Indexing linkers

• US 5,508,169 A
• Filed: 10/28/1994
• Issued: 04/16/1996
• Est. Priority Date: 04/06/1990
• Status: Expired due to Fees

• Current Assignee: Atomic Energy of Canada Limited (Government of Canada), Queen's University At Kingston
• Original Assignee: Atomic Energy of Canada Limited (Government of Canada), Queen's University At Kingston
• Inventors: Deugau, Kenneth V., Unrau, Paul
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/331,152
• Time in Patent Office: 536 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 536/23.1, 536/24.33, 536/25.3, 935/1, 935/8, 935/16, 935/76, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Synthetic nucleic acid molecules are provided which are called indexing linkers and which can be selectively linked to unknown, or non-identical cohesive ends of nucleic acid fragments. Such indexing linkers are useful for the selective isolation, identification, amplification, labelling, and modification, of nucleic acid fragments, especially subsets of such fragments released by cleavage using Type IIS restriction endonucleases or restriction endonucleases recognizing interrupted palindromic sequences. Sets of such indexing linker molecules are described which are useful for indexing fragments released by cleavage of a genome. Methods are described for distinguishing such fragments using said indexing linker molecules for effecting detection, or further modification of such fragments; and for amplifying such fragments by the polymerase chain reaction.

Claims

1. A method for obtaining indexed DNA fragments from a mixture of DNA fragments, for identifying, isolating, mapping, amplifying, or sequencing said fragments, said method comprising:

(i) treating DNA with a restriction endonuclease which is selected from the group consisting of Type IIS restriction endonucleases and restriction endonucleases which recognize interrupted palindromic sequences, to produce DNA fragments having at least one 3'"'"'- or 5'"'"'-protruding single strand;

(ii) adding said DNA fragments to a set of indexing linkers;

each said indexing linker being a DNA duplex having one 3'"'"'- or 5'"'"'-protruding single strand of a length corresponding to the length of the 3'"'"'- or 5'"'"'-protruding single strand of the cleavage site of said restriction endonuclease, wherein said set comprises a collection of said indexing linkers whose 3'"'"'- or 5'"'"'-protruding single strands collectively encode up to all possible permutations and combinations of nucleotides, A, C, G and T, and wherein each indexing linker in said set is physically separated from every other indexing linker in said set;

(iii) contacting said DNA fragments added to said set of indexing linkers with a DNA ligase to cause a selective ligation of those indexing linkers whose 3'"'"'- or 5'"'"'-protruding single strands are fully complementary to the 3'"'"'- or 5'"'"'-protruding single strands of the DNA fragments; and

(iv) obtaining indexed DNA fragments.

2. The method of claim 1, wherein said process further comprises a step (v) of identifying an indexed DNA fragment.

3. The method of claim 1, wherein said process further comprises a step (v) of isolating an indexed DNA fragment.

4. The method of claim 1, wherein said process further comprises a step (v) of mapping an indexed DNA fragment.

5. The method of claim 1, wherein said process further comprises a step of (v) subjecting an indexed DNA fragment to an amplification reaction to obtain the selective amplification of said DNA fragment.

6. The method of claim 5, wherein said amplification is preformed by cloning.

7. The method of claim 5, wherein said amplification is preformed by the polymerase chain reaction.

8. The method of claim 5, wherein said process further comprises a step (vi) of sequencing said amplified DNA fragment.

9. The method of claim 1, wherein said indexing linkers are immobilized.

10. The method of claim 1, wherein said indexing linkers are covalently labelled with a fluorescent tag.

11. The method of claim 1, wherein said indexing linkers are covalently labelled with a radioisotope label.

12. The method of claim 1, wherein said indexing linkers are covalently labelled with an affinity ligand.

13. A method for obtaining indexed DNA fragments from a mixture of DNA fragments, for identifying, isolating, mapping, amplifying, or sequencing said fragments, said method comprising:

