US6284465B1
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Apparatus, systems and method for locating nucleic acids bound to surfaces
Assignee: Agilent Technologies Inc
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Filing: 1999-04-15
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Granted: 2001-09-04
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Expires: 2019-04-15
Abstract
Abstract An apparatus, systems and method for locating nucleic acids in an array on a substrate have self-locating nucleic acid features. The nucleic acid features produce nucleotide-dependent location signals or optically detectable contrast between nucleotide-bound regions and non-nucleotide-bound regions of the substrate when scanned by an optical scanner. When used as analytical tools for monitoring gene expression and mutations in gene sequences, the nucleotide features are hybridized with nucleic acids of known or unknown sequences. The apparatus, systems and method locate both weakly and strongly hybridized nucleotide features on the substrate for identification of target nucleic acid sequences. The nucleotide feature signals or contrast are independent of the optical signals conventionally produced by the hybridized nucleotides. Therefore, the apparatus, systems and method locate all of the nucleotide features, hybridized or not, independently of the extent of hybridization. The present invention advantageously self-locates both bright and dim hybridized features on an array substrate and is therefore, independent of the random and systematic errors associated with the manufacturing equipment and processes. Moreover, the present invention provides a powerful quality control tool to the in situ synthesis process. The present invention provides information about what part or percentage of each feature contains full-length probes. The present optical scanning system detects optical signals from the nucleotide features independently of the signals from the hybridized nucleotides using essentially conventional scanning technology. The independently detected signals are processed such that all features are located and the hybridized features are accurately detected and analyzed.
Inventors
- 👤 Paul K. Wolber
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 11:21
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/17; G01N21/25; G01N21/251; G01N21/253; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6834; C12Q1/6837; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
167 words
An apparatus with self-locating nucleic acid positions in an array comprising: a substrate having a surface; a first plurality of nucleotide sequences bound to the surface of the substrate; regions on the substrate surface that are devoid of the first plurality of nucleotide sequences, wherein the first plurality of nucleotide sequences comprise a first optically detectable contrast from the regions; and a second plurality of nucleotide sequences hybridized to complementary nucleotide sequences of the first plurality of nucleotide sequences, the second plurality of nucleotide sequences comprising a second optically detectable contrast distinguishable from the first optically detectable contrast, wherein the first optically detectable contrast comprises a first optical signal and the second optically detectable contrast comprises a second optical signal when scanned with an optical scanner, the first optical signal being independent of the second optical signal, such that the first optical signal indicates the location of the first plurality of nucleotide sequences in the array independently of the hybridization by the second plurality of nucleotide sequences.
Claim 2
Independent Claim
167 words
An apparatus with self-locating nucleic acid positions in an array comprising: a substrate having a surface; a first plurality of nucleotide sequences bound to the surface of the substrate; regions on the substrate surface that are devoid of the first plurality of nucleotide sequences, wherein the first plurality of nucleotide sequences comprise a first optically detectable contrast from the regions; and a second plurality of nucleotide sequences hybridized to complementary nucleotide sequences of the first plurality of nucleotide sequences, the second plurality of nucleotide sequences comprising a second optically detectable contrast distinguishable from the first optically detectable contrast, wherein the first optically detectable contrast comprises a first optical signal and the second optically detectable contrast comprises a second optical signal when scanned with an optical scanner, the first optical signal being independent of the second optical signal, such that the first optical signal indicates the location of the first plurality of nucleotide sequences in the array independently of the hybridization by the second plurality of nucleotide sequences.
Claim 3
Dependent Claim
33 words
The apparatus of claim 1 , wherein the first plurality of nucleotide sequences comprises a plurality of oligonucleotide probes and the second plurality of nucleotide sequences comprises a plurality of target nucleotide sequences.
Claim 4
Dependent Claim
45 words
The apparatus of claim 1 , wherein the first plurality of nucleotide sequences comprises a first signal producing system to produce the first optical signal, and wherein the second plurality of nucleotide sequences comprises a second signal producing system to produce the second optical signal.
Claim 5
Dependent Claim
26 words
The apparatus of claim 3 , wherein the first signal producing system comprises a first label and the second signal producing system comprises a second label.
