Nucleic acid indexing

• US 6,280,948 B1
• Filed: 09/03/1999
• Issued: 08/28/2001
• Est. Priority Date: 03/11/1997
• Status: Expired due to Fees

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Guilfoyle, Richard A., Guo, Zhen
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): SIU, STEPHEN C
• Application Number:
  US09/380,786
• Time in Patent Office: 725 Days
• Field of Search: 435/6, 435/5, 435/91.1, 536/24.3, 536/24.2
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags

Abstract

A restriction site indexing method for selectively amplifying any fragment generated by a Class II restriction enzyme includes adaptors specific to fragment ends containing adaptor indexing sequences complementary to fragment indexing sequences near the termini of fragments generated by Class II enzyme cleavage A method for combinatorial indexing facilitates amplification of restriction fragments whose sequence is not known. Profiling methods and other methods for characterizing polynucleotides are presented.

Claims

1. A method for indexing polynucleotides, comprising the steps of:

(A) combining under base-pairing conditions;

(i) one or more distinguishable sets of indexing adaptors, each adaptor comprising a sequence for amplification and at least one terminus having a single-stranded region characterized by, in non-overlapping order inward from the end;

(a) an indexing sequence n bases long and (b) a sequence characteristic of cleavage by a Class II restriction endonuclease, wherein n is an integer and wherein further each set of adaptors comprises one or more indexing sequences, and

(ii) one or more corresponding polynucleotides, each of which comprises at least one terminus characterized by, in non-overlapping order inward from the end;

(a) a region having a sequence characteristic of cleavage by a Class II restriction endonuclease and (b) a double-stranded region having on one strand a sequence that base-pairs with an indexing sequence of an adaptor and on the other strand a sequence complementary thereto;

(B) base-pairing at least one adaptor terminus and at least one corresponding polynucleotide terminus to form at least one strand-displaced structure wherein the indexing sequence of the single-stranded region of the adaptor terminus is base-paired with the sequence that base-pairs with an indexing sequence of the terminus of the corresponding polynucleotide, and the complementary sequence on the other strand of the polynucleotide terminus is displaced from base-pairing thereto;

(C) for each adaptor set, amplifying the corresponding polynucleotides that form strand-displaced structures using the sequence for amplification; and

(D) distinguishing the amplified corresponding polynucleotides thereby indexing the polynucleotides by their base-pairing to the distinguishable sets of adaptors.

2. A method according to claim 1, wherein n is 1 to 4.

3. A method according to claim 2 wherein n is 2, 3 or 4.

4. A method according to claim 1, wherein the indexing adaptors in all the sets of adaptors together comprise all possible sequences of A, T, G and C n bases long.

5. A method according to claim 1, wherein the sequence for amplification is selected from a group consisting of a PCR primer, a T3 promoter, a T7 promoter, an SP6 promoter, and a sequence complementary to any of the same.

6. A method according to claim 1, wherein the amplification is an exponential amplification.

7. A method according to claim 1, further comprising the step of ligating, in a strand-displaced structure, the end of the adaptor strand comprising the indexing sequence to the end of the abutting strand of the double-stranded region of the corresponding polynucleotide.

8. A method according to claim 7, wherein strand-displaced structures are formed at both ends of at least one corresponding polynucleotide.

9. A method according to claim 8, wherein the sequence for amplification is a PCR primer.

10. A method according to claim 1, wherein the sequence characteristic of cleavage by a Class II restriction endonuclease has a 3′

-terminated single-stranded region.

11. A method according to claim 1, wherein the sequence characteristic of cleavage by a Class II restriction endonuclease has a 5′

-terminated single-stranded region.

12. A method according to claim 1,, wherein the sequence characteristic of cleavage by a Class II restriction endonuclease has a blunt end.

