Capturing sequences adjacent to Type-IIs restriction sites for genomic library mapping

• US 5,710,000 A
• Filed: 06/07/1995
• Issued: 01/20/1998
• 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): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/485,606
• Time in Patent Office: 958 Days
• Field of Search: 435/6, 435/172.3, 435/18
• 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 method of identifying sequences in a polynucleotide sequence, comprising:

(a) cleaving the polynucleotide sequence with a first type-IIs endonuclease;

(b) ligating a first adapter sequence to the polynucleotide sequence cleaved in step (a), said first adapter having a recognition site for a second type-IIs endonuclease, the recognition site being oriented so that the second type-IIs endonuclease has a cleavage site in the polynucleotide sequence;

(c) cleaving the polynucleotide sequence resulting from step (b), with the second type-IIs endonuclease;

(d) ligating a second adapter sequence to the polynucleotide sequence cleaved in step (c); and

determining the sequence of nucleotides of the polynucleotide sequence between the first and second adapter sequences.

2. 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; and

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

3. The method of claim 2, wherein

the first type-IIs endonuclease is EarI;

and

the second type-IIs endonuclease is HgaI.

4. The method of claim 1, wherein said first and second adapter sequences comprise primer sequences.

5. The method of claim 4, wherein prior to said determining step, the sequence of nucleotides in the polynucleotide between the first and second adapter sequences is amplified.

6. The method of claim 1, wherein the sequence of nucleotides between the first and second adapter sequences is determined by hybridization to an oligonucleotide probe.

7. The method of claim 6, 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.

8. A method of generating an ordered map of a library of polynucleotide fragments, the method comprising:

identifying sequences in each of the polynucleotide fragments in the library, according to the method of claim 1;

comparing the sequences identified in each fragment with the sequences identified in each other fragment to obtain a level of correlation between each fragment and each other fragment; and

ordering the fragments according to their level of correlation.

9. A method of identifying polymorphisms in a target polynucleotide sequence, the method comprising:

identifying sequences in a wild-type polynucleotide sequence, according to the method of claim 1,

repeating said identifying step on the target polynucleotide sequence; and

determining differences in the sequences identified in each of said identifying steps, the differences being indicative of a polymorphism.

10. The method of claim 1, wherein said sequences in a polynucleotide sequence are adjacent to a polymorphism, said polymorphism resulting in a presence of a type-IIs restriction endonuclease recognition site in said polynucleotide sequence.

11. A method of identifying a source of a biological sample, the method comprising:

identifying a plurality of sequences in a polynucleotide sequence derived from the sample, according to the method of claim 1; and

comparing the plurality of sequences identified in said identifying step with a plurality of sequences identically identified from a polynucleotide derived from a known source, identity of the plurality of sequences identified from the sample with the plurality of sequences identified from the known source being indicative that the sample was derived from the known source.