(i) treating DNA with a restriction endonuclease which is a member selected from the group consisting of Type IIS restriction endonucleases and restriction endonucleases which recognize interrupted palindromic sequences, to produce DNA fragments having at least one 3'"'"'- or 5'"'"'-protruding single strand;

(ii) adding said DNA fragments to two sets of indexing linkers, wherein;

each member of a set of indexing linkers is a common DNA duplex having one 3'"'"'- or 5'"'"'-protruding single strand of a length corresponding to the length of the 3'"'"'- or 5'"'"'-protruding single strand of the cleavage site of said restriction endonuclease, said 3'"'"'- or 5'"'"'-protruding single strand of the member of a set encoding collectively up to all permutations and combinations of nucleotides, A, C, G and T;

said members of a set being physically separated from each other;

wherein the common DNA duplex of each of said two sets of indexing linkers is different; and

wherein at least one indexing linker from one set of indexing linkers is physically present with at least one indexing linker from the other set of indexing linkers;

(iii) contacting said DNA fragments added to said two sets of indexing linkers with a DNA ligase to cause a selective ligation of those indexing linkers whose 3'"'"'- or 5'"'"'-protruding single strands are fully complementary to the 3'"'"'or 5'"'"'-protruding single strands of the DNA fragments; and

(iv) obtaining indexed DNA fragments.

14. The method of claim 13, wherein said process further comprises a step of (v) of identifying an indexed DNA fragment.

15. The method of claim 13, wherein said process further comprises a step (v) of isolating an indexed DNA fragment.

16. The method of claim 13, wherein said process further comprises a step (v) of mapping an indexed DNA fragment.

17. The method of claim 13, wherein said process further comprises a step of (v) subjecting an indexed DNA fragment to an amplification reaction to obtain the selective amplification of said DNA fragment.

18. The method of claim 17, wherein said amplification is preformed by cloning.

19. The method of claim 17, wherein said amplification is preformed by the polymerase chain reaction.

20. The method of claims 17, wherein said process further comprises a step (vi) of sequencing said amplified DNA fragment.

21. The method of claim 13, wherein said indexing linkers are immobilized.

22. The method of claim 13, wherein said indexing linkers are covalently labelled with a fluorescent tag.

23. The method of claim 13, wherein said indexing linkers are covalently labelled with a radioisotope label.

24. The method of claim 13, wherein said indexing linkers are covalently labelled with an affinity ligand.

25. A kit for obtaining indexed DNA fragments from a mixture of DNA fragments, for identifying, isolating, mapping, amplifying, or sequencing said fragments,

said kit comprising two sets of indexing linkers, wherein:

(i) each member of a set of indexing linkers is a common DNA duplex having one 3'"'"'- or 5'"'"'-protruding single strand of a length corresponding to the length of a 3'"'"'- or 5'"'"'-protruding single strand of the cleavage site of a Type IIS restriction endonuclease or restriction endonuclease recognizing interrupted palindromic sequences;

(ii) said 3'"'"'- or 5'"'"'-protruding single strands of the members of at least one set encode collectively at least eight different permutations and combinations of the nucleotides, A, C, G and T;

(iii) said members of a set being physically separated from each other on the basis of the identity of their 3'"'"'- or 5'"'"'-protruding end;

(iv) said two sets of indexing linkers being distinguished from each other on the basis of the identity of their DNA duplexes; and

(v) said indexing linkers being arranged in pairwise combinations on the basis of the identity of their DNA duplexes.