13. A method for characterizing a population of polynucleotides, comprising the steps of:

(A) combining under base-pairing conditions;

(i) one or more distinguishable sets of indexing adaptors, each adaptor comprising a sequence for amplification and at least one terminus having a single-stranded region characterized by, in non-overlapping order inward from the end;

(a) an indexing sequence n bases long and (b) a sequence characteristic of cleavage by a Class II restriction endonuclease, wherein n is an integer and wherein further each set of adaptors comprises one or more indexing sequences, and

(ii) a population of polynucleotides that includes one or more corresponding polynucleotides, each of which comprises at least one terminus characterized by, in non-overlapping order inward from the end;

(a) a region having a sequence characteristic of cleavage by a Class II restriction endonuclease and (b) a double-stranded region having on one strand a sequence that base-pairs with an indexing sequence of an adaptor and on the other strand a sequence complementary thereto;

(B) base-pairing at least one adaptor terminus to at least one corresponding polynucleotide terminus to form at least one strand-displaced structure wherein the indexing sequence of the single-stranded region of the adaptor terminus is base-paired with the sequence that base-pairs with an indexing sequence of the terminus of the corresponding polynucleotide, and the complementary sequence on the other strand of the polynucleotide terminus is displaced from base-pairing thereto;

(C) for each adaptor set, amplifying corresponding polynucleotides that form strand-displaced structures using the sequence for amplification;

(D) determining one or more amplified corresponding polynucleotides or the absence thereof; and

(E) characterizing the population of polynucleotides by the amplified corresponding polynucleotides that form strand-displaced structures using the sequence for amplification, or the absence thereof.

14. A method according to claim 13, wherein n is 1 to 5.

15. A method according to claim 13, wherein the population is a population of cDNAs or other polynucleotides representative of mRNAs.

16. A method according to claim 15, wherein the characterization is indicative of gene expression in a sample from which the population was derived.

17. A method according to claim 13, wherein the population is a population of genomic DNAs or polynucleotides representative of genomic DNAs.

18. A method according to claim 17, wherein the characterization comprises characterizing mutations or the absence thereof in:

(a) the sequence characteristic of cleavage by a Class II restriction endonuclease;

(b) the indexing sequence or both (a) and (b) of one or more corresponding polynucleotides in the population.

19. A method according to claim 18, wherein the absence or presence of a mutation thus characterized in one or more corresponding polynucleotides is diagnostic of a potential to develop one or more diseases, or of one or more diseases.

20. A method according to claim 13, further comprising the step of ligating, in a strand-displaced structure, the end of the adaptor-strand comprising the indexing sequence to the end of the abutting strand of the double-stranded region of the corresponding polynucleotide.

21. A method according to claim 20 wherein strand-displaced structures are formed at both ends of at least one corresponding polynucleotide.

22. A method according to claim 21, wherein the sequence for amplification is a PCR primer.

23. A method according to claim 22, further comprising the step of amplifying by PCR corresponding polynucleotides having adaptor strands ligated to each terminus, using the sequences for amplification either as a primer or as a primer binding site.

24. A method according to claim 13, further comprising, for at least one adaptor set, resolving from one another at least two corresponding polynucleotides that form strand-displaced structures.

25. A method according to claim 24, wherein the population is a population of cDNAs or other polynucleotides representative of mRNAs in a sample, the size and the quantity of the separated corresponding polynucleotides is determined, each separated corresponding polynucleotide is identified by the indexing sequence and the Class II restriction endonuclease characteristic sequence of the adaptor set with which it formed a strand-displaced structure and by its size, and the quantities of the thus identified corresponding polynucleotides for at least two adaptor sets provides a profile of gene expression in the source from which the cDNA was derived.

26. A method according to claim 25, wherein corresponding polynucleotides for at least one set of adaptors are resolved by size by electrophoresis.

27. A method according to claim 25, wherein the sequences characteristic of a Class II restriction endonuclease and the indexing sequences of the adaptor sets together subdivide the cDNAs or other polynucleotides representative of the mRNAs into sets of corresponding polynucleotides in each of which corresponding polynucleotides can be individually differentiated by electrophoresis.

28. A method for comparing mRNA in two or more samples, comprising the steps of claim 25 to generate a profile of gene expression of a first sample and, independently, a profile of gene expression in a second sample and comparing the profiles of the first and second samples.

29. A method according to claim 13, wherein the sequence for amplification is selected from a group consisting of a PCR primer, a T3 promoter, a T7 promoter, an SP6 promoter, and a sequence complementary to same.