Methods for analyzing nucleic acids using a type IIs restriction endonuclease

• US 6,509,160 B1
• Filed: 08/27/2001
• Issued: 01/21/2003
• Est. Priority Date: 09/16/1994
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Sapolsky, Ronald J., Lipshutz, Robert J., Gingeras, Thomas R.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/940,868
• Publication Number:
  US 20030008292A1
• Time in Patent Office: 512 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/196
• US Class Current: 506/9
• 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 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• 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 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention relates to novel methods for sequencing and mapping genetic markers in polynucleotide sequences using Type-IIs restriction endonucleases. The methods herein described result in the “capturing” and determination of specific oligonucleotide sequences located adjacent to Type-IIs restriction sites. The resulting sequences are useful as effective markers for use in genetic mapping, screening and manipulation.

Claims

1. A method for identifying differences between a first and second nucleic acid sample, comprising:

fragmenting the samples with a first type IIs endonuclease;

(a) ligating to the fragments a first adapter sequence comprising a second type-IIs endonuclease recognition site;

(b) fragmenting the samples with a third endonuclease and ligating a second adapter sequence to the fragments;

(c) amplifying the fragments;

(d) marking the amplified fragments from the first sample;

(e) mixing the first and second samples under conditions that will allow heteroduplex formation between sequences in the first and second samples;

(f) selectively degrading one strand from heteroduplexes which contain a mismatch and isolating the remaining single-stranded fragments;

(g) creating double-stranded DNA from the single-stranded fragments and amplifying the double-stranded DNA;

(h) cleaving the DNA with the second type-IIs endonuclease; and

determining the sequence of polynucleotides between the first and second type-IIs endonuclease recognition sites.

2. The method of claim 1, wherein the first and second adapter sequences comprise primer sequences.

3. The method of claim 1, wherein the amplified fragments from the first sample are marked by methylation.

4. The method of claim 1, wherein the first type IIs endonuclease is selected from the group consisting of BsmAI, EarI, MnlI, PleI, AlwI, BbsI, BsaI, BspMI, Esp3I, HgaI, SapI, SfaNI, BseRI, HphI and MboII;

the second type-IIs endonuclease is selected from the group consisting of HgaI, BbvI, BspMI, BsmFI and FokI; and

the third endonuclease cleaves more frequently than the first type-IIs endonuclease.

5. The method of claim 4, wherein the first type IIs endonuclease is BseRI;

the second type-IIs endonuclease is Fok; and

the third endonuclease is HaeIII.

6. The method of claim 1, wherein prior to said determining step a third adapter sequence comprising a primer is ligated to the cleavage product of step (i) and the sequence of nucleotides in the polynucleotide between the first and third adapter sequences is amplified.

7. The method of claim 1, wherein the sequence of nucleotides between the first and second type-IIs endonuclease recognition sites is determined by hybridization to an oligonucleotide probe.

8. The method of claim 7, wherein said oligonucleotide probe is a positionally distinct probe on an oligonucleotide array, a position of the probe being indicative of the sequence of the probe.

9. The method of claim 1, wherein the mixture of step (f) is treated with a mismatch repair enzyme and an exonuclease.

10. The method of claim 9, wherein the mismatch repair enzyme is MutLSH and the exonuclease is ExoIII.

11. A method of reducing the complexity of a nucleic acid sample comprising:

fragmenting the sample using a first type-IIs restriction enzyme to produce fragments;

ligating a first adapter sequence to at least some of the fragments;

fragmenting the samples with an endonuclease;

ligating a second adapter sequence to at least some of the fragments; and

amplifying at least some of the fragments.

12. The method of claim 11, wherein the first and second adapter sequences comprise primer sequences.

13. The method of claim 11, wherein the first type-IIs endonuclease is selected from the group consisting of BsmAI, EarI, MnlI, PleI, AlwI, BbsI, Bsal, BspMI, Esp3I, HgaI, SapI, SfaNI, BseRI, HphI, MboII, HgaI, BbvI, BspMI, BsmFI and FokI.

14. The method of claim 11, wherein the endonuclease is a type IIs endonuclease.

15. The method of claim 14, wherein the endonuclease is selected from the group consisting of BsmAI, EarI, MnlI, PleI, AlwI, BbsI, BsaI, BspMI, Esp3I, HgaI, SapI, SfaNI, BseRI, HphI, MboII, HgaI, BbvI, BspMI, BsmFI and FokI.

16. A method of reducing the complexity of a nucleic acid sample comprising:

fragmenting the sample using a type-IIs restriction enzyme to produce fragments;

ligating an adapter sequence to at least some of the fragments; and

amplifying at least some of the fragments.

17. The method of claim 16, wherein the type-IIs endonuclease is selected from the group consisting of BsmAI, EarI, MnlI, Plel, AlwI, BbsI, BsaI, BspMI, Esp3I, HgaI, SapI, SfaNI, BseRI, HphI, MboII, HgaI, BbvI, BspMI, BsmFI and FokI.

18. The method of claim 16, wherein the adapter sequence comprises a primer sequence.