Methods and compositions for transferring DNA sequence information among vectors

• US 5,989,872 A
• Filed: 08/12/1997
• Issued: 11/23/1999
• Est. Priority Date: 08/12/1997
• Status: Expired due to Fees

• Current Assignee: Clontech Laboratories Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Clontech Laboratories Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Hua, Shaobing, Luo, Ying, Zhu, Li
• Primary Examiner(s): Guzo, David
• Application Number:
  US08/909,525
• Time in Patent Office: 833 Days
• Field of Search: 435/5, 435/91.2, 435/91.4, 435/91.41, 435/455, 435/463, 435/465, 435/325, 435/354, 435/366, 536/23.1, 536/24.1, 536/24.2, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/81 for yeasts

Abstract

Methods and compositions are provided for transferring DNA sequence information from a first vector to a second vector. In the subject methods, a first vector comprising a region of DNA having a sequence of interest is contacted with a set of three pairs of oligonucleotide primers under conditions sufficient to produce three different PCR products, where each product corresponds to a different reading frame. The oligonucleotide primers comprise a first region of sequence identity with the first vector and a second region permissive of site specific recombination with the second vector. The resultant PCR products are combined with the second vector under conditions sufficient for site specific recombination to occur. Also provided are kits for use in performing the subject methods.

Claims

1. A method of transferring DNA sequence information from a first vector to a second vector, said method comprising:

contacting said first vector with a set of three pairs of oligonucleotide primers under conditions sufficient to produce three different PCR products, wherein each oligonucleotide primer comprises a first region of sequence identity with said first vector and a second region which does not hybridize with said first vector and provides for homologous recombination with said second vector, whereby three different PCR products are produced; and

contacting said three different PCR products with said second vector under conditions sufficient for homologous recombination to occur;

whereby said DNA sequence information is transferred from said first vector to said second vector.

2. The method according to claim 1, wherein said DNA sequence information is the sequence of an EST clone.

3. The method according to claim 1, wherein said three different PCR products correspond to three different reading frames.

4. The method according to claim 1, wherein each pair of said set of oligonucleotide primers is contacted with said first vector at substantially the same time.

5. The method according to claim 1, wherein each pair of said set of oligonucleotide primers is contacted with said first vector at different times.

6. A second vector produced according to claim 1, wherein each of the first regions in said set of primers is the same length.

7. A method of transferring DNA sequence information from a first plasmid to a second plasmid, said method comprising:

contacting a first plasmid with a set of three pairs of oligonucleotide primers under conditions sufficient to produce three different PCR products, wherein each PCR product corresponds to a different reading frame and each oligonucleotide primer comprises a first region of sequence identity with said first plasmid and a second region which does not hybridize with said first plasmid and provides for homologous recombination with said second plasmid, whereby three different PCR products are produced; and

contacting said three different PCR products, with said second plasmid under conditions sufficient for homologous recombination to occur;

whereby said DNA information is transferred from said first plasmid to said second plasmid.

8. The method according to claim 7, wherein each pair of said set of oligonucleotide primers is contacted with said first plasmid at substantially the same time.

9. The method according to claim 7, wherein each pair of said set of oligonucleotide primers is contacted with said first plasmid at different times.

10. The method according to claim 7, wherein said second plasmid is a DNA sequence encoding either a DNA binding domain or an activation domain of a eukaryotic transcriptional activator.

11. A second plasmid produced according to claim 7, wherein each of the first regions in said set of primers is the same length.

12. A method of transferring DNA sequence information from a first plasmid into an expression plasmid, said method comprising:

contacting said first plasmid with a set of three pairs of oligonucleotide primers under conditions sufficient to produce three different PCR products, wherein each PCR product corresponds to a different reading frame and each oligonucleotide primer comprises a first region of sequence identity with said first plasmid and a second region which does not hybridize with said first plasmid and provides for homologous recombination with said expression plasmid, whereby three different PCR products are produced; and

co-transforming said three different PCR products and said expression plasmid into a yeast host whereby homologous recombination occurs, wherein said expression plasmid comprises a DNA sequence encoding either a DNA binding domain or an activation domain of a eukaryotic transcriptional activator;

whereby said DNA sequence information is transferred into said expression plasmid.

13. The method according to claim 12, wherein each pair of said set of oligonucleotide primers is contacted with said first plasmid at substantially the same time.

14. The method according to claim 12, wherein each pair of said set of oligonucleotide primers is contacted with said first plasmid at different times.

15. The method according to claim 12, wherein said eukaryotic transcriptional activator is GAL4.

16. The method according to claim 12, wherein said second region ranges in length from 20 to 80 bp.

17. An expression plasmid produced according to claim 12, wherein each of the first regions in said set of primers is the same length.

18. The method according to claim 16, wherein said second region does not exceed 50 bp in length.

19. A kit for use in transferring DNA sequence information from a first vector to a second vector, said kit comprising:

a set of three pairs of oligonucleotide primers, wherein each primer comprises a first region of sequence identity with said first vector and a second region which does not hybridize with said first vector and provides for homologous recombination with said second vector.

20. The kit according to claim 19, wherein said second vector is an expression plasmid comprising a sequence encoding either a DNA binding domain or an activation domain of a eukaryotic transcriptional activator.

21. The kit according to claim 20, wherein said kit further comprises two different expression plasmids, wherein each plasmid comprises said sequence encoding either said DNA binding domain or said activation domain.

22. A kit for use in transferring DNA sequence information from a first plasmid to an expression plasmid, said kit comprising:

an expression plasmid comprising a sequence encoding either a GAL4 DNA binding domain or a GAL4 activation domain;

a set of three pairs of oligonucleotide primers, wherein each primer comprises a first region of sequence identity with a first plasmid and a second region which does not hybridize with said first plasmid and provides for homologous recombination with said expression plasmid.