US5149625A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Multiplex analysis of DNA
Assignee: Harvard University
•
Filing: 1990-03-28
•
Granted: 1992-09-22
•
Expires: 2010-03-28
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
US
Data Source
google_patents
Last Synchronized
2026-10-31 14:29
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S436/00; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 27
Claim 1
Independent Claim
82 words
A kit comprising at least two vectors, each vector of said set having at least one cloning site and having at least one tag sequence comprising at least 15 base pairs, each of said tag sequences of any one of the vectors being incapable of hybridizing with any of said tag sequences of any other of the vectors 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
82 words
A kit comprising at least two vectors, each vector of said set having at least one cloning site and having at least one tag sequence comprising at least 15 base pairs, each of said tag sequences of any one of the vectors being incapable of hybridizing with any of said tag sequences of any other of the vectors 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
15 words
The kit of claim 1, wherein said tag sequences do not self-hybridize to form hairpins.
Claim 4
Dependent Claim
28 words
The kit of claim 1, wherein said vectors are produced from a parental vector by providing a parental vector and ligating said tag sequences into said parental vector.
Claim 5
Dependent Claim
29 words
The kit of claim 3, wherein said tag sequences are random sequences formed in a DNA synthesizer which is supplied with at least two nucleotides at each addition step.
Claim 6
Dependent Claim
21 words
The kit of claim 1, further comprising: a first oligonucleotide probe able to hybridize specifically with one of said tag sequences.
Claim 7
Dependent Claim
22 words
The kit of claim 5, further comprising a second oligonucleotide probe able to hybridize specifically with a second of said tag sequences.
Claim 8
Dependent Claim
70 words
A kit comprising a plurality of containers, each container comprising a different analysis vector produced by providing a parental vector and ligating a set of tag oligonucleotides into said parental vector, each tag being incapable of hybridizing to any other tag nucleotide in said set or to any oligonucleotide complementary to any other tag nucleotide in said set, and wherein each tag oligonucleotide is at least 15 bases in length.
Claim 9
Dependent Claim
70 words
A kit comprising a plurality of containers, each container comprising a different analysis vector produced by providing a parental vector and ligating a set of tag oligonucleotides into said parental vector, each tag being incapable of hybridizing to any other tag nucleotide in said set or to any oligonucleotide complementary to any other tag nucleotide in said set, and wherein each tag oligonucleotide is at least 15 bases in length.
Claim 10
Dependent Claim
25 words
The kit of claim 1 or 7, wherein said stringent hybridization conditions comprise conditions in which mismatched duplexes are melted to form single stranded molecules.
Claim 11
Dependent Claim
47 words
A kit useful for multiplex analysis of sample DNA, said kit comprising a plurality separate containers of oligonucleotides, each of said oligonucleotides comprising a primer sequence and a multiplexing detection component, each detection component in a given container being different from each detection component in other containers.
Claim 12
Dependent Claim
47 words
A kit useful for multiplex analysis of sample DNA, said kit comprising a plurality separate containers of oligonucleotides, each of said oligonucleotides comprising a primer sequence and a multiplexing detection component, each detection component in a given container being different from each detection component in other containers.
Claim 13
Dependent Claim
21 words
The kit of claim 9, wherein each of said detection components comprises a tag oligonucleotide sequence of at least 15 bases.
Claim 14
Independent Claim
261 words
A method for analyzing a DNA specimen comprising (a) providing a set of at least two vectors, each vector of said set comprising a cloning site and including at least one tag sequence, each tag sequence in each vector differing from each tag sequence of every other vector of said set, and each being incapable of hybridizing to said DNA specimen under stringent conditions, (b) providing a first and a second DNA sequence from said DNA specimen, said first DNA sequence being different from said second DNA sequence, (c) ligating said first DNA sequence into the cloning site of one of said vectors, and said second DNA sequence into the cloning site of a second of said vectors, thereby producing a plurality of hybrid vectors, (d) providing a pool of said hybrid vectors, (e) treating separate aliquots of said pool 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 in one of said vessels differ from said fixed known base or bases in another of said vessels, (f) separating said fragments comprising tag sequences from each said vessel according to their size, (g) hybridizing the separated fragments of step (e) under stringent conditions with a first oligonucleotide probe able to hybridize specifically with one of said tag sequences, and (h) detecting the pattern of hybridization wherein said pattern reflects the nucleotide sequence or restriction map of said DNA specimen.