Claim 6
Dependent Claim
33 words
The apparatus of claim 4 , wherein the first label comprises a specific binding pair comprising biotin and colloidal gold chemically conjugated to streptavidin, and wherein the first optical signal comprises scattered light.
Claim 7
Dependent Claim
36 words
The apparatus of claim 4 , wherein the first label comprises a first fluorophore and wherein the first optical signal comprises a spectrally shifted fluorescence produced by the first fluorophore relative to the second optical signal.
Claim 8
Dependent Claim
45 words
The apparatus of claim 4 , wherein the first label comprises a specific binding pair comprising biotin and plastic microspheres chemically conjugated to streptavidin, the microspheres containing a fluorescent material, and wherein the first optical signal comprises fluorescence spectrally shifted from the second optical signal.
Claim 9
Dependent Claim
35 words
The apparatus of claim 1 , wherein the first plurality of nucleotide sequences are surrounded by dried crystallized salts on the substrate, and wherein the first optical signal comprises scattered light from the crystallized salts.
Claim 10
Dependent Claim
56 words
The apparatus of claim 1 , wherein the first plurality of nucleotide sequences reflects light along a plan e of polarization, and wherein the first optical signal comprises a reflected signal in which the plane of polarization of the reflected light has been rotated by interaction with chiral portions of the first plurality of nucleotide sequences.
Claim 11
Dependent Claim
29 words
The apparatus of claim 1 , wherein the second plurality of nucleotide sequences comprises a fluorophore label, and wherein the second optical signal comprises fluorescence produced by the fluorophore.
Claim 12
Independent Claim
185 words
A system for locating nucleic acid positions in an array comprising: a substrate having a surface; a first plurality of nucleotide sequences bound to the surface of the substrate; regions on the substrate surface that are devoid of the first plurality of nucleotide sequences, wherein the first plurality of nucleotide sequences comprise a first optically detectable contrast from the regions; a second plurality of nucleotide sequences hybridized to at least some sequences of the first plurality of nucleotide sequences, the second plurality of nucleotide sequences comprising a second optically detectable contrast distinguishable from the first optically detectable contrast; and an optical system for optically scanning the surface of the substrate, such that the first optically detectable contrast comprises a first optical signal and the second optically detectable contrast comprises a second optical signal independently of the first optical signal, for detecting the first optical signal independently of the second optical signal, for analyzing the first optical signal to locate the first plurality of nucleotide sequences, and independently for analyzing the second optical signal for hybridization by the second plurality of nucleotide sequences on the substrate.
Claim 13
Independent Claim
185 words
A system for locating nucleic acid positions in an array comprising: a substrate having a surface; a first plurality of nucleotide sequences bound to the surface of the substrate; regions on the substrate surface that are devoid of the first plurality of nucleotide sequences, wherein the first plurality of nucleotide sequences comprise a first optically detectable contrast from the regions; a second plurality of nucleotide sequences hybridized to at least some sequences of the first plurality of nucleotide sequences, the second plurality of nucleotide sequences comprising a second optically detectable contrast distinguishable from the first optically detectable contrast; and an optical system for optically scanning the surface of the substrate, such that the first optically detectable contrast comprises a first optical signal and the second optically detectable contrast comprises a second optical signal independently of the first optical signal, for detecting the first optical signal independently of the second optical signal, for analyzing the first optical signal to locate the first plurality of nucleotide sequences, and independently for analyzing the second optical signal for hybridization by the second plurality of nucleotide sequences on the substrate.
Claim 14
Dependent Claim
33 words
The system of claim 11 , wherein the first plurality of nucleotide sequences comprises a plurality of oligonucleotide probes and the second plurality of nucleotide sequences comprises a plurality of target nucleotide sequences.
Claim 15
Dependent Claim
45 words
The system of claim 11 , wherein the first plurality of nucleotide sequences comprises a first signal producing system to produce the first optical signal, and wherein the second plurality of nucleotide sequences comprises a second signal producing system to produce the second optical signal.
Claim 16
Dependent Claim
36 words
The system of claim 11 , wherein the first plurality of nucleotide sequences are surrounded by dried crystallized salts on the substrate, and wherein the first optical signal comprises scattered light from the dried crystallized salts.
