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 - 34 . (canceled) 
     
     
         35 . A one tube method for the directional introduction of a nucleic acid segment into a vector, the method comprising:
 (a) preparing a reaction mixture comprising:
 (i) a starting nucleic acid molecule having two Type IIs restriction sites, 
 (ii) a linear vector comprising a first terminus with a 3′ overhang and a second terminus with a 5′ overhang wherein the 3′ and 5′ overhangs differ in nucleotide sequence, 
 (iii) a Type IIs restriction endonuclease, and 
 (iv) a ligase; and 
   (b) incubating the reaction mixture prepared in (a) under conditions suitable for:
 (i) cleavage of the termini of the starting nucleic acid molecule by the Type IIs restriction endonuclease to generate the nucleic acid segment comprising a first terminus with 5′ overhang and a second terminus with a 3′ overhang wherein the 5′ and 3′ overhangs differ in nucleotide sequence, 
 (ii) hybridization of complementary termini of the nucleic acid segment and the linear vector, and 
 (iii) ligation of the resulting hybridized complementary termini formed in (ii), 
   wherein the nucleic acid segment generated in step (b) (i) does not contain recognition sites for the Type IIs restriction endonuclease,   wherein the 3′ overhang of the first terminus of the linear vector is complementary to the 5′ overhang of the first terminus of the nucleic acid segment, and   wherein the 5′ overhang of the second terminus of the linear vector is complementary to the 3′ overhang of the second terminus of the nucleic acid segment.   
     
     
         36 . The method of  claim 35 , wherein the Type IIs restriction endonuclease is SapI. 
     
     
         37 . The method of  claim 35 , wherein the starting nucleic acid molecule is generated by polymerase chain reaction. 
     
     
         38 . The method of  claim 35 , wherein the starting nucleic acid molecule is a vector.