Claim 15
Independent Claim
261 words
A method for analyzing a DNA specimen comprising (a) providing a set of at least two vectors, each vector of said set comprising a cloning site and including at least one tag sequence, each tag sequence in each vector differing from each tag sequence of every other vector of said set, and each being incapable of hybridizing to said DNA specimen under stringent conditions, (b) providing a first and a second DNA sequence from said DNA specimen, said first DNA sequence being different from said second DNA sequence, (c) ligating said first DNA sequence into the cloning site of one of said vectors, and said second DNA sequence into the cloning site of a second of said vectors, thereby producing a plurality of hybrid vectors, (d) providing a pool of said hybrid vectors, (e) treating separate aliquots of said pool 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 in one of said vessels differ from said fixed known base or bases in another of said vessels, (f) separating said fragments comprising tag sequences from each said vessel according to their size, (g) hybridizing the separated fragments of step (e) under stringent conditions with a first oligonucleotide probe able to hybridize specifically with one of said tag sequences, and (h) detecting the pattern of hybridization wherein said pattern reflects the nucleotide sequence or restriction map of said DNA specimen.
Claim 16
Dependent Claim
73 words
The method of claim 11, wherein the pattern detected in step (h) reflects the nucleotide sequence of said first DNA sequence and said method further comprises the steps of rehybridizing the separate fragments of step (f) under stringent conditions with a second oligonucleotide probe different from the first and detecting a second pattern of rehybridization wherein said second pattern of hybridization reflects the nucleotide sequence or restriction map of said second DNA sequence.
Claim 17
Dependent Claim
17 words
The method of claim 11 or 12, in which each said vector comprises two said tag sequences.
Claim 18
Dependent Claim
20 words
The method of claim 13, in which said two tag sequences on the same vector are different from each other.
Claim 19
Dependent Claim
23 words
The method of claim 11 or 12, further comprising binding the fragments of step (e) to a solid support prior to said hybridizing.
Claim 20
Dependent Claim
25 words
The method of claim 11 or 12, wherein said stringent hybridization conditions comprise conditions in which mismatched duplexes are melted to form single stranded molecules.
Claim 21
Independent Claim
397 words
A method for simultaneously analyzing multiple DNA molecules comprising, (a) providing a pool of individually different DNA tag sequences, each tag sequence in said pool being foreign to, and incapable of hybridizing under stringent conditions to, any of said DNA molecules to be analyzed; (b) from separate aliquots of said pool of tag sequences, generating a plurality of sets of hybrid DNA fragments in separate vessels, said hybrid fragments in each vessel differing in length from one another and terminating in a fixed known base or bases, wherein said fixed known base or bases in one of said vessels differ from said fixed known base or bases in another of said vessels; i) each of said fragments comprising at least one of said tag sequences and at least part of one of said DNA molecules to be analyzed, each of said fragments that includes part of any given one said DNA molecules to be analyzed having a tag sequence that is different from each of the tag sequences in fragments that include part of any other one of said DNA molecules to be analyzed, ii) each fragment of a first set of said hybrid DNA fragments comprising a first one of said tags, said first set of fragments comprising fragments of different lengths of a first one of said DNA molecules to be analyzed; and each fragment of a second set of said sets fragments comprising a second one of said tags, said second set of fragments comprising fragments of different lengths of a second one of said DNA molecules to be analyzed, (c) separating said first and said second set of fragments according to their size, yielding size-separated fragments, (d) hybridizing said size-separated fragments under stringent conditions with a first oligonucleotide probe able to hybridize specifically with said first one of said tag sequences, (e) detecting a first pattern of hybridization with said first probe, wherein said first pattern reflects the nucleotide sequence or restriction map of at least a part of said first DNA molecule to be analyzed, (f) hybridizing said size separating fragments under stringent conditions with a second oligonucleotide probe able to hybridize specifically with said second one of said tag sequences, and (g) detecting a second pattern of hybridization with said second probe, wherein said second pattern reflects the nucleotide sequence or restriction map of said second DNA molecule to be sequence.
