Methods for cloning amplified nucleic acid molecules
View Patent ↗The present invention is directed generally to methods facilitating the cloning of nucleic acid molecules. In particular, the invention relates to the use of polymerase inhibitors, including but not limited to anti-polymerase antibodies (such as anti-Taq antibodies) and fragments thereof, to inactivate residual polymerase activity remaining after the amplification (particularly via PCR) of a target nucleic acid molecule. The invention further provides compositions, particularly storage-stable compositions, comprising one or more components, such as one or more restriction endonucleases and one or more polymerase inhibitors, that are useful in cloning amplified or synthesized nucleic acid molecules by the above-described methods. The invention also relates to nucleic acid molecules produced by these methods, and to genetic constructs (such as vectors) and host cells comprising these nucleic acid molecules.
1. A method for cloning one or more nucleic acid molecules having a first 3′ overhang into one or more vectors having a second 3′ overhang, comprising:
(a) forming a mixture comprising the one or more nucleic acid molecules having the first 3′ overhang, the one or more vectors having the second 3′ overhang and one or more antibodies or fragments thereof which inhibit a polymerase; and
(b) ligating the nucleic acid molecules having the first 3′ overhang to the one or more vectors having the second 3′ overhang to form one or more genetic constructs, thereby cloning the one or more nucleic acid molecules having the first 3′ overhang.
2. The method of claim 1 , wherein the first 3′ overhang is a dA overhang.
3. The method of claim 1 , wherein the second 3′ overhang is a dT overhang.
4. The method of claim 1 , wherein the one or more antibodies or fragments thereof are anti-Taq antibodies.
5. A method of ligating amplified or synthesized nucleic acid molecules having a first 3′ overhang into one or more vectors having a second 3′ overhang, comprising:
(a) forming a mixture comprising the amplified or synthesized nucleic acid molecules having the first 3′ overhang, one or more vectors having the second 3′ overhang, and one or more antibodies or fragments thereof which inhibit a polymerase; and
(b) ligating the amplified or synthesized nucleic acid molecules having the first 3′ overhang to the one or more vectors having the second 3′ overhang to form one or more genetic constructs.
6. The method of claim 5 , wherein the first 3′ overhang is a dA overhang.
7. The method of claim 5 , wherein the second 3′ overhang is a dT overhang.
8. The method of claim 5 , wherein the one or more antibodies or fragments thereof are anti-Taq antibodies.