Claim 17
Dependent Claim
55 words
The system of claim 11 , wherein the first plurality of nucleotide sequences reflects light along a plane of polarization, and wherein the first optical signal comprises a reflected signal in which the plane of polarization of the reflected light has been rotated by interaction with chiral portions of the first plurality of nucleotide sequences.
Claim 18
Dependent Claim
25 words
The system of claim 11 , wherein the second plurality of nucleotide sequences comprises a fluorophore label, and wherein the second optical signal comprises fluorescence.
Claim 19
Dependent Claim
120 words
The system of claim 11 , wherein the optical scanning system comprises a first detection subsystem for detecting the first optical signal having a first input and a first output; a second detection subsystem for detecting the second optical signal independently of the first detection subsystem and having a second input and a second output; and an analysis subsystem having inputs and outputs, wherein the first output from the first detection subsystem and second output from the second detection subsystem are received by the inputs of the analysis subsystem, and wherein the outputs of the analysis subsystem comprise the location of the first plurality of nucleotide sequences and the location of the second plurality of nucleotide sequences on the substrate.
Claim 20
Dependent Claim
68 words
The system of claim 17 , wherein the optical scanning system further comprises a signal filtering device and a full reflector, the signal filtering device comprising one or more of a dichroic reflector, an interference filter and a monochrometer, wherein the signal filtering device filters the first optical signal from the second optical signal and the full reflector directs the first optical signal into the first detection subsystem.
Claim 21
Dependent Claim
35 words
The system of claim 17 , wherein the optical scanning system further comprises a partial reflecting device for reflecting the first optical signal into the first detection subsystem and for reflecting signal noise absorbing device.
Claim 22
Independent Claim
172 words
A method for locating nucleic acid positions on a substrate comprising the steps of: providing a first plurality of nucleotide sequences on a substrate surface in a pattern such that there are regions on the substrate surface that are devoid of the first plurality of nucleotide sequences, the first plurality of nucleotide sequences comprising a first optically detectable contrast from the regions; hybridizing at least some of the first plurality of nucleotide sequences with a second plurality of nucleotide sequences, the second plurality of nucleotide sequences comprising a second optically detectable contrast distinguishable from the first optically detectable contrast; optically scanning the substrate surface such that the first optically detectable contrast comprises a first optical signal and the second optically detectable contrast comprises a second optical signal; independently detecting the first optical signal and the second optical signal; and determining the locations of the first plurality of nucleotide sequences and the second plurality of nucleotide sequences on the substrate based on the detected first optical signal and the detected second optical signal.
Claim 23
Independent Claim
172 words
A method for locating nucleic acid positions on a substrate comprising the steps of: providing a first plurality of nucleotide sequences on a substrate surface in a pattern such that there are regions on the substrate surface that are devoid of the first plurality of nucleotide sequences, the first plurality of nucleotide sequences comprising a first optically detectable contrast from the regions; hybridizing at least some of the first plurality of nucleotide sequences with a second plurality of nucleotide sequences, the second plurality of nucleotide sequences comprising a second optically detectable contrast distinguishable from the first optically detectable contrast; optically scanning the substrate surface such that the first optically detectable contrast comprises a first optical signal and the second optically detectable contrast comprises a second optical signal; independently detecting the first optical signal and the second optical signal; and determining the locations of the first plurality of nucleotide sequences and the second plurality of nucleotide sequences on the substrate based on the detected first optical signal and the detected second optical signal.
Claim 24
Dependent Claim
33 words
The method of claim 20 , wherein the first plurality of nucleotide sequences comprises a plurality of oligonucleotide probes and the second plurality of nucleotide sequences comprises a plurality of target nucleotide sequences.
Claim 25
Dependent Claim
51 words
The method of claim 20 , wherein the step of providing comprises the step of incorporating a first signal producing system on the first plurality of nucleotide sequences, and wherein the step of hybridizing comprises the step of incorporating a second signal producing system on the second plurality of nucleotide sequences.
Claim 26
Dependent Claim
57 words
The method of claim 22 , wherein the step of incorporating a first signal producing system comprises the step of labeling the first plurality of nucleotide sequences with a first label, and wherein the step of incorporating a second signal producing system comprises the step of labeling the second plurality of nucleotide sequences with a second label.
