Iterative and regenerative DNA sequencing method

• US 6,258,533 B1
• Filed: 03/05/1998
• Issued: 07/10/2001
• Est. Priority Date: 11/01/1996
• Status: Expired due to Fees

• Current Assignee: University of Iowa Research Foundation
• Original Assignee: University of Iowa Research Foundation
• Inventors: Jones, Douglas H.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/035,183
• Time in Patent Office: 1,223 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/149 Sequential reactions

Abstract

An iterative and regenerative method for sequencing DNA is described. This method sequences DNA in discrete intervals starting at one end of a double stranded DNA segment. This method overcomes problems inherent in other sequencing methods, including the need for gel resolution of DNA fragments and the generation of artifacts caused by single-stranded DNA secondary structures. A particular advantage of this invention is that it can create offset collections of DNA segments and sequence the segments in parallel to provide continuous sequence information over long intervals. This method is also suitable for automation and multiplex automation to sequence large sets of segments.

Claims

1. A sequencing method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:

a) digesting said double stranded nucleic acid segment with a restriction enzyme whose cleavage site is separate from its recognition site to produce a double stranded molecule having a single stranded overhang sequence corresponding to an enzyme cut site;

b) providing an adaptor having a cycle identification tag, a restriction enzyme recognition domain and a sequence identification region;

c) hybridizing said adaptor to said double stranded nucleic acid having said single-stranded overhang sequence to form a ligated molecule;

d) amplifying said ligated molecule from step (c) with a labeled primer specific for said cycle identification tag, restriction enzyme recognition domain, and a portion of said sequence identification region of said adaptor;

e) identifying said nucleotide n by identifying said primer incorporated into the amplification product; and

f) repeating steps (a) through (e) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

3. The method of claim 1 or 2, wherein said enzyme cut site is the cut site located the farthest away from said recognition domain.

4. The method of claim 1 or 2, wherein said restriction enzyme of step (a) is a class-IIS restriction endonuclease.

5. The method of claim 4, wherein said class-IIS restriction endonuclease is selected from the group consisting of AccBSI, AceIII, AciI, AclWI, AlwI, Alw26I, AlwXI, Asp26HI, Asp27HI, Asp35HI, Asp36HI, Asp40HI, Asp50HI, AsuHPI, BaeI, BbsI, BbvI, BbvII, Bbv16II, Bce83I, BcefI, BcgI, Bco5I, Bco116I BcoKI, BinI, Bli736I, BpiI, BpmI, Bpu10I, BpuAI, Bsal, BsaMI, Bsc9II, BscAI, BscCI, BseII, Bse3DI, BseNI, BseRI, BseZI, BsgI, BsiI, BsmI, BsmAI, BsmBI, BsmFI, Bsp24I, Bsp423I, BspBS3II, BspIS4I, BspKT5I, BspLU11III, BspMI, BspPI, BspST5I, BspTS514I, BsrI, BsrBI, BsrDI, BsrSI, BssSI, Bst11I, Bst71I, Bst2BI, BstBS32I, BstD102I, BstF5I, BstTS5I, Bsu6I, CjeI, CjePI, Eam1104I, EarI, Eco31I, Eco57I, EcoA4I, EcoO44I, Esp3I, FauI, FokI, GdiII, GsuI, HgaI, HphI, Ksp632I, MboII, MlyI, MmeI, Mn1I, Mva1269I, PhaI, PieI, RleAI, SapI, SfaNI, SimI, StsI, TaqII, TspII, TspRI, Tth111II, and VpaK32I.

6. The method of claim 1 or 2, wherein a nucleic acid ligase is used to attach at least one strand of said restriction enzyme recognition domain of step (b) to said nucleic acid segment.

7. The method of claim 1 or 2, wherein said method further comprises blocking an enzyme recognition domain lying outside said enzyme recognition domain of step (b).

8. The method of claim 7, wherein said blocking occurs through an in vitro primer extension.

9. The method of claim 8, wherein said in vitro primer extension is DNA amplification in vitro.

10. The method of claim 9, wherein said DNA amplification in vitro occurs during said amplification in step (d).

11. The method of claim 8, wherein said in vitro primer extension occurs following said amplification in step (d).

12. The method of claim 8, wherein said method further comprises hemi-methylating an enzyme recognition domain lying outside said enzyme recognition domain of step (b).

13. The method of claim 12, wherein said hemi-methylation occurs through an in vitro primer extension using a primer having a portion of said enzyme recognition domain that blocks enzyme recognition if it is hemi-methylated.

14. The method of claim 13, wherein said primer extension occurs with a methylated nucleotide.

15. The method of claim 8, wherein said restriction endonuclease recognizes a hemi-methylated recognition domain, and the primer contains at least one methylated nucleotide in a methylated portion of said recognition domain.

16. The method of claim 1 or 2, wherein said nucleic acid segment is a genomic DNA.

17. The method of claim 1 or 2, wherein said nucleic acid segment is a cDNA.

18. The method of claim 1 or 2, wherein said nucleic acid segment is a product of an in vitro DNA amplification.

19. The method of claim 1 or 2, wherein said nucleic acid segment is a PCR product.

20. The method of claim 1 or 2, wherein said nucleic acid segment is a product of a strand displacement amplification.

21. The method of claim 1 or 2, wherein said nucleic acid segment is a vector insert.

22. The method of claim 1 or 2, wherein said labeled primer comprises one or more labels selected from the group consisting of fluorescent, near infra-red, radionucleotide and chemiluminescent labels.

23. The method of claim 1 or 2, wherein said nucleic acid segment is attached to a solid matrix.

24. The method of claim 23, wherein said solid matrix is a magnetic streptavidin.

25. The method of claim 23, wherein said solid matrix is a magnetic glass particle.

26. The method of claim 1 or 2, wherein said adaptor of step (b) is attached to a solid matrix.

27. The method of claim 26, wherein said solid matrix is a magnetic streptavidin.

28. The method of claim 26, wherein said solid matrix is a magnetic glass particle.

2. A sequencing method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:

a) digesting said double stranded nucleic acid segment with a restriction enzyme whose cleavage site is separate from its recognition site, resulting in a trimmed end in said double stranded molecule;

b) providing an adaptor having a cycle identification tag and a restriction enzyme recognition domain;

c) ligating said adaptor to the trimmed end of said double stranded nucleic acid to form a ligated molecule;

d) amplifying said ligated molecule from step (c) with a labeled primer specific for said cycle identification tag and said restriction enzyme recognition domain of the adaptor, and for a nucleotide in said trimmed end in said double stranded molecule;

e) identifying said nucleotide n by identifying said primer incorporated into the amplification product; and

f) repeating steps (a) through (e) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

29. The method of claim 2, wherein said step (a) is modified to generate a blunt end in said nucleic acid segment.

30. The method of claim 29, wherein said step (b) is modified to identify a nucleotide in said blunt end of said nucleic acid segment by using a 3′

exonuclease activity of a DNA polymerase to generate a single nucleotide long single-stranded nucleic acid template.

31. The method of claim 30, said method further comprising sequencing said nucleotide by a template-directed polymerization with a labeled nucleotide or nucleotide terminator.

32. The method of claim 31, wherein said template-directed polymerization is followed by identification of an incorporated label.