EP0303459A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Multiplex sequencing
Assignee: Harvard University
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Filing: 1988-08-10
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Granted: —
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Expires: 2008-08-10
Abstract
Abstract This invention features vectors and a method for sequencing DNA. The method includes the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.
Inventors
- 👤 George M. Church
- 👤 Stephen Kieffer-Higgins
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-30 05:52
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; C12Q1/6813; C12Q1/6874; Y; Y10; Y10S; Y10S435/00; Y10S435/803
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
89 words
A set comprising at least two vectors, each said vector of said set characterised by having a DNA construct having at least one restriction endonuclease site and having at least one tag sequence comprising at least 15 base pairs, each tag sequence being located within 50 bases of one said site, each said tag sequence being incapable of hybridizing with another of said tag sequences under stringent hybridization conditions, each said vector of said set differing from each other said vector of said set only at said tag sequence.
Claim 2
Dependent Claim
89 words
A set comprising at least two vectors, each said vector of said set characterised by having a DNA construct having at least one restriction endonuclease site and having at least one tag sequence comprising at least 15 base pairs, each tag sequence being located within 50 bases of one said site, each said tag sequence being incapable of hybridizing with another of said tag sequences under stringent hybridization conditions, each said vector of said set differing from each other said vector of said set only at said tag sequence.
Claim 3
Dependent Claim
108 words
A vector for sequencing genomic DNA of an organism, said vector characterised by a DNA construct having at least two restriction endonuclease sites, each said site being recognised by a first restriction endonuclease, wherein treatment of said vector with said first endonuclease produces a discrete DNA fragment consisting of the DNA extending between said sites comprising two tag sequences separated from each other by a second restriction endonuclease site, wherein said tag sequences each comprise at least 15 bases and each end of said tag sequences is located within 50 bases of the nearest said first and second restriction site, said tag sequences being unique in said vector.
Claim 4
Dependent Claim
108 words
A vector for sequencing genomic DNA of an organism, said vector characterised by a DNA construct having at least two restriction endonuclease sites, each said site being recognised by a first restriction endonuclease, wherein treatment of said vector with said first endonuclease produces a discrete DNA fragment consisting of the DNA extending between said sites comprising two tag sequences separated from each other by a second restriction endonuclease site, wherein said tag sequences each comprise at least 15 bases and each end of said tag sequences is located within 50 bases of the nearest said first and second restriction site, said tag sequences being unique in said vector.
Claim 5
Dependent Claim
18 words
A set as claimed in claim 1, wherein said tag sequences have no cytosine residues in one strand.
Claim 6
Dependent Claim
18 words
A vector as claimed in claim 2, wherein said tag sequences have no cytosine residues in one strand.
Claim 7
Dependent Claim
26 words
A set as claimed in claim 1 wherein said vectors are produced from a parental vector having randomly formed tag sequences ligated into said parental vector.
Claim 8
Dependent Claim
28 words
A set as claimed in claim 5 wherein said randomly formed sequences are formed in a DNA synthesizer supplied with at least two nucleotides at each addition step.
Claim 9
Dependent Claim
57 words
A set of tag oligonucleotides, characterised in that each said tag oligonucleotide of said set is unique in said set and neither said tag oligonucleotide nor oligonucleotide homologous to said tag oligonucleotide will hybridize under stringent hybridization conditions to other said tag oligonucleotides in said set, wherein each tag oligonucleotide is at least 15 bases in length.
Claim 10
Dependent Claim
57 words
A set of tag oligonucleotides, characterised in that each said tag oligonucleotide of said set is unique in said set and neither said tag oligonucleotide nor oligonucleotide homologous to said tag oligonucleotide will hybridize under stringent hybridization conditions to other said tag oligonucleotides in said set, wherein each tag oligonucleotide is at least 15 bases in length.
Claim 11
Dependent Claim
22 words
A set of vectors produced by ligation into a parental vector of the set of tag oligonucleotides as claimed in claim 7.
Claim 12
Dependent Claim
32 words
A set as claimed in any one of claims 1, 3, 5, 6 or 7 wherein said hybridization conditions comprise conditions in which mismatched duplexes are melted to form single stranded molecules.
Claim 13
Dependent Claim
21 words
A set as claimed in claim 9 wherein said hybridization conditions comprise heating at 42°C in 500-1000 mM sodium phosphate buffer.
Claim 14
Independent Claim
165 words
A method for sequencing a DNA specimen, said method characterised by the steps of: a) ligating said DNA specimen into a vector comprising a tag sequence, said tag sequence comprising at least 15 bases, wherein said tag sequence will not hybridize to said DNA specimen under stringent hybridization conditions and is unique in said vector, to form a hybrid vector, b) treating separate aliquots of said hybrid vector in a plurality of vessels to produce fragments comprising said tag sequence, wherein said fragments in each vessel differ in length from each other and all terminate at a fixed known base or bases, wherein said fixed known base or bases differ from those in other said vessels, c) separating said fragments from each said vessel according to their size, d) hybridizing said fragments with an oligonucleotide able to hybridize specifically with said tag sequence, and e) detecting the pattern of hybridization of said tag sequence, wherein said pattern reflects the nucleotide sequence of said DNA specimen.
