Nucleic acid adapters containing a type IIs restriction site and methods of using the same

• US 6,027,894 A
• Filed: 01/16/1998
• Issued: 02/22/2000
• Est. Priority Date: 09/16/1994
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lipshutz, Robert J., Sapolsky, Ronald J., Gingeras, Thomas R.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/008,094
• Time in Patent Office: 767 Days
• Field of Search: 536/24.1, 536/24.2, 536/24.33, 435/91.41, 435/6
• US Class Current: 435/6.13
• 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 nucleic acid adapter comprising a type IIs endonuclease recognition sequence positioned in the adapter such that on joining the adapter to a second nucleic acid, and treating with the type IIs endonuclease, the type IIs endonuclease cuts within both strands of the second nucleic acid without cutting the adapter.

2. The adaptor of claim 1, wherein the type IIs endonuclease is selected from the group consisting of HgaI, BbvI, BspMI, BsmFI and FokI.

3. The adapter of claim 1, further comprising a primer or promoter sequence.

4. A method of isolating a fragment of a target nucleic acid, comprising:

ligating the target nucleic acid to a nucleic adapter comprising a type IIs endonuclease recognition sequence to form a recombinant nucleic acid, wherein the type IIs recognition sequence is positioned in the adapter such that on joining the adapter to the target nucleic acid, and treating with the type IIs endonuclease, the type IIs endonuclease cuts the target nucleic acid without cutting the adapter; and

cleaving the recombinant nucleic acid with the type IIs restriction endonuclease to form a further recombinant nucleic acid comprising the adapter linked to a fragment of the target.

5. The method of claim 4, wherein the type IIs endonuclease is selected from the group consisting of HgaI, BbvI, BspMI, BsmFI and FokI.

6. The method of claim 4, wherein the adapter further comprises a primer or promoter, and wherein the method further comprises amplifying the further nucleic acid.

7. The method of claim 4, further comprising hybridizing the further recombinant nucleic acid to an array of probes.

8. The method of claim 4, wherein each of a population of target nucleic acids are joined to the adapter, and the cleaving step forms a population of further nucleic acids.

9. The method of claim 8, further comprising hybridizing the population of further nucleic acids to an array of probes to generate a hybridization pattern.

10. The method of claim 9, further comprising repeating the joining, cleaving and hybridizing steps with a second population of differentially generated target sequences to generate a second hybridization pattern, and comparing the hybridization pattern and the second hybridization pattern.