Process for categorizing nucleotide sequence populations

• US 5,728,524 A
• Filed: 01/13/1995
• Issued: 03/17/1998
• Est. Priority Date: 07/13/1992
• Status: Expired due to Fees

• Current Assignee: Medical Research Council (Government of United Kingdom)
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Sibson, David Ross
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/367,266
• Time in Patent Office: 1,159 Days
• Field of Search: 435/5, 435/6, 435/91.1, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• 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 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: F02B 2075/027 four

Abstract

A process for the categorization of nucleic acid sequences in which said sequences are linked to a population of adaptor molecules each exhibiting specificity for linking to a sequence including a predetermined nucleotide base, categorization of the resulting linked sequences being based upon selection for the base.

Claims

1. A process for categorizing uncharacterized nucleic acid by sorting said nucleic acid into sequence-specific subsets comprising:

(a) optionally, cleaving said uncharacterized nucleic acid by the action of a reagent, which reagent cleaves said nucleic acid to produce smaller size cleavage products;

(b) reacting either the uncharacterized nucleic acid or said cleavage products from (a) with a first population of adaptor molecules to generate first adaptored products, each of which first adaptor molecules comprises a nucleic acid sequence end recognition means, wherein said population of first adaptor molecules comprises a range of such molecules having sequence end recognition means linking, when present, to a first predetermined subset of said uncharacterized nucleic acid sequences to form first adaptored products;

(c) selecting and separating said first adaptored products resulting from (b);

(d) cleaving adaptors from the selected and separated first adaptored products from (c) to form first selected nucleic acids;

(e) reacting said first selected nucleic acids resulting from (d) with a second population of adaptor molecules to generate second adaptored products, each of which second adaptor molecules comprises nucleic acid end recognition means, wherein said second population of adaptor molecules comprises sequence end recognition means linking to a second predetermined subset of nucleic acid sequences to form second adaptored products; and

(f) selecting and separating said second adaptored products resulting from (e).

2. A process of claim 1, wherein the cleavage reagent is an endonuclease.

3. A process of claim 1, wherein the first or second adaptor molecules include oligonucleotides in which the nucleic acid sequence end recognition means comprises a single stranded end of known nucleotide composition.

4. A process of claim 3, wherein the single stranded end exhibits complementarity to a single predetermined nucleotide.

5. A process of claim 1, wherein the first or second population of adaptor molecules includes individual molecules such that both ends of the uncharacterized nucleic acid or cleavage products can be linked thereto.

6. A process of claim 1, wherein at least some of the first and/or second adaptor molecules are adapted to be immobilized on a solid phase.

7. A process of claim 1, wherein selection of first and/or second adaptor products is based upon selecting those adaptor molecules having a single predetermined base.

8. A process of claim 1, wherein optional step (a) is effected using an endonuelease specific to double stranded nucleic acid.

9. A process of claim 8, wherein the endonuclease is selected from the group consisting of BstNI, Ddel, Hgal, Hinfl and Mnll.

10. A process of claim 1, wherein at least some of the first or second adaptor molecules also comprise a known sequence permitting hybridization with a PCR primer.

11. A process of claim 9, wherein selection of first or second adaptored products is based upon selecting products subjected to priming of nucleic acid synthesis in which a primer has a single predetermined base.

12. A process of categorizing uncharacterized nucleic acid, comprising:

(a) cleaving said uncharacterized nucleic acid using an endonuclease which has cleavage and recognition site separated to cleave said nucleic acid to produce double stranded cleavage products, the individual strands of which overlap at cleaved ends to leave a single strand extending to a known extent;

b) ligating the cleavage products from (a) with first adaptor molecules to generate first adaptoted cleavage products, each of which first adaptor molecules has a cleavage product end recognition sequence wherein the thus-used first adaptor molecules comprise a range of adaptor molecules having recognition sequences complementary to a first predetermined subset of the sequences of the cleavage generated extending single strands resulting from (a);

