Restriction enzyme mediated adapter

• US 6,258,539 B1
• Filed: 04/30/1999
• Issued: 07/10/2001
• Est. Priority Date: 08/17/1998
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: The Perkin-Elmer Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Richards, John H., Hunkapiller, Michael W.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Taylor, Janell E.
• Application Number:
  US09/303,774
• Time in Patent Office: 802 Days
• Field of Search: 435/6, 435/5, 435/7, 435/91.1, 435/91.2, 436/501, 536/26, 536/27, 536/28, 536/77, 536/78, 514/100
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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 2525/161 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

Abstract

The present invention relate to methods and compositions for simultaneously analyzing multiple different polynucleotides of a polynucleotide composition comprising multiple diverse polynucleotide sequences. The subject methods and compositions may also be applied to analyze or identify single polynucleotides; however, the subject methods and compositions are particularly useful for analyzing large diverse populations of polynucleotides, e.g., cDNA libraries. Most embodiments of the invention involve hybridizing terminus probes (of known base sequence) to adapter-modified restriction fragment generated from polynucleotide for analysis, and subsequently joining the terminus probes and internal fragment probes to each other. The terminus probe hybridizes to bases of restriction endonuclease recognition site present at the terminus of a restriction fragment generated from the polynucleotide for analysis. The terminus probes and internal fragment probes may be marked so as to facilitate the simultaneous testing of multiple polynucleotides for the presence of many possible nucleotide base sequences. The identity or expression of a particular polynucleotide of interest may be ascertained (or at least partially determined) by producing a short identifier sequence derived from the nucleotide base sequence information obtained from (1) the hybridization of a terminus probe, and (2) the recognition site of a restriction endonuclease used to generate the polynucleotide molecule of interest. Multiple identification sequences may be obtained in parallel, thereby permitting the rapid characterization of a large number of diverse polynucleotides. Parallel processing may be achieved by differentially marking terminus probes or internal fragment probes. Parallel processing may be achieved by using ordered arrays of oligonucleotides that are terminus probes.

Claims

1. A method of analyzing a polynucleotide, said method comprising

forming a restriction fragment from the polynucleotide, wherein the restriction fragment has a first and second terminus and at least one of the termini is generated by a restriction endonuclease,

joining a first adapter to a terminus of the restriction fragment, whereby an adapter-modified restriction fragment is produced, and

hybridizing a terminus probe to a single strand of the adapter-modified restriction fragment at a position including the terminus generated by the restriction endonuclease, wherein the terminus probe has a constant and a variable region.

2. The method according to claim 1, comprising,

combining (i) the nucleotide sequence information from the terminus probe with (ii) the nucleotide sequence of the recognition site of the restriction endonuclease used to produce the terminus, so as to produce an identifier sequence.

3. The method according to claim 1 wherein the polynucleotide is a cDNA.

4. The method of claim 1, wherein the polynucleotide is derived from genomic. DNA.

5. The method of claim 3, wherein the restriction fragment is a representative restriction fragment.

6. The method of claim 1, wherein the identifier sequence comprises an SNP.

7. A method according to claim 1, wherein the terminus probe is a feature of an oligonucleotide array.

8. The method of claim 6, wherein the variable region of the terminus probes are 1 to 10 nucleotides in length.

9. The method of claim 1, said method further comprising the step of extending the terminus probe.

10. The method of claim 9, wherein the terminus probe is extended with a chain-terminating nucleotide.

11. The method of claim 10, wherein the chain-terminating nucleotide is fluorescently labeled.

12. The method of claim 10, said method comprising,

combining (i) the nucleotide sequence information from the terminus probe with, (ii) the nucleotide sequence of the recognition site of the restriction endonuclease used to produce the terminus, so as to produce an identifier sequence, and (iii) the base information from a chain terminating nucleotide incorporated in the chain extension reaction.

13. A method according to claim 1, wherein the terminus probe comprises an array sorting signal.

14. The method of claim 13, further comprising the step of extending the terminus probe with a chain-terminating nucleotide.

15. The method of claim 14, wherein the chain-terminating nucleotide is fluorescently labeled.

16. The method of claim 11 further comprising contacting the array-sorting signal with a sorting signal receptor array.

17. The method of claim 13, wherein the array sorting signal is a polynucleotide.

18. The method of claim 16, further comprising the step of extending the terminus probe with a chain-terminating nucleotide, wherein the extension takes place on a terminus probe that is bound to the sorting signal array.

19. The method of claim 13, said method comprising,

combining (i) the nucleotide sequence information from the terminus probe with, (ii) the nucleotide sequence of the recognition site of the restriction endonuclease used to produce the terminus, so as to produce an identifier sequence, and (iii) the base information from a chain terminating nucleotide incorporated in the chain extension reaction.

20. A method according to claim 5, wherein the representative restriction fragment is generated by a method comprising the steps,

immobilizing the polynucleotide on a solid support,

contacting the polynucleotide with a first restriction endonuclease, whereby an immobilized restriction fragment is produced, and

purifying the immobilized restriction fragment.

21. The method of claim 20, further comprising,

contacting the immobilized restriction fragment with a second restriction endonuclease, whereby the representative restriction fragment is produced, and purifying the representative restriction fragment.

22. The method according to claim 21, further comprising,

joining a linker to the terminus produced by the first restriction enzyme on the immobilized restriction fragment, whereby an adapter-modified immobilized restriction fragment was produced, and

contacting the adapter-modified immobilized restriction fragment with a second restriction endonuclease, whereby the representative restriction fragment is produced.

23. The method of claim 24, wherein the adapter comprises a type IIs restriction site and the second restriction endonuclease is a type IIs restriction endonuclease recognizes the type IIs restriction site in the adapter and cleaves within the immobilized restriction fragment.

24. The method of claim 1, said method further comprising,

joining a second adapter to the second terminus of the restriction fragment, amplifying the restriction fragment, wherein the amplification process uses a first amplification primer that anneals to the first adapter and a second amplification primer that anneals to the second adapter, wherein the amplification process takes place prior to the hybridization of the terminus probe.

25. The method of claim 24, wherein the at least one of the amplification primers is a selective primer.

26. The method of claim 25, wherein the first and second primers are selective primers.

27. The method of claim 1, wherein the polynucleotide for analysis is produced by amplifying a portion of a cDNA preparation or a portion of a genomic DNA preparation.