Claim 27
Dependent Claim
92 words
The method of claim 23 , wherein the step of labeling comprises labeling the first plurality of nucleotide sequences with a first fluorophore, the step of hybridizing comprises the step of labeling the second plurality of nucleotide sequences with a second fluorophore, the first fluorophore having a first fluorophore having first fluorescence that is spectrally shifted from a second fluorescence from the second fluorophore, and wherein the step of detecting comprises detecting the second fluorescence as the second optical signal and detecting the spectrally shifted first fluorescence as the first optical signal.
Claim 28
Dependent Claim
102 words
The method of claim 20 , wherein the step of providing comprises the step of incorporating a first member of a specific binding pair on the first plurality of nucleotide sequences, and before the optical scanning step, the method further comprises the step of adding a second member of the specific binding pair to the first member, the step of hybridizing comprises the step of labeling the second plurality of nucleotide sequences with a fluorophore, and wherein the step of independently detecting comprises detecting fluorescence as the second optical signal and independently detecting the first optical signal from the specific binding pair.
Claim 29
Dependent Claim
83 words
The method of claim 20 , wherein the step of hybridizing comprises the step of labeling the second plurality of nucleotide sequences with a fluorophore, and wherein the method further comprises, after the step of hybridizing, the step of drying the substrate such that crystallized salts surround the plurality of nucleotide sequences, and wherein the step of detecting comprises detecting a scattered light first optical signal from the crystallized salts and a fluorescence second optical signal from the second plurality of nucleotide sequences.
Claim 30
Dependent Claim
66 words
The method of claim 20 , wherein the first plurality of nucleotide sequences reflects light along a plane of polarization, and wherein the step of detecting comprises detecting the first optical signal comprising a reflected signal in which the plane of polarization of the reflected light has been rotated by interaction with chiral portions of the first plurality of nucleotide sequences bound to the substrate surface.
Claim 31
Dependent Claim
36 words
The method of claim 20 , wherein the step of hybridizing comprises the step of labeling the second plurality of nucleotide sequences with a fluorophore and the second optical signal comprises fluorescence produced by the fluorophore.
Claim 32
Dependent Claim
87 words
The method of claim 20 , wherein the step providing comprises the step of synthesizing a plurality of full-length polynucleotide sequences in situ on the substrate for each of the first plurality of nucleotide sequences, wherein the step of synthesizing comprises steps of: a plurality of sequential steps of adding a nucleotide to a single end of each polynucleotide sequence; and after the last step of adding, the step of labeling a last nucleotide of the full-length polynucleotide sequences with a member of a signal producing system.
Claim 33
Dependent Claim
67 words
The method of claim 29 , wherein the step of labeling comprises the step of adding a label as the member of the signal producing system to the last nucleotide of the full-length polynucleotide sequences and wherein the step of determining comprises further determining what percentage of the first plurality of nucleotide sequences contains the full-length polynucleotide sequences from the first optical signal produced by the member.
Claim 34
Dependent Claim
31 words
The method of claim 30 , wherein the step of adding a label comprises adding a member of a specific binding pair to the last nucleotide of the full-length polynucleotide sequences.
Claim 35
Dependent Claim
36 words
The method of claim 31 , wherein the step of hybridizing comprises the step of adding a complementary member to the member of the specific binding pair on the last nucleotide of the full-length polynucleotide sequences.
Claim 36
Dependent Claim
37 words
The method of claim 31 , further comprising, after the step of hybridizing, the step of adding a complementary member to the member of the specific binding pair on the last nucleotide of the full-length polynucleotide sequences.
Claim 37
Dependent Claim
38 words
The method of claim 20 , wherein the steps of determining comprises the step of identifying the locations of the first plurality of nucleotide sequences regardless of the extent of hybridization by the second plurality of nucleotide sequences.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2004-09-03 | 2005-03-05 | 2005-09-04 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2008-09-03 | 2009-03-05 | 2009-09-04 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2012-09-03 | 2013-03-05 | 2013-09-04 | $7700.00 | Window Closed / Lapsed |