(c) selecting and separating the first adaptored cleavage products from (b) which carry an adaptor of first known recognition sequence;

(d) cleaving first adaptor molecules from the selected and separated products from (c) to form first selected nucleic acids;

(e) ligating the first selected nucleic acids from (d) with second adaptor molecules to generate second adaptoted cleavage products, each of which second adaptor molecules has a cleavage product end recognition sequence wherein the thus-used second adaptor molecules comprise a range of adaptor molecules having recognition sequences complementary to a second predetermined subset of the sequences of the cleavage-generated extending single strands resulting from (d); and

(f) selecting and separating the second adaptoted cleavage products from (e) which comprise an adaptor of second known recognition sequence.

13. A process of claim 12, wherein in step (b) or step (e) a number of separate reactions are performed in each of which a subset of the range of adaptor molecules is ligated.

14. A process of claim 12, wherein the uncharacterized nucleic acid is a single stranded nucleic acid which is first converted to double stranded nucleic acid.

15. A process of claim 12, wherein an endonuclease is employed in step (a) which is chosen from Class II restriction endonueleases acting on cleavage sites which are asymmetrically spaced across the two strands of a double stranded substrate, and the specificity of which is not affected by the nature of the bases adjacent to a cleavage site.

16. A process of claim 15 wherein the endonuelease is Fok 1.

17. A process of claim 15, wherein the cleavage products from (a) have extending single strands which are from 1 to 10 bases in length.

18. A process of claim 17, wherein said extending single strands resulting from (a) are from 4 to 6 bases in length.

19. A process of claim 12, wherein the first and/or second adaptor molecules have cleavage product end recognition sequences which comprise extending single strands which are complementary to the cleavage product extending single strands resulting from step (a) and/or step (e).

20. A process of claim 19, wherein at least some first and/or second adaptor molecule cleavage product end recognition sequences end with a 5'"'"' hydroxyl group.

21. A process of claim 12, wherein first or second adaptor molecule cleavage product end recognition sequences are at least three nucleotides in length with unselected random bases present at each position two or more nucleotides in from the end.

22. A process of claim 12, wherein the first or second adaptor molecules also comprise a portion permitting separation when a nucleic acid sequence is attached to its adaptor.

23. A process of claim 22, wherein the portion permitting separation includes a biotin residue.

24. A process of claim 12, wherein selection of first and/or second adaptored products is based upon selecting those first or second adaptor molecules having a single predetermined base.

25. A process of claim 12, wherein the first or second adaptor molecules also comprise a predetermined core sequence so that nucleic acid sequences which have been linked to such molecules can be amplified by the use of a primer exhibiting complementarily to the core sequence.

26. A process of claim 25, wherein selection of adaptored products in (c) and/or (f) is based upon selecting products subjected to priming of nucleic acid synthesis in which a primer has a single predetermined base.

27. A process of claim 12, wherein adaptor molecules are employed in (b) which include a range of adaptor molecules having recognition sequences complementary to all possible members of a chosen subset of cleavage products present after step (a).

28. A process of claim 12, wherein adaptor molecules are employed in (e) which include a range of adaptor molecules having recognition sequences complementary to all possible members of a chosen subset of cleavage products present after step (d).

29. A process of claim 12, wherein at least some of the first and/or second adaptor molecules carry a predetermined recognition sequence which differs from that of all other adaptors present.

30. A categorizing nucleotide sequence population kit, comprising a population of adaptor molecules in the form of double stranded oligonucleotides having a single stranded end extension as a nucleic acid sequence end recognition means and primers for use in a nucleic acid amplification process, optionally wherein at least some of the adaptor molecules includes a sequence for primer hybridization thereto, wherein the single stranded end extension of said adaptor molecules have recognition means complementary to all possible members of a chosen subset of nucleic acids to be categorized.

