Covalently linked complementary oligodeoxynucleotides as universal nucleic acid sequencing primer linkers

Claim 2

In a method for chain termination sequencing of a DNA sequence, which comprises replicating a template chain with mixtures of nucleotides, 'where each of the mixtures has a 'different composition resulting in a mixture of partial sequence strands terminating at different nucleotides in the sequence, separating the partial sequence strands into individual length strands and determining the nucleotide sequence as a result of the site of termination of the strands, the improvement which comprises: joining only one of the termini at at least one of the ends-of a double-stranded DNA fragment to be sequenced to a splinker, wherein said splinker is characterized by having a complementary region of at least four base pairs and a selectively cleavable link; denaturing the splinker-linked double-stranded DNA to provide for a splinker-linked single-stranded DNA as a template fragment, where said splinker acts as a primer; and enzymatically extending the primer with said mixtures of nucleotides for sequencing.

Claim 3

A method according to Claim 1, wherein said selectively cleavable link is a restriction site.

Claim 4

A method according to Claim 1, wherein said selectively cleavable link is a ribonucleotide.

Claim 5

4 . In a method for chain termination sequencing of a DNA sequence which comprises replicating a template chain with mixtures of nucleotides where each of the mixtures has a different dideoxy nucleotide, resulting in a mixture of partial sequence strands terminating at different nucleotides in the sequence, separating the partial sequence strands by electrophoresis into individual strand lengths and determining the nucleotide sequence as.a result of the site of termination of the strands, the improvement which comprises: joining a double-stranded DNA fragment to be sequenced having hydroxyl termini to a splinker, wherein said splinker is characterized by having two complementary regions proximal to opposite termini capable of duplexing to form a double strand of at least five base pairs, each of said complementary regions joined by a single strand of at least three nucleotides, so that the entire splinker defines a hairpin, said duplex region defining a restriction site; denaturing the splinker-linked double-stranded DNA to provide for a splinker-linked single-stranded DNA as a template fragment, where said splinker acts as a primer; and enzymatically extending the primer with said chain terminating mixtures of nucleotides for sequencing.

Claim 6

4 . In a method for chain termination sequencing of a DNA sequence which comprises replicating a template chain with mixtures of nucleotides where each of the mixtures has a different dideoxy nucleotide, resulting in a mixture of partial sequence strands terminating at different nucleotides in the sequence, separating the partial sequence strands by electrophoresis into individual strand lengths and determining the nucleotide sequence as.a result of the site of termination of the strands, the improvement which comprises: joining a double-stranded DNA fragment to be sequenced having hydroxyl termini to a splinker, wherein said splinker is characterized by having two complementary regions proximal to opposite termini capable of duplexing to form a double strand of at least five base pairs, each of said complementary regions joined by a single strand of at least three nucleotides, so that the entire splinker defines a hairpin, said duplex region defining a restriction site; denaturing the splinker-linked double-stranded DNA to provide for a splinker-linked single-stranded DNA as a template fragment, where said splinker acts as a primer; and enzymatically extending the primer with said chain terminating mixtures of nucleotides for sequencing.

Claim 7

A method according to Claim 4, wherein said splinker has a label which remains bound to said partial sequences after restriction at said splinker . restriction site.

Claim 8

A composition of matter comprising a single-stranded DNA of at least 12 nucleotides comprising complementary regions capable of duplexing proximal to the termini joined by a single-stranded sequence of at least three nucleotides.

Claim 9

A composition of matter comprising a single-stranded DNA of at least 12 nucleotides comprising complementary regions capable of duplexing proximal to the termini joined by a single-stranded sequence of at least three nucleotides.

Claim 10

A composition of matter according to Claim 6 , wherein said single strand is of up to and including 30 nucleotides and wherein said complementary region is at least five base pairs and at least one of the complementary regions is at the terminus.

Claim 11

A composition of matter according to Claim 7, wherein the 5' terminus of said single-stranded sequence is phosphorylated.

Claim 12

A composition of matter according to Claim 6, wherein the 5' or 3' strand has a two nucleotide overhang, into at least one 3' overhang of a restriction site.

Claim 13

A probe for detecting a nucleic acid sequence of interest comprising a composition of matter according to Claim 6 joined at one terminus to a DNA sequence complementary to said sequence of interest.