US 5,508,169 A · Indexing linkers

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.

Claims33

Dependent claims

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.

3 more elements

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.

6 more elements

Dependencies are inferred from the claim text.

Indexing linkers

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Indexing linkers

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Abstract

Claims33