Method for controlling length of overhang of double stranded DNA
AssigneeEnzynomics Co. Ltd.
InventorsYong Keol Shin, Yunil Suh, Min Young Lee, Ah Rong Jeong
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Abstract
Disclosed are methods and kits for controlling the length of overhangs at the ends of double stranded DNA based on a competitive inhibition. Further provided are ligase-independent methods for joining the two ends of DNA strands using the same. The present methods efficiently control the overhangs of ds DNA. This user-controlled overhangs supply a tool for efficient ligation in a ligase independent way and can be advantageously used in DNA recombinant technology such as cloning gene or DNA fragments of interest or PCR products.
Claims9
A method of controlling overhang length of a double strand DNA comprising:
- providing a linear double strand DNA (ds DNA);
- and
- treating the ds DNA with a first DNA polymerase having a 3′-5′ exonuclease activity and a second DNA polymerase without a 3′-5′ exonuclease activity in the absence of dNTPs,
- wherein the 3′-5′ exonuclease activity of the first polymerase is competitively inhibited by the second DNA polymerase,
- and wherein each end of the linear ds DNA is either of 5′ overhang, 3′ overhang or blunt-ended.
A method of joining two ends of double strand DNA (ds DNA), comprising:
- providing at least two linear ds DNA molecules, each DNA molecule having at least one region located at either or both end(s) thereof that is complementary to other region in the DNA molecules, wherein each end of the two linear ds DNA is either of 5′ overhang, 3′ overhang or blunt-ended;
- and
- treating the at least two ds DNA molecules with a first DNA polymerase having an 3′-5′ exonuclease activity and a second DNA polymerase with no3′-5′ exonuclease activity in the absence of dNTPs under a condition to produce overhangs of 5 to 100 nucleotide (nt) in length at the complementary region, whereby the at least two linear ds DNA molecules treated are joined at the produced overhangs by complementarity,
- wherein the 3′-5′ exonuclease activity of the first polymerase is competitively inhibited by the second DNA polymerase.
Dependencies are inferred from the claim text.