Generation of single-strand circular DNA from linear self-annealing segments

• US 6,498,023 B1
• Filed: 11/28/2000
• Issued: 12/24/2002
• Est. Priority Date: 12/02/1999
• Status: Expired due to Fees

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Molecular Staging Inc. (Qiagen NV)
• Inventors: Abarzúa, Patricio
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Spiegler, Alexander H.
• Application Number:
  US09/723,685
• Time in Patent Office: 756 Days
• Field of Search: 435/91.2, 435/6, 435/91.41, 435/91.1, 435/91.4, 435/91.5
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

The present invention provides a method for the rapid simultaneous production of a plurality of single-stranded DNA circles having a predetermined size and nucleotide sequence using pre-designed hairpin oligonucleotides containing complementary sequences for directing ligation to form dumbbell-shaped monomers followed by heat denaturation to yield single-stranded DNA circles.

Claims

1. A process for preparing multiple copies of single-stranded circular DNA, comprising:

(a) contacting at least two hairpin oligonucleotides, wherein each said hairpin oligonucleotide contains a segment that does not hybridize with any segment of the same or another of said hairpin oligonucleotides and wherein each of said at least two hairpin oligonucleotides comprises a single stranded terminal sequence complementary to that of the other hairpin oligonucleotide, and under conditions permitting said complementary terminal sequences to hybridize,

(b) ligating the resulting hybridized hairpin oligonucleotides to form a monomer comprising a duplex linear segment containing at least one endonuclease restriction site with a single-stranded hairpin loop at each end,

(c) denaturing said monomer to form a single-stranded circle,

(d) contacting said single stranded circle of (c) with a plurality of deoxynucleoside triphosphates (dNTPs), at least one primer oligonucleotide complementary to at least one segment of the single stranded circle formed in step (c) and a DNA polymerase capable of supporting rolling circle amplification (RCA) under conditions promoting the formation of RCA product,

(e) contacting said RCA product of (d) with an endonuclease specific for said endonuclease restriction site of (b) to form a new set of hairpin oligonucleotides, and

(f) repeating steps (a) through (c) using the hairpin oligonucleotides formed in step (e),

thereby generating multiple copies of single-stranded circular DNA.

2. The process of claim 1 wherein the hairpin oligonucleotides of step (a) are of different lengths.

3. The process of claim 1 wherein at least one of the hairpin oligonucleotides contains a pre-selected nucleotide sequence.

4. The process of claim 1 wherein the endonuclease restriction site contains a phosphorothioate derivative.

5. The process of claim 1 wherein the ligation step employs an enzyme.

6. The process of claim 5 wherein the enzyme is selected from the group consisting of T4 ligase, Ampligase and E. coli ligase.

7. The process of claim 1 further comprising ligation at a temperature of between 4°

C. to 65°

C.

8. The process of claim 1 wherein the ligation step is non-enzymatic.

9. The process of claim 8 wherein the ligation step requires the formation of a phosphorothioate derivative of a hairpin oligonucleotide.

10. The process of claim 1 wherein said DNA polymerase is a member selected from the group consisting of E. coli DNA polymerase I, Klenow fragment, T4 or T7 DNA polymerases, and Taq polymerase.

11. The process of claim 1 wherein said denaturation is accomplished by heat denaturation.

12. The method of claim 11 wherein the heat denaturation is carried out at a temperature of between 70°

C. and 100°

C.

13. The method of claim 12 wherein the temperature is at least 95°

C.

14. The process of claim 1 wherein said restriction site is a BamHI restriction site and said endonuclease is BamHI.

15. The method of claim 1 wherein said double-stranded linear segment formed in step (b) comprises at least two endonuclease restriction sites.

16. The process of claim 1 further comprising repeating steps (d) through (f) at least once.

17. A process for preparing multiple copies of single-stranded circular DNA, comprising:

(a) contacting a non-circular DNA duplex oligonucleotide and at least 2 hairpin oligonucleotides, wherein each said hairpin oligonucleotide contains a segment that does not hybridize with any other segment of the same or another of said hairpin oligonucleotides and wherein each of said hairpin oligonucleotides comprises a single-stranded terminal sequence and wherein said non-circular DNA duplex contains a single-stranded terminal sequence at each end and wherein each single stranded terminal sequence of said non-circular DNA duplex is complementary to a single stranded terminal sequence of at least one of said at least two hairpin oligonucleotides under conditions promoting hybridization of said single stranded terminal sequences,

(b) ligating the hybridized hairpin oligonucleotides and non-circular duplex to form a monomer comprising a linear duplex segment containing at least one endonuclease restriction site with a single-stranded loop at each end,

(c) denaturing said monomer to form a single-stranded circle,

(d) contacting the single stranded circle formed in step (c) with a plurality of deoxynucleoside triphosphates (dNTPs), at least one primer oligonucleotide complementary to at least one segment of the single stranded circle formed in step (c) and a DNA polymerase capable of supporting rolling circle amplification under conditions promoting the formation of RCA product,

(e) contacting said RCA product with an endonuclease specific for said endonuclease restriction site of step (b) to form a new set of hairpin oligonucleotides for reaction in step (a), and

(f) repeating steps (a) through (c) using the hairpin oligonucleotides formed in step (e),

thereby generating multiple copies of single-stranded circular DNA.

18. The process of claim 17 wherein the hairpin oligonucleotides of step (a) are of different lengths.

19. The process of claim 17 wherein at least one of the hairpin oligonucleotides contains a pre-selected nucleotide sequence.

20. The process of claim 17 wherein the single stranded terminal sequences of the hairpin oligonucleotides of step (a) are different from each other.

21. The process of claim 17 wherein the single stranded terminal sequences of the non-circular DNA duplex oligonucleotide of step (a) are different from each other.

22. The process of claim 17 wherein the ligation of the duplex segment with each of the hairpin oligonucleotides is carried out as separate steps.

23. The process of claim 17 wherein the ligation step employs an enzyme.

24. The process of claim 23 wherein the enzyme is selected from the group consisting of T4 ligase, Ampligase and E. coli ligase.

25. The process of claim 17 further comprising ligation at a temperature of between 4°

C. to 65°

C.

26. The process of claim 17 wherein the ligation step is non-enzymatic.

27. The process of claim 17 wherein at least one endonuclease restriction site comprises a phosphorothioate derivative.

28. The process of claim 17 wherein said denaturation is accomplished by heat denaturation.

29. The method of claim 28 wherein the heat denaturation is carried out at a temperature of between 70°

C. and 100°

C.

30. The method of claim 29 wherein the temperature is at least 95°

C.

31. The method of claim 17 wherein said double-stranded linear segment formed in step (b) comprises at least two endonuclease restriction sites.

32. The process of claim 17 wherein said non-circular DNA duplex oligonucleotide comprises at least one restriction enzyme target site.

33. The process of claim 32 wherein said endonuclease restriction site does not contain a single stranded terminal sequence.

34. The process of claim 17 wherein said non-circular DNA duplex oligonucleotide comprises at least two restriction enzyme target sites.

35. The process of claim 17 wherein said at least one endonuclease restriction site is a BamHl site and said endonuclease is BamHI.

36. The process of claim 35 further comprising repeating steps (d) through (f) at least once.