Cell extract promoted cloning
View Patent ↗Methods are disclosed for assembling a plurality of double-stranded DNA fragments into DNA molecules in a single in vitro recombination reaction comprising contacting the plurality of double-stranded DNA fragments with a bacterial extract derived from a RecA deficient bacterial strain so as to assemble the plurality of DNA fragments into DNA molecules.
1. A method of assembling a plurality of double-stranded DNA fragments into DNA molecules in a single in vitro recombination reaction comprising contacting in vitro the plurality of double-stranded DNA fragments with a bacterial extract from a RecA deficient bacterial strain so as to assemble the plurality of DNA fragments into DNA molecules, wherein each DNA fragment has a 3′ end and a 5′ end, wherein fragments assemble with each other when the 5′ end of one fragment has 20 bp to 100 bp that are homologous with the 3′ end of another fragment, wherein heterologous sequences, when present, of up to 1000 bp that flank the homologous region are removed prior to assembling the DNA fragments into DNA molecules, and wherein the in vitro recombination reaction is performed without the addition of any of an exogenous DNA restriction enzyme, an exogenous DNA modifying enzyme, or an exogenous DNA ligase.
2. The method of claim 1 , wherein fragments assemble with each other when the 5′ end of one fragment has 30 bp to 52 bp that are homologous with the 3′ end of another fragment.
3. The method of claim 1 , wherein the DNA fragments comprise heterologous sequences of up to 1,000 bp adjacent to the homologous end of the fragment.
4. The method of claim 1 , wherein the DNA fragments are produced by digesting a plasmid vector or a bacterial artificial chromosome (BAC) with restriction enzymes.
5. The method of claim 1 , wherein the DNA fragments are produced by amplifying DNA using polymerase chain reaction (PCR).
6. The method of claim 1 , wherein redα, redβ and gam genes are introduced into the genome of the RecA deficient bacteria prior to obtaining the bacterial extract.
7. The method of claim 1 , wherein the bacteria are a RecA deficient Escherichia coli strain.
8. The method of claim 1 , which comprises transforming the assembled recombinant DNA molecules into host bacteria to amplify the DNA molecules.
9. The method of claim 1 , wherein the DNA fragments that are assembled are chemically synthesized DNA fragments.
10. The method of claim 1 , wherein fragments assemble with each other when the 5′ end of one fragment has 20 bp to 52 bp that are homologous with the 3′ end of another fragment.
11. The method of claim 1 , wherein the DNA fragments are produced by amplifying DNA.
12. A method of assembling a plurality of double-stranded DNA fragments into DNA molecules in a single in vitro recombination reaction comprising contacting in vitro the plurality of double-stranded DNA fragments with a bacterial extract from a RecA deficient bacterial strain so as to assemble the plurality of DNA fragments into DNA molecules, wherein each DNA fragment has a 3′ end and a 5′ end, and wherein fragments assemble with each other when the 5′ end of one fragment has 20 bp to 100 bp that are homologous with the 3′ end of another fragment, and wherein the DNA fragments are assembled into DNA molecules without the use of an exogenous DNA polymerase.
13. The method of claim 12 , wherein the exogenous DNA polymerase is T4 DNA polymerase.
14. A method of assembling a plurality of double-stranded DNA fragments into DNA molecules in a single in vitro recombination reaction comprising contacting in vitro the plurality of double-stranded DNA fragments with a bacterial extract from a RecA deficient bacterial strain so as to assemble the plurality of DNA fragments into DNA molecules, wherein each DNA fragment has a 3′ end and a 5′ end, and wherein fragments assemble with each other when the 5′ end of one fragment has 20 bp to 100 bp that are homologous with the 3′ end of another fragment, and wherein more than 34,500 colony forming units are obtained per nanogram of DNA.
15. The method of claim 14 , wherein more than 68,000 colony forming units are obtained per nanogram of DNA.
16. The method of claim 14 , wherein more than 124,000 colony forming units are obtained per nanogram of DNA.
17. The method of claim 14 , wherein more than 162,000 colony forming units are obtained per nanogram of DNA.
18. The method of claim 14 , wherein more than 632,000 colony forming units are obtained per nanogram of DNA.
19. The method of claim 14 , wherein redα, redβ and gam genes are introduced into the genome of the RecA deficient bacteria prior to obtaining the bacterial extract.