Method to protect DNA ends
A method of protecting the 3′ end of a DNA molecule from nuclease damage is disclosed. In one embodiment, the method comprises the step of exposing the DNA molecule to a preparation of DdrA protein.
1. A method of protecting a 3′ end of a DNA molecule from exonuclease damage comprising the step of exposing the DNA molecule to an amount of a pure preparation of DdrA protein effective to decrease exonuclease damage in relation to natural exonuclease damage that would occur in the absence of DdrA, wherein the DdrA protein consists of SEQ ID NO:2.
2. A method of protecting a 3′ end of a DNA molecule from exonuclease damage comprising the step of exposing the DNA molecule to an amount of a PURE preparation of DdrA protein effective to decrease exonuclease damage in relation to natural exonuclease damage that would occur in the absence of DdrA, wherein the DdrA protein consisting of SEQ ID NO:2, and wherein the DdrA protein is incubated with at least a first and a second linear duplex DNA each with a 3′-ending strand and a 5′-ending strand, wherein the first linear duplex DNAs comprises an end that is complementary to an end of the second linear duplex DNA.
3. The method of claim 2 further comprising the step of exposing the first and second DNA molecule to a exonuclease, wherein the DdrA protein protects the 3′ ending strands while allowing the 5′ ending strands to be degraded, thereby producing single-stranded extensions with 3′ ends on both first and second DNAs.
4. The method of claim 3 further comprising the steps of annealing the single stranded extensions of the first and second DNAs, thereby producing a joined first and second DNA.
5. The method of claim 4 further comprising the steps of processing the joined DNA with a nuclease, a DNA polymerase, and a DNA ligase to make the joined DNA contiguous.
6. The method of claim 5 , wherein the method is used to join the first and second DNAs of at least 1 Kb each.
7. The method of claim 6 , wherein the method reconstructs the first and second DNAs from mixtures of multiple DNA fragments of greater than 1 Kb.
8. The method of claim 1 additionally including the step of exposing the DNA molecule to DrSSB.
9. The method of claim 1 wherein the DNA is between 0.1–100 nanomolar.
10. The method of claim 1 wherein the DdrA protein is between 1 nanomolar–100 micromolar.
11. The method of claim 10 wherein the concentration of DdrA is 1–10 micromolar.
12. The method of claim 1 wherein the DNA is protected for at least 30 minutes.
13. The method of claim 2 additionally including the step of exposing the DNA molecule to DrSSB.
14. The method of claim 5 wherein DNA is between 0.1–100 nanomolar.
15. The method of claim 5 wherein the DdrA protein is between 1 nanomolar–100 micromolar.