Methods and compositions for generating recombinant nucleic acid molecules
View Patent ↗A method of generating a double stranded (ds) recombinant nucleic acid molecule covalently linked in both strands by contacting two or more ds nucleotide sequences with a topoisomerase under conditions such that both termini of at least one end of a first ds nucleotide sequence are covalently linked by the topoisomerase to both termini of at least one end of a second ds nucleotide sequence is provided. Also provided is a method for generating a ds recombinant nucleic acid molecule covalently linked in one strand, by contacting two or more ds nucleotide sequences with a type IA topoisomerase under conditions such that one strand, but not both strands, of one or both ends of a first ds nucleotide sequence are covalently linked by the topoisomerase. Compositions for performing such methods, and compositions generated from such methods also are provided, as are kits containing components useful for conveniently practicing the methods.
1. A reaction mixture comprising:
(a) a first double-stranded DNA molecule which comprises a promoter,
(b) a second double-stranded DNA molecule, and
(c) a recombinant nucleic acid molecule formed from the first double-stranded DNA molecule and the second double-stranded DNA molecule,
wherein at least one end of at least one strand of the first double-stranded DNA molecule is topoisomerase-charged,
wherein at least one end of at least one strand of the second double-stranded DNA molecule is topoisomerase-charged, and
wherein the topoisomerase-charged ends of the first double-stranded DNA molecule and the second double-stranded DNA molecule are capable of hybridizing to each other such that in the recombinant nucleic acid molecule formed from the first double-stranded DNA molecule and the second double-stranded DNA molecule each strand of the first double-stranded DNA molecule is covalently linked to each strand of the second double-stranded DNA molecule and the recombinant nucleic acid molecule-does not contain a nick in either strand at the position where the first and second nucleic acid molecules are joined.
2. The reaction mixture of claim 1 , wherein the first double-stranded DNA molecule comprises a T7 promoter.
3. The reaction mixture of claim 1 , wherein the topoisomerase is a type IB topoisomerase or a catalytic domain of a type IB topoisomerase.
4. The reaction mixture of claim 1 , wherein the topoisomerases are of different types.
5. The reaction mixture of claim 1 , wherein the second double-stranded DNA molecule is the product of a polymerase chain reaction.
6. The reaction mixture of claim 1 , wherein the first double-stranded DNA molecule is a vector.
7. The reaction mixture of claim 1 , wherein the second double-stranded DNA molecule is a cDNA.
8. The reaction mixture of claim 1 , wherein the second double-stranded DNA molecule is genomic DNA.
9. The reaction mixture of claim 1 , further comprising one or more components selected from the group consisting of buffers, salts and polyamines.
10. The reaction mixture of claim 1 , wherein the second double-stranded DNA molecule is synthetic DNA.