Method for the preparation of nucleic acid

• US 20040040053A1
• Filed: 11/22/2002
• Published: 02/26/2004
• Est. Priority Date: 11/22/2001
• Status: Abandoned Application

• Current Assignee: Naoki Goshima, Nobuo Nomura, Saki Sono, Yasutomo Kisu
• Original Assignee: Naoki Goshima, Nobuo Nomura, Saki Sono, Yasutomo Kisu
• Inventors: Nomura, Nobuo, Sono, Saki, Goshima, Naoki, Kisu, Yasutomo
• Application Number:
  US10/301,849
• Publication Number:
  US 20040040053A1
• Time in Patent Office: Days
• US Class Current: 800/278
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The present invention provides materials and methods for the rapid cloning and mutagenesis of nucleic acid molecules. The present invention permits simultaneous introduction of a one or more point mutations and adapter sequences into a nucleic acid of interest.

Claims

1. A method of preparing a plurality of nucleic acid molecules, comprising:

contacting a template nucleic acid molecule with at least a first, second and third primer and a polypeptide having DNA polymerase activity to form a mixture;

incubating the mixture under conditions sufficient to extend the primers, wherein both the second and the third primers comprise a sequence that anneals to a sequence on the template and, wherein the sequence of at least one of the second and third primers comprises at least one base that does not base pair with the sequence of the template.

2. A method of amplifying a double-stranded DNA molecule comprising:

(a) providing at least a first, second and third primer, wherein the first primer is at least partially complementary to a sequence of the second strand of the DNA molecule and the second and third primers are at least partially complementary to a sequence of the first strand of the DNA molecule;

(b) hybridizing the first primer to the second strand and the second and third primers to the first strand in the presence of a polypeptide having DNA polymerase activity, under conditions such that a third DNA strand complementary to the second strand and a fourth and a fifth DNA strand complementary to the first strand are synthesized, wherein at least one of the second and third primers contains nucleotide that does not base pair to the sequence on the first strand of the DNA molecule to which it hybridizes.

3. A method according to claim 2, comprising:

(c) denaturing the product of (b); and

(d) repeating (a) to (c) one or more times to produce third strands comprising a sequence complementary to either the second or the third primer and to produce fourth and fifth strands comprising a sequence complementary to the first primer.

4. A method according to claim 3, wherein (a) to (c) are repeated from 1 to 25 times.

5. A method according to claim 3, wherein (a) to (c) are repeated from 1 to 15 times.

6. A method according to claim 3, wherein (a) to (c) are repeated from 3 to 10 times.

7. A method according to claim 3, wherein (a) to (c) are repeated from 3 to 8 times.

8. A method according to claim 3, wherein (a) to (c) are repeated 4 to 6 times.

9. A method according to claim 3, wherein the first primer comprises a first adapter sequence, the second primer comprises a second adapter sequence and/or the third primer comprises a third adapter sequence.

10. A method according to claim 9, wherein the adapter sequence of the second and the third primer are the same.

11. A method according to claim 9, wherein the adapter sequence of the second and the third primer are different.

12. A method according to claim 3, comprising:

(e) contacting the product of (d) with at least a fourth, fifth and sixth primer, wherein the fourth primer is at least partially complementary to the fourth and fifth strands and the fifth and sixth primers are at least partially complementary to the third strands; and

(f) hybridizing the fourth primer to the fourth and fifth strands and the fifth and sixth primers to the third strands in the presence of a polypeptide having DNA polymerase activity, under conditions such that a sixth DNA strand complementary to the fourth strand, a seventh DNA strand complementary to the fifth strand, an eighth DNA strand complementary to the thirds strands, and a ninth DNA strand complementary to the third strands are synthesized.

13. A method according to claim 12, comprising

(g) denaturing the product of (f);

and

(h) repeating (e) to (g) one or more times to produce sixth strands comprising a sequence complementary to the fifth primer, seventh strands comprising a sequence complementary to the sixth primer, eighth strands comprising a sequence complementary to the fourth primer and ninth strands comprising a sequence complementary to the fourth primer.

14. A method according to claim 13, wherein at least one of the fourth, fifth or sixth primer comprises an adapter sequence.

15. A method according to claim 13, wherein the fourth primer comprises a fourth adapter sequence, the fifth primer comprises a fifth adapter sequence and the sixth primer comprises a sixth primer sequence.

16. A method according to claim 13, wherein the adapter sequences of the fifth and sixth primers are the same.

17. A method according to claim 13, wherein the adapter sequences of the fifth and sixth primers are different.

18. A method according to claim 15, wherein each adapter sequences comprises a sequence independently selected from the group consisting of restriction enzyme recognition sites, topoisomerase recognition sites, recombination sites, and transposition sites.

19. A method according to claim 18, wherein the recombination sites are selected from a group consisting of lox sites and att sites.

20. A method according to claim 18, wherein the att sites are selected from a group consisting of attB1, attP1, attL1, attR1, attB2, attP2, attL2, attR2, attB3, attP3, attL3, attR3, attB5, attP5, attL5, and attR5.

21. A method according to claim 13, comprising

(i) contacting the product of (h) with one or more polypeptides and one or more vectors under conditions sufficient to insert all or a portion of the product of (h) into the vector.

22. A method according to claim 21, comprising

(j) transforminng a competent cell with the product of (i);

and

(k) selecting for cells comprising the product of (i).

23. A method according to claim 21, wherein one or more vectors comprise at least one recombination site and at least one polypeptide is a recombination protein.

24. A method according to claim 21, wherein the product of (h) is contacted with one or more polypeptides and at least two different vectors under conditions sufficient to insert all or a portion of the product of (h) into the vectors.

25. A method according to claim 24, comprising selecting for one of the vectors.