gene, a sacB gene, an ASK1 gene, a ΦX174 E gene and a DpnI gene. 
     
     
         9 . The method of  claim 1 , wherein said first nucleic acid molecule and/or said second nucleic acid molecule further comprises one or more recombination sites. 
     
     
         10 . The method of  claim 9 , wherein said first nucleic acid molecule and/or said second nucleic acid molecule further comprises one or more topoisomerase recognition sites and/or one or more topoisomerases. 
     
     
         11 . The method of  claim 10 , wherein said first nucleic acid molecule and/or said second nucleic acid molecule comprises two or more recombination sites. 
     
     
         12 . The method of  claim 11 , wherein said topoisomerase recognition site, if present, is flanked by said two or more recombination sites. 
     
     
         13 . The method of  claim 12 , wherein said recombination sites are selected from the group consisting of attB sites, attP sites, attL sites, attR sites, lox sites, psi sites, tnpI sites, dif sites, cer sites, frt sites, and mutants, variants and derivatives thereof. 
     
     
         14 . The method of  claim 10 , wherein said topoisomerase recognition site, if present, is recognized and bound by a type I topoisomerase. 
     
     
         15 . The method of  claim 14 , wherein said type I topoisomerase is a type IB topoisomerase. 
     
     
         16 . The method of  claim 15 , wherein said type IB topoisomerase is selected from the group consisting of eukaryotic nuclear type I topoisomerase and a poxvirus topoisomerase. 
     
     
         17 . The method of  claim 1 , wherein said expressed interfering RNA is between 35-60 nucleotides in length. 
     
     
         18 . The method of  claim 17 , wherein said expressed interfering RNA forms a hairpin loop. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . A vector comprising:
 (a) one or more toxic genes;   (b) one or more type IIs restriction enzyme recognition sites; and   (c) one or more site-specific recombination sites.   
     
     
         22 . The vector of  claim 21 , wherein said type IIs restriction enzyme recognition sites are selected from the group consisting of BsaI, BbsI, BbvII, BsmAI, BspMI, Eco3II, BsmBI, BaeI, FokI, HgaI, SlaNI and Sth132I. 
     
     
         23 . The vector of  claim 21 , wherein said recombination sites are selected from the group consisting of attB sites, attP sites, attL sites, attR sites, lox sites, psi sites, tnpI sites, dif sites, cer sites, frt sites, and mutants, variants and derivatives thereof. 
     
     
         24 . The vector of  claim 21 , wherein said vector further comprises one or more topoisomerase recognition sites and/or one or more topoisomerases. 
     
     
         25 . The vector of  claim 24 , wherein said molecule comprises two or more recombination sites. 
     
     
         26 . The vector of  claim 24 , wherein said topoisomerase recognition site, if present, is flanked by said two or more recombination sites. 
     
     
         27 . The vector of  claim 24 , wherein said topoisomerase recognition site, if present, is recognized and bound by a type I topoisomerase. 
     
     
         28 . The vector of  claim 27 , wherein said type I topoisomerase is a type IB topoisomerase. 
     
     
         29 . A method of regulating the expression of one or more genes in a transgenic cell or a transgenic animal using interfering RNA, comprising:
 (a) identifying one or more target nucleic acid sequences in said cell or animal;   (b) preparing one or more nucleic acid molecules which encode one or more interfering RNAs, wherein said interfering RNAs bind to said one or more target nucleic acid sequences;   (c) combining   (i) one or more first nucleic acid molecules encoding one or more interfering RNAs flanked by one or more first type IIs restriction enzyme recognition sites;   (ii) one or more second nucleic acid molecules comprising one or more selectable markers flanked by one or more second type IIs restriction enzyme recognition sites; and   (iii) one or more site-specific type IIs restriction enzymes; and   (d) incubating said combination under conditions sufficient to join one or more of said one or more nucleic acid molecules encoding one or more interfering RNAs and one or more of said second nucleic acid molecules, thereby producing one or more desired product nucleic acid molecules;   (e) inserting said one or more interfering RNA-containing product nucleic acid molecules into said cell or one or more cells of said animal, under conditions such that said one or more interfering RNAs bind to said one or more target nucleic acid sequences, thereby regulating expression of said one or more genes.   
     
     
         30 . The method of  claim 29 , wherein said expressed interfering RNA is between 35-60 nucleotides in length. 
     
     
         31 . The method of  claim 30 , wherein said expressed interfering RNA forms a hairpin loop. 
     
     
         32 - 33 . (canceled) 
     
     
         34 . The method of  claim 29 , wherein said regulation results in decreased expression of said one or more genes.