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

• US 20070015182A1
• Filed: 05/05/2006
• Published: 01/18/2007
• Est. Priority Date: 12/02/1999
• Status: Abandoned Application

• Current Assignee: Patricio Abarzua
• Original Assignee: Patricio Abarzua
• Inventors: Abarzua, Patricio
• Application Number:
  US11/429,549
• Publication Number:
  US 20070015182A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-20. -20. (canceled)

21. A method for simultaneous synthesis of multiple copies of different single-stranded circular DNA molecules, comprising:

(a) contacting a plurality of universal hairpin oligonucleotides with at least two different target 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 hairpin oligonucleotides comprises a single stranded terminal non-palindromic sequence (segment C) such that said segment C of each universal hairpin oligonucleotide is complementary to the segment C of each target hairpin oligonucleotide but said segment C of any target oligonucleotide is not complementary to segment C of the same or another target hairpin oligonucleotide, and wherein said contacting occurs under conditions promoting said contacting and promoting hybridization of said segments C,

(b) ligating the resulting hybridized hairpin oligonucleotides to form a population of monomers each comprising a duplex linear segment with a single-stranded loop at each end.

22. The method of claim 21 further comprising amplifying the monomers via rolling circle amplification.

23. The method of claim 22 wherein said duplex linear segment of step (b) comprises at least one endonuclease restriction site, the method further comprising contacting the product of rolling circle amplification with an endonuclease specific for said endonuclease restriction site to form a new set of hairpin oligonucleotides and then repeating steps (a) through (c) using said new set of hairpin oligonucleotides.

24. The method of claim 21 wherein the target hairpin oligonucleotides of step (a) are of different sizes.

25. The method of claim 21 wherein at least one of the hairpin oligonucleotides contains a pre-selected nucleotide sequence.

26. The method of claim 21 wherein said duplex linear segment of step (b) comprises at least one endonuclease restriction site.

27. The method of claim 26 wherein the endonuclease restriction site contains a phosphorothioate derivative.

28. The process of claim 21 wherein said restriction site is a BamHI restriction site and said endonuclease is Bam HI.

29. The method of claim 21 wherein the ligation step employs an enzyme.

30. The method of claim 29 wherein the enzyme is selected from the group consisting of T4 ligase, Ampligase and E. coli ligase.

31. The method of claim 21 further comprising ligation at a temperature of between 4°

C. to 65°

C.

32. The method of claim 21 wherein the ligation step is non-enzymatic.

33. The process of claim 32 wherein the ligation step requires the formation of a phosphorothioate derivative of a hairpin oligonucleotide.

34. The method of claim 21 wherein said double-stranded linear segment formed in step (b) comprises at least two endonuclease restriction sites.

35. The method of claim 21 wherein said single-stranded circular DNA molecules are produced in a ratio equal to that of the respective starting target hairpin oligonucleotides used to form said circular DNA molecules.

36. The method of claim 21 wherein at least 3 different target hairpin oligonucleotides are contacted with said universal hairpin oligonucleotides.

37. The method of claim 21 wherein at least 4 different target hairpin oligonucleotides are contacted with said universal hairpin oligonucleotides.

38. A method for simultaneous synthesis of multiple copies of different single-stranded circular DNA molecules, comprising:

(a) contacting a plurality of universal hairpin oligonucleotides with at least two different target 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 hairpin oligonucleotides comprises a single stranded terminal non-palindromic sequence (segment C) such that said segment C of each universal hairpin oligonucleotide is complementary to the segment C of each target hairpin oligonucleotide but said segment C of any target oligonucleotide is not complementary to segment C of the same or another target hairpin oligonucleotide, and wherein said contacting occurs under conditions promoting said contacting and promoting hybridization of said segments C,

(b) ligating the resulting hybridized hairpin oligonucleotides to form a population of monomers each comprising a duplex linear segment with a single-stranded loop at each end,

(c) contacting said monomers with a plurality of deoxynucleoside triphosphates (dNTPs), multiple primer oligonucleotides comprising at least one oligonucleotide complementary to at least one segment of each of the monomers and a DNA polymerase capable of supporting rolling circle amplification (RCA), said contacting occurring under conditions promoting said contacting and promoting said rolling circle amplification to form an RCA product, thereby simultaneously generating multiple copies of different single-stranded circular DNA molecules.