Nucleic acid indexing

• US 6,228,999 B1
• Filed: 05/07/1999
• Issued: 05/08/2001
• 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: Guo, Zhen, Guilfoyle, Richard A.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/307,392
• Time in Patent Office: 732 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/25.3, 536/24.33
• US Class Current: 536/22.1
• 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.

Claims

1. A set of adaptors, each adaptor being at least partially double-stranded and comprising at least one single-stranded terminus having an end, the single-stranded terminus characterized by, in non-overlapping order inward from the end:

(a) an indexing sequence n bases long contiguous with (b) a sequence characteristic of cleavage by a Class II restriction endonuclease, wherein n is an integer, and wherein the set comprises indexing sequences that can base pair to all the sequences said n bases long that can be formed by the bases A, C, G and T.

2. A set of adaptors according to claim 1, wherein n is 1, 2, 3, 4 or 5.

3. A set of adaptors according to claim 2, wherein n is 2, 3 or 4.

4. A set of adaptors according to claim 1, wherein the bases are A, C, G and T.

5. A set of adaptors according to claim 1, wherein the bases are selected from the group consisting of A, C, G, T and modified bases.

6. A set of adaptors according to claim 1, comprising adaptors with all the indexing sequences said n bases long that can be formed by the bases A, C, G and T.

7. A set of adaptors according to claim 6, wherein n is 1, 2, 3, 4 or 5.

8. A set of adaptors according to claim 7, wherein n is 2, 3 or 4.

9. A set of adaptors according to claim 1, comprising adaptors with all the indexing sequences said n bases long that can be formed by the bases A, C, G, T and a nucleoside base analog other than A, C, T or G that can form specific base-pairs with A, C, G or T in DNA.

10. A set of adaptors as claimed in claim 1, wherein the adaptors in the set are grouped into one or more kits in one or more containers.

11. A set of adaptors as claimed in claim 10, wherein the sequence characteristic of cleavage by a Class II restriction endonuclease is selected from the group consisting of a sequence having a 5′

overhang and a sequence having a 3′

overhang.

12. A set of adaptors as claimed in claim 11, wherein the Class II restriction endonuclease is selected from the group consisting of BclI, NotI, DpnII, BamHI, HindIII, AvrII, ApaLI, KpnI, SphI, NsiI, and SacI.

13. A set of adaptors as claimed in claim 11, wherein n is 1, 2, or 3.

14. A set of adaptors as claimed in claim 9, wherein the base-pairing specificity of each base of the adaptor indexing sequences is selected from the group consisting of A, C, G, T, Py, Pu and N, wherein Py denotes base-pairing to A and G, Pu denotes base-pairing to C and T and N denotes base-pairing to A, C, G and T.

15. A set of adaptors as claimed in claim 14, wherein N is the modified base 3′

-nitropyrrole or the modified base 5′

-nitroindole.

16. A set of adaptors, each adaptor comprising at least one single-stranded terminus having an end, the single-stranded terminus characterized by, in non-overlapping order inward from the end:

(a) an indexing sequence n bases long contiguous with (b) a sequence characteristic of cleavage by a Class II restriction endonuclease, wherein n is an integer, and wherein the set comprises indexing sequences that can base pair to all the sequences said n bases long that can be formed by the bases A, C, G and T and wherein at least one base in one or more of the indexing sequences is a nucleoside base analog other than A, C, T or G that can form specific base-pairs with A, C, G or T in DNA.

17. A set of adaptors as claimed in claim 16, wherein the base-pairing specificity of each base of the adaptor indexing sequences is selected from the group consisting of A, C, G, T, Py, Pu and N, wherein Py denotes base-pairing to A and G, Pu denotes base-pairing to C and T and N denotes base-pairing to A, C, G and T.

18. A set of adaptors as claimed in claim 17, wherein N is the modified base 3′

-nitropyrrole or the modified base 5′

-nitroindole.