Oligonucleotide constructs and methods for the generation of sequence signatures from nucleic acids

• US 5,962,221 A
• Filed: 06/09/1995
• Issued: 10/05/1999
• Est. Priority Date: 01/19/1993
• Status: Expired due to Fees

• Current Assignee: The University Of Tennessee Research Corporation (University of Tennessee)
• Original Assignee: The University Of Tennessee Research Corporation (University of Tennessee)
• Inventors: Caetano-Anolles, Gustavo
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/489,269
• Time in Patent Office: 1,579 Days
• Field of Search: 435/5, 435/6, 435/91.1, 435/91.2, 435/810, 536/22.1, 536/23.1, 536/24.1, 536/24.3-24.33, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2535/139 Random amplification polymorphism detection [RAPD]
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Novel oligonucleotides for amplification and profiling of nucleic acid templates are disclosed. Enhancements of nucleic acid fingerprinting methods are disclosed.

Claims

1. An oligonucleotide construct comprising a structure selected from the group consisting of (B_x)_n (N)_m and (N)_m (B_x)_n, wherein B is A, T, G, or C and wherein each B may be the same as or different than any other B in the construct so long as at least one B differs from another B, x is a whole number from 2 to 5, n is a whole number from 2 to 10, N is a degenerate base, and m is a whole number from 1 to 10.

2. The construct of claim 1 wherein n is 3 to 6.

3. The construct of claim 1 wherein m is 2 to 5.

4. The construct of claim 3 wherein m is 2 or 3.

5. The construct of claim 1 which further comprises an arbitrary sequence having the formula (R)_y, wherein R is selected from the group consisting of C, G, T, and A, and y is a whole number from 1 to 25, and which arbitrary sequence (R)_y is connected to (B_x)_n, (N)_m, or both (B_x)_n and (N)_m, or is within (B_x)_n or (N)_m.

6. The construct of claim 5 wherein y is 2 to 10.

7. An oligonucleotide mini-hairpin primer having a 3'"'"' single stranded end and a 5'"'"' end which primer comprises a double stranded stem of at least 2 complementary annealed base pairs, the double stranded stem extending to the 5'"'"' end of the primer and the other end of the stem extending as the single strand of at least 3 bases to the 3'"'"' end of the primer, and intermediate to the 5'"'"' and 3'"'"' ends of the stem, a single stranded loop of at least 3 bases.

8. The primer of claim 7 wherein the loop consists of 3 or 4 bases.

9. The primer of claim 7 wherein the stem consists of 2 base pairs.

10. The primer of claim 7 wherein the base pairs are only GC base pairs.

11. The primer of claim 7 wherein the single strand consists of 3 to 8 bases.

12. An oligonucleotide primer having a 3'"'"' single stranded end and a 5'"'"' end which primer comprises a double stranded stem of 2 complementary annealed base pairs, the double stranded stem extending to the 5'"'"' end of the primer and the other end of the stem extending as the single strand of at least 3 bases to the 3'"'"' end of the primer, and intermediate to the 5'"'"' and 3'"'"' ends of the stem, a single stranded loop of 3 or 4 bases.

13. The oligonucleotide primer of claim 12 wherein the loop has 3 bases.

14. The oligonucleotide primer of claim 12 wherein the loop has 4 bases.

15. The oligonucleotide primer of claim 12 wherein one of the bases of the loop is A.

16. The oligonucleotide primer of claim 12 wherein the length of the single strand is of 3 to 8 nucleotides.

17. The oligonucleotide primer of claim 12 which is a deoxyribonucleotide sequence.

18. The oligonucleotide primer of claim 12 which is a ribonucleotide sequence.

19. The oligonucleotide primer of claim 12 wherein the double stranded stem has two complementary GC pairs.

20. The oligonucleotide primer of claim 13 wherein the nucleotides of the loop are GAA or GTT.

21. The oligonucleotide primer of claim 18 wherein the nucleotides of the loop are GAAA.

22. The oligonucleotide primer of claim 14 which is a DNA primer, and wherein the loop comprises a nucleotide sequence selected from the group consisting of GAA, GTTA, and GNAA, wherein N is A, G, C, and T.

23. The oligonucleotide primer of claim 22 wherein the loop comprises the nucleotide sequence GNAA, wherein N is G, A, C, and T.

24. The oligonucleotide primer of claim 13 wherein the single strand comprises a sequence of nucleotides selected from the group consisting of CTG, GCTG, NCTG, GAGCTG, NAGCTG, and CCGAGCTG, wherein N is A, G, C, and T.

25. The oligonucleotide primer of claim 24 wherein N is G.

26. The oligonucleotide primer of claim 14 wherein the single strand has a sequence selected from the group consisting of GCTG and GAGCTG.

27. The oligonucleotide primer of claim 12 wherein the primer has an extended 5'"'"' end.

28. The oligonucleotide primer of claim 12 wherein the primer has a sequence selected from the group consisting of GCGAAGCGCTG, GCGAAGCGAGCTG, and GCGAAGCCCGAGCTG.

29. The oligonucleotide primer of claim 12 wherein one or more nucleotides at the 3'"'"' or 5'"'"' end of the primer has been replaced by a base selected from the group consisting of hypoxanthine, 6H,8H03,4-dihydropyrimido(4,5-c) (1,2)oxazin-7-one, and 2-amino-6-methoxyaminopurine.

30. A combination of a multiplicity of the oligonucleotide primers of claim 12, wherein the 3'"'"' single stranded end extending from the double stranded stem comprises 3 nucleotides, wherein the nucleotides constitute combinations of the known 4 bases of DNA, and wherein the portion of the oligonucleotide primers other than the 3'"'"' single stranded end extending from the double stranded stem contain identical sequences in each of the primers.

32. The primer of claim 12 which is annealed to a DNA template.

33. The primer of claim 12 wherein the first three bases of the 3'"'"' end are determinative to produce reproducible fingerprints.

34. The primer of claim 33 wherein the three bases at the 3'"'"' end of the primer are arbitrary.

31. An oligonucleotide primer having a 3'"'"' single stranded end and a 5'"'"' end which primer comprises a double stranded stem of at least 2 complementary annealed base pairs, the double stranded stem extending to the 5'"'"' end of the primer and the other end of the stem extending as the single strand of at least 3 bases to the 3'"'"' end of the primer, and intermediate to the 5'"'"' and 3'"'"' ends of the stem, a single stranded loop.

35. The oligonucleotide primer of claim 31 wherein the loop has 3 or 4 bases, wherein at least one of the bases is A.

36. An oligonucleotide primer having a 3'"'"' single stranded end and a 5'"'"' end which primer comprises a double stranded stem of at least 2 complementary annealed base pairs, the double stranded stem extending to the 5'"'"' end of the primer and the other end of the stem extending as the single strand of at least 3 bases to the 3'"'"' end of the primer, and intermediate to the 5'"'"' and 3'"'"' ends of the stem, a single stranded loop.

37. The primer of claim 36 wherein the first three bases of the 3'"'"' end are determinative to produce reproducible fingerprints.

38. The primer of claim 37 wherein the three bases at the 3'"'"' end of the primer are arbitrary.

39. The oligonucleotide primer of claim 36 wherein the loop has 3 or 4 bases, wherein at least one of the bases is A.