METHODS FOR NUCLEIC ACID MAPPING AND IDENTIFICATION OF FINE-STRUCTURAL-VARIATIONS IN NUCLEIC ACIDS

• US 20090325239A1
• Filed: 07/10/2009
• Published: 12/31/2009
• Est. Priority Date: 01/04/2006
• Status: Active Grant

• Current Assignee: Versitech Limited (Government of Hong Kong Special Administrative Region)
• Original Assignee: Versitech Limited (Government of Hong Kong Special Administrative Region)
• Inventors: LOK, Si
• Granted Patent: US 8,329,400 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• 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

A method of juxtaposing sequence tags (GVTs) that are unique positional markers along the length of a population of target nucleic acid molecules is provided, the method comprising: fragmenting the target nucleic acid molecule to form target DNA insert; ligating the target DNA insert to a DNA vector or backbone to create a circular molecule; digesting the target DNA insert endonuclease to cleave the target DNA insert at a distance from each end of the target DNA insert yielding two GVTs comprising terminal sequences of the target DNA insert attached to an undigested linear backbone; recircularizing the linear backbone with the attached GVTs to obtain a circular DNA containing a GVT-pair having two juxtaposed GVTs; and recovering the GVT-pair DNA by nucleic acid amplification or digestion with endonuclease having sites flanking the GVT-pair. Cosmid vectors are provided for creating GVT-pairs of ˜45- to 50-kb separation sequencable by next-generation DNA sequencers.

Claims

1. A method for producing juxtaposing sequence tags (GVTs), where two constituent members of a sequence tag pair (GVT-pair) located along the length of a population of target nucleic acid molecules are positional markers of a defined separation distance or are positional markers of two adjacent and cleavable restriction endonuclease sites of one or more restriction endonucleases, the method comprising:

fragmenting a large nucleic acid molecule to form target DNA insert;

ligating the target DNA insert to a linear DNA backbone at terminal cloning sites of the target DNA insert, leading to the creation of a circular molecule comprising the target DNA insert;

digesting the target DNA insert within the circular molecule using at least one endonuclease cleaving the target DNA insert at a distance from each of the insert'"'"'s terminal cloning sites thereby creating a linear molecule comprising two sequence tags (GVTs) comprising the terminal sequences of the target DNA insert, one of the two GVTs attached to each ends of the undigested DNA backbone;

recircularizing the linear DNA backbone with the attached GVTs to create a circular DNA molecule thereby creating a GVT-pair comprising two juxtaposed GVTs that are the same relative orientation as the target DNA insert;

isolating the created GVT-pair by nucleic acid amplification from primer sites on the DNA backbone or by digestion with endonuclease at sites that are on the DNA backbone and are flanking the created GVT-pair.

2. A method according to claim 1, wherein the individual GVTs of the isolated GVT-pair are made to adopt an inverted orientation relative to the target DNA insert by the method further comprising:

recircularizing the isolated and created GVT-pair by intra-molecular ligation;

digesting the resulting circular molecule with a restriction endonuclease, that cleaves the GVT-pair to yield a linear molecule with the GVTs in an inverted orientation.

3. A method according to claim 1, wherein the created GVT-pair comprises two end regions of a target DNA insert having a separation distance that is less than 250-kb in separation, less than 100-kb in separation, less than 50-kb separation, less than 25-kb in separation, less than 10-kb in separation, less than 5-kb in separation, or less than 2.5-kb in separation.

4. A method according to claim 1, wherein the target DNA insert is either genomic DNA, genomic DNA from isolated chromosomes, DNA isolated from isolated chromosome regions, cDNA, mitochondrial DNA, chloroplast DNA, viral DNA, microbial DNA, plastid DNA, chemically synthesized DNA, DNA product of nucleic acid amplification, or DNA transcribed from RNA.

5. A method according to claim 1, wherein the nucleic acid molecule is fragmented randomly to form a target DNA insert by the selected application of mechanical force, partial digestion with one or more nuclease enzymes alone or in combination, or by complete digestion using one or more nuclease enzymes alone or in combination.

6. A method according to claim 1, wherein the target nucleic acid molecule is fragmented to form a target DNA insert by the use of one or more nucleases that are sensitive to DNA methylation status.

7. A method according to claim 1, wherein the target DNA insert is size fractionated.

8. A method according to claim 1, wherein the target DNA insert is not size fractionated.

9. A method according to claim 1, wherein the target DNA insert is at least 250-kb, 100-kb, at least 50-kb, at least 25-kb, at least 10-kb, at least 5-kb, or at least 2.5-kb in length.