31. A kit as claimed in claim 30, wherein:

(a) the adaptor molecule nucleic acid sequence end recognition means recognizes a predetermined base, the presence of which base can form the basis for selection, and/or

(b) the primers include within their sequence a predetermined base, presence of which base can form the basis for selection.

32. A kit of claim 30, wherein the adaptor molecules include nucleotide sequence which, in use, not only permits indexing of nucleic acid sequences linked to said adaptor molecules but also establishes directionaiity in said nucleic acid sequences.

33. A kit of claim 30, wherein at least some of the adaptor molecules also include means permitting immobilization on a solid phase of such adaptor molecules when linked to nucleic acid sequences.

41. A kit of claim 30 further comprising of adaptor molecules, wherein the adaptor molecules comprise a nucleic acid sequence end recognition means recognizing a single predetermined base, thereby permitting categorization of nucleic acid sequences linked to said adaptor molecules on the basis of selecting for a subset in which said single predetermined base has been recognized.

34. A kit of 30, and also including a nucleic acid cleavage reagent.

35. A process for the categorization of nucleic acid sequences in which said sequences are linked to a population of adaptor molecules each exhibiting specificity for linking to a sequence including a single predetermined nucleotide base, categorization of the resulting linked sequences being based upon selection for the single predetermined nucleotide base.

36. A process of categorizing uncharacterized nucleic acid comprising:

(a) cleaving said uncharacterized nucleic acid of an endonuclease having cleavage and recognition site separated, to produce double stranded cleavage products having individual strands of which overlap at cleaved ends to leave a single strand extending to a known length;

(b) ligating the cleavage products from (a) with adaptor molecules to generate adaptored cleavage products, each of which adaptor molecules has a cleavage product end recognition sequence, wherein the thus-used adaptor molecules comprise a range of adaptor molecules having recognition sequences complementary to a predetermined subset of the sequences of the cleavage-generated extending single strands; and

(c) selecting and separating adaptored cleavage products resulting from (b) which comprise an adaptor of known recognition sequence wherein said selecting is based upon a chosen base or bases in said recognition sequence which is/are other than the entirety of said recognition sequence.

37. A process for categorizing uncharacterized nucleic acid by sorting said nucleic acid into sequence-specific subsets comprising:

(a) optionally, initially cleaving said uncharacterized nucleic acid with a reagent, which cleaves said nucleic acid to produce smaller size cleavage products;

(b) reacting either the uncharacterized nucleic acid or cleavage products from (a) with a population of adaptor molecules to generate adaptored products, each of which adaptor molecules comprises a nucleic acid sequence end recognition means, wherein said population of adaptor molecules comprise a range of such molecules having sequence end recognition means linking, when present, to a predetermined subset of nucleic acid sequences; and

(c) selecting and separating adaptored products resulting from (b) which include an adaptor of chosen nucleic acid sequence end recognition means, wherein said selecting is based upon a chosen base or bases in the nucleic acid sequence which is recognized by said nucleic acid end recognition means, which base or bases is/are other than the entirety of said recognized nucleic acid sequence.

38. A method of categorizing or sorting a mixture of uncharacterized nucleic acids into a predetermined subset of nucleic acid sequences, comprising:

linking, when present, a predetermined subset of nucleic acid sequences of said uncharacterized nucleic acids to a population of adaptor molecules, wherein each adaptor molecule contains a nucleic acid sequence end recognition means recognizing a single and same predetermined base, to form an adaptored product, thereby permitting categorization of said mixture of uncharacterized nucleic acid sequences on the basis of selecting for a subset in which said same and single predetermined base has been recognized, and

selecting and separating said adaptored product.

39. A process of claim 38, wherein the adaptor molecules are double stranded oligonucleotides having a single stranded extension portion at an end which serves as the nucleic acid sequence end recognition means.

40. A process of claim 38, wherein the adaptor molecules also include a sequence permitting hybridization with a primer for nucleic acid amplification of any nucleic acid sequence linked to the adaptor molecules.