Claim 15
Independent Claim
165 words
A method for sequencing a DNA specimen, said method characterised by the steps of: a) ligating said DNA specimen into a vector comprising a tag sequence, said tag sequence comprising at least 15 bases, wherein said tag sequence will not hybridize to said DNA specimen under stringent hybridization conditions and is unique in said vector, to form a hybrid vector, b) treating separate aliquots of said hybrid vector in a plurality of vessels to produce fragments comprising said tag sequence, wherein said fragments in each vessel differ in length from each other and all terminate at a fixed known base or bases, wherein said fixed known base or bases differ from those in other said vessels, c) separating said fragments from each said vessel according to their size, d) hybridizing said fragments with an oligonucleotide able to hybridize specifically with said tag sequence, and e) detecting the pattern of hybridization of said tag sequence, wherein said pattern reflects the nucleotide sequence of said DNA specimen.
Claim 16
Dependent Claim
26 words
A method as claimed in claim 11 further comprising ligating said DNA to a plurality of said vectors to form a plurality of said hybrid vectors.
Claim 17
Dependent Claim
34 words
A method as claimed in claim 12 wherein each said vector differs at said tag sequence and each said tag sequence is unable to hybridize under stringent hybridization conditions to other said tag sequences.
Claim 18
Dependent Claim
21 words
A method as claimed in claim 11 further comprising rehybridizing said fragments with each said tag sequence of each said vector.
Claim 19
Dependent Claim
16 words
A method as claimed in claim 11, wherein each said vector comprises two said tag sequences.
Claim 20
Dependent Claim
20 words
A method as claimed in claim 11 further comprising binding said fragments to a solid support prior to said hybridizing.
Claim 21
Dependent Claim
28 words
A method as claimed in claim 11 or claim 13 wherein said stringent hybridization conditions comprise conditions in which mismatched duplexes are melted to form single stranded molecules.
Claim 22
Independent Claim
84 words
A method for repeatedly hybridizing a solid support, comprising nucleic acid, with a plurality of labels, characterised by the steps of: a) enclosing said support within a container comprising material thin enough to allow detection of hybridization of said nucleic acid with said labels, b) inserting hybridization fluid comprising a first said label into said container, c) removing said hybridization fluid from said container, and d) detecting hybridization of said first label to said solid support, while said solid support remains in said container.
Claim 23
Independent Claim
84 words
A method for repeatedly hybridizing a solid support, comprising nucleic acid, with a plurality of labels, characterised by the steps of: a) enclosing said support within a container comprising material thin enough to allow detection of hybridization of said nucleic acid with said labels, b) inserting hybridization fluid comprising a first said label into said container, c) removing said hybridization fluid from said container, and d) detecting hybridization of said first label to said solid support, while said solid support remains in said container.
Claim 24
Dependent Claim
23 words
A method as claimed in claim 18, further comprising after said detecting step, repeating steps b,c and d using a second said label.
Claim 25
Dependent Claim
24 words
A method as claimed in claim 18, wherein steps b,c and d are repeated at least 20 times with at least 20 said labels.
Claim 26
Dependent Claim
34 words
A method as claimed in claim 11, further comprising prior to said separating step, the steps of providing a molecular weight marker in a said vessel and detecting said markers after said separating step.
Claim 27
Dependent Claim
30 words
A method as claimed in claim 16 wherein said solid support comprises said vector or said oligonucleotide, wherein said vector or oligonucleotide act as an identifying marker for said support.
Claim 28
Independent Claim
95 words
A method for determining the DNA sequence of a DNA specimen characterised by the steps of: a) performing a first sequencing reaction on said DNA specimen; b) performing a second sequencing reaction on a known DNA specimen; c) placing corresponding products of said first and second sequencing reactions in the same lanes of a DNA sequencing gel; d) running the products into said gel; e) detecting the location of said products in said gel, and f) using the location of said known DNA products to aid calculation of the DNA sequence of said DNA specimen.
Claim 29
Independent Claim
95 words
A method for determining the DNA sequence of a DNA specimen characterised by the steps of: a) performing a first sequencing reaction on said DNA specimen; b) performing a second sequencing reaction on a known DNA specimen; c) placing corresponding products of said first and second sequencing reactions in the same lanes of a DNA sequencing gel; d) running the products into said gel; e) detecting the location of said products in said gel, and f) using the location of said known DNA products to aid calculation of the DNA sequence of said DNA specimen.
Claim 30
Dependent Claim
78 words
An automated hybridization device, for repeatedly hybridizing a solid support comprising nucleic acid, with a plurality of labels, characterised by: a container enveloping said solid support, comprising material thin enough to allow detection of hybridization of said nucleic acid with a said label by placement of a label-sensitive film adjacent said container, said container including an inlet and outlet for introducing and withdrawing hybridization fluid, and comprising means for automatically regulating introduction and removal of said hybridization fluid.
Claim 31
Dependent Claim
78 words
An automated hybridization device, for repeatedly hybridizing a solid support comprising nucleic acid, with a plurality of labels, characterised by: a container enveloping said solid support, comprising material thin enough to allow detection of hybridization of said nucleic acid with a said label by placement of a label-sensitive film adjacent said container, said container including an inlet and outlet for introducing and withdrawing hybridization fluid, and comprising means for automatically regulating introduction and removal of said hybridization fluid.
Claim 32
Dependent Claim
19 words
An automated hybridization device as claimed in claim 24 comprising inflatable means for contacting said container with said film.
Claim 33
Dependent Claim
18 words
An automated hybridization device as claimed in claim 24 or 25 comprising a lightproof box enveloping said container.
Claim 34
Dependent Claim
23 words
An automated hybridization device as claimed in claim 24 or 25 comprising a plurality of said containers, each separated by a label-opaque shelf.
References Cited (Backward Citations)
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