10. A method according to claim 1, wherein the restriction endonuclease used to digest the target DNA insert to create the GVTs is a restriction endonuclease that recognizes a four or more base pair recognition sequence.

11. A method according to claim 1, wherein the restriction endonuclease used to digest the target DNA insert to create the GVTs is a type II restriction endonuclease.

12. A method according to claim 11, wherein the type II restriction endonuclease is FspB I, Csp6 I, or any of their isoschizomers or neoschizomers, alone or in combination.

13. A method according to claim 1, wherein the DNA backbone is less than 25-kb in length, less than 10-kb in length, less than 5-kb in length, less than 1-kb in length, less than 500-bp in length, less than 250-bp in length, less than 100-bp in length, or less than 50-bp in length.

14. A method of claim 1, wherein the target DNA insert is first ligated to an adaptor at each end and then the adaptor-ligated target DNA insert is ligated to a DNA backbone.

15. A method according to claim 1, wherein the DNA backbone is assembled from two, three or more DNA segments before, during or after ligating to the target DNA insert.

16. A method according to claim 1, wherein the DNA backbone contains functional adaptor sequence at one or both termini to facilitate the ligation of the DNA backbone to the target DNA insert.

17. A method according to claim 16, wherein the adaptor is 8-bp to 100-bp in length.

18. A method according to claim 1, wherein the DNA backbone contains one or more sequences that are capable of directing DNA amplification of the created GVT-pair.

19. A method according to claim 18, wherein the DNA backbone contains one or more sequences that are capable of directing DNA amplification of the created GVT-pair on a solid support.

20. A method according to claim 19, wherein the DNA backbone contains one or more sequences that are capable of directing isothermal DNA amplification of the created GVT-pair on a solid support.

21. A method according to claim 1, wherein the adaptor contains one or more sequences that are capable of binding DNA sequencing primers for sequencing the created GVT-pair.

22. A method according to claim 1, wherein the produced GVT-pair is one of a plurality of similarly produced GVT-pairs representative of a library of linked genomic tags from a population of target DNA.

23. A method according to claim 1, wherein the DNA backbone contains one or more nucleotides conjugated to a moiety that is capable of generating a detectable signal that can be read by an instrument or by visual inspection.

24. A method according to claim 1, wherein the DNA backbone contains one or more nucleotides that are conjugated to an affinity purification tag.

25. A method according to claim 24, wherein the affinity purification tag is biotin.

26. A method according to claim 25, comprising the step of isolating nucleic acid fragments by affinity purification with an avidin or streptavidin coated solid support.

27. A method according to claim 1, wherein the DNA backbone is free of palindromic sequences of four bases or greater in length.

28. A method according to claim 1, wherein the DNA backbone is free of recognition and cleavage sites for the type II restriction endonuclease FspB I, Csp6 I, or any of their their isoschizomers or neoschizomers.

29. A method according to claim 1, wherein methylation of the DNA backbone prevents cleavage by one or more restriction endonucleases.

30. A method according to claim 1, wherein the termini of the DNA backbone are created by digestion with a type IIS restriction endonuclease that produces nucleotide overhangs to facilitate ligation of a target DNA insert bearing the complementary nucleotide overhangs.

31. A method according to claim 30, wherein the termini of the DNA backbone are created by digestion with a type IIS restriction endonuclease that produces a single base 3′

-nucleotide overhang at each terminus to facilitate ligation of the target DNA insert bearing a complementary 3′

-nucleotide overhang to create a circular molecule.

32. A method according to claim 30, wherein the 3′

-single nucleotide extension of the DNA backbone is a thymine base and the complementary 3′

-single nucleotide extension on the target DNA insert is an adenine base.

33. A method according to claim 30, wherein the type IIS restriction endonuclease is BciVI or any of its isoschizomers.

34. A method according to claim 30, wherein the DNA backbone is free of the recognition site of the type IIS restriction endonuclease BciVI, or any of its isoschizomers or neoschizomers.

35. A method according to claim 1, wherein the DNA backbone is a DNA vector that is capable of propagation in a cell.

36. A method according to claim 1, wherein the DNA backbone is a bacterial artificial chromosome vector or a yeast artificial chromosome vector.

37. A method according to claim 1, wherein the DNA backbone is a DNA vector selected from a group consisting of plasmid, phagemid, cosmid, and fosmid.

38. A method according to claim 1, wherein the DNA backbone contains one or more sequences that are capable of mediating bacteria phage packaging.

39. A method according to claim 38, wherein the bacteria phage packaging sequence is the COS sequence derived from bacteria phage lambda.

40. A method according to claim 1, wherein the DNA backbone comprises a selection marker gene.