26. The kit according to claim 25, wherein said indexing linkers are immobilized.

27. The kit according to claim 25, wherein said indexing linkers are covalently labelled with a fluorescent tag.

28. The kit according to claim 25, wherein said indexing linkers are covalently labelled with a radioisotope label.

29. The kit according to claim 25, wherein said indexing linkers are covalently labelled with an affinity ligand.

30. A method for obtaining indexed DNA fragments from a mixture of DNA fragments, for identifying, isolating, mapping, amplifying, or sequencing said fragments, said method comprising:

(i) treating DNA with a restriction endonuclease which is a member selected from the group consisting of Type IIS restriction endonucleases and restriction endonucleases which recognize interrupted palindromic sequences, to produce DNA fragments having at least one 3'"'"'- or 5'"'"'-protruding single strand;

(ii) combining said DNA fragments with a set of indexing linkers and DNA ligase;

wherein each said indexing linker being a DNA duplex having one 3'"'"'- or 5'"'"'-protruding single strand of a length corresponding to the length of the 3'"'"'- or 5'"'"'-protruding single strand of the cleavage site of said restriction endonuclease, wherein said set comprises a collection of said indexing linkers whose 3'"'"'- or 5'"'"'-protruding single strands collectively encode up to all possible permutations and combinations of nucleotides, A, C, G and T, and wherein said indexing linkers are physically separated from each other;

such that indexing linkers whose 3'"'"'- or 5'"'"'-protruding single strands are fully complementary to said 3'"'"'- or 5'"'"'-protruding single strands of each DNA fragments are ligated; and

(iii) obtaining indexed DNA fragments.

31. A method for obtaining indexed DNA fragments from a mixture of DNA fragments, for identifying, isolating, mapping, amplifying, or sequencing said fragments, said method comprising:

(i) treating DNA with a restriction endonuclease which is a member selected from the group consisting of Type IIS restriction endonucleases and restriction endonucleases which recognize interrupted palindromic sequences, to produce DNA fragments having at least one 3'"'"'- or 5'"'"'-protruding single strand;

(ii) combining said DNA fragment with a set of indexing linkers and DNA ligase;

wherein each said indexing linker being a DNA duplex having one 3'"'"'- or 5'"'"'-protruding single strand of a length corresponding to the length of the 3'"'"'- or 5'"'"'-protruding single strand of the cleavage site of said restriction endonuclease, wherein said set comprises a collection of said indexing linkers whose 3'"'"'- or 5'"'"'-protruding single strands collectively encode up to all possible permutations and combinations of nucleotides, A, C, G and T, and wherein said indexing linkers are physically separated from each other;

such that indexing linkers whose 3'"'"'- or 5'"'"'-protruding single strands are fully complementary to said 3'"'"'- or 5'"'"'-protruding single strands of each DNA fragments are ligated to form a mixture of indexed dsDNA fragments; and

(iii) amplifying at least one of said indexed DNA fragments.

32. The method of claim 31, wherein said amplification step (iii) comprises:

(a) denaturing said indexed dsDNA fragments to obtain indexed ssDNA fragments;

(b) contacting said mixture of indexed ssDNA fragments with two oligonucleotide primers which are fully complementary to each indexing linker ligated at the 3'"'"'- and 5'"'"'-ends of said indexed ssDNA fragment,

(c) treating the reaction mixture of step (b) with DNA polymerase to obtain a new dsDNA molecule which is identical to said indexed dsDNA fragment obtained in step (ii),

(d) denaturing the dsDNA obtain in step (c) and repeating steps (a)-(c).

33. In a polymerase chain reaction kit comprising:

heat source, oligonucleotide primers, DNA polymerase and a mixture of all four deoxynucleotide precursors, wherein the improvement comprises;

a panel for obtaining indexed DNA fragments from a mixture of DNA fragments, for identifying, isolating, mapping, amplifying, or sequencing said fragments,

said panel comprising a set of indexing linkers, each said indexing linker being a DNA duplex having one 3'"'"'- or 5'"'"'-protruding single strand of a length corresponding to the 3'"'"'- or 5'"'"'-protruding single strand of the cleavage site of a Type IIS restriction endonuclease or a restriction endonuclease recognizing interrupted palindromic sequences, wherein said set comprises a collection of indexing linkers whose 3'"'"'- or 5'"'"'-protruding single strands collectively encode up to all possible permutations and combinations of the nucleotides, A, C, G and T, and wherein said indexing linkers are physically separated from each other on the basis of the identity of their 3'"'"'- or 5'"'"'-protruding single strand.