Claim 22
Independent Claim
397 words
A method for simultaneously analyzing multiple DNA molecules comprising, (a) providing a pool of individually different DNA tag sequences, each tag sequence in said pool being foreign to, and incapable of hybridizing under stringent conditions to, any of said DNA molecules to be analyzed; (b) from separate aliquots of said pool of tag sequences, generating a plurality of sets of hybrid DNA fragments in separate vessels, said hybrid fragments in each vessel differing in length from one another and terminating in a fixed known base or bases, wherein said fixed known base or bases in one of said vessels differ from said fixed known base or bases in another of said vessels; i) each of said fragments comprising at least one of said tag sequences and at least part of one of said DNA molecules to be analyzed, each of said fragments that includes part of any given one said DNA molecules to be analyzed having a tag sequence that is different from each of the tag sequences in fragments that include part of any other one of said DNA molecules to be analyzed, ii) each fragment of a first set of said hybrid DNA fragments comprising a first one of said tags, said first set of fragments comprising fragments of different lengths of a first one of said DNA molecules to be analyzed; and each fragment of a second set of said sets fragments comprising a second one of said tags, said second set of fragments comprising fragments of different lengths of a second one of said DNA molecules to be analyzed, (c) separating said first and said second set of fragments according to their size, yielding size-separated fragments, (d) hybridizing said size-separated fragments under stringent conditions with a first oligonucleotide probe able to hybridize specifically with said first one of said tag sequences, (e) detecting a first pattern of hybridization with said first probe, wherein said first pattern reflects the nucleotide sequence or restriction map of at least a part of said first DNA molecule to be analyzed, (f) hybridizing said size separating fragments under stringent conditions with a second oligonucleotide probe able to hybridize specifically with said second one of said tag sequences, and (g) detecting a second pattern of hybridization with said second probe, wherein said second pattern reflects the nucleotide sequence or restriction map of said second DNA molecule to be sequence.
Claim 23
Dependent Claim
17 words
The method of claim 14, in which each said hybrid DNA fragment comprises two said tag sequences.
Claim 24
Dependent Claim
16 words
The method of claim 18, in which said two tag sequences are different from each other.
Claim 25
Dependent Claim
23 words
The method of claim 18 or 19, further comprising binding the fragments of step (e) to a solid support prior to said hybridizing.
Claim 26
Dependent Claim
25 words
The method of claim 18 or 19, wherein said stringent hybridization condition comprises conditions in which mismatched duplexes are melted to form single stranded molecules.
Claim 27
Dependent Claim
126 words
The method of claim 17, in which said pool of DNA tag sequences is provided by providing a set of at least two vectors, each vector of said set comprising a cloning site and including at least one of said tag sequences at a position within 50 bases of said cloning site, each tag sequence in each vector differing from each tag sequence of every other vector of said set, ligating said first one of said DNA molecules to be analyzed into the cloning site of one of said vectors, and ligating said second one of said DNA molecules to be analyzed into the cloning site of a second of said vectors, and then providing a pool of the resulting hybrid vectors comprising said tag sequences.
References Cited (Backward Citations)
US4942124A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-09-22 | 1996-03-23 | 1996-09-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-09-22 | 2000-03-23 | 2000-09-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-09-22 | 2004-03-23 | 2004-09-22 | $7700.00 | Window Closed / Lapsed |