Apparatus, systems and method for locating nucleic acids bound to surfaces

• US 6,284,465 B1
• Filed: 04/15/1999
• Issued: 09/04/2001
• Est. Priority Date: 04/15/1999
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Wolber, Paul K.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/292,289
• Time in Patent Office: 873 Days
• Field of Search: 435/6, 250/559.29, 422/82.05, 436/94
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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.

Claims

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. The system of claim 11, wherein the second plurality of nucleotide sequences comprises a fluorophore label, and wherein the second optical signal comprises fluorescence.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

22. 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.

23. 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.

24. 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.

25. 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.

26. 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.

27. 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.

28. 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.

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

34. 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.