Method for detecting and classifying nucleic acid hybridization

• US 6,340,568 B2
• Filed: 04/19/2001
• Issued: 01/22/2002
• Est. Priority Date: 02/02/1998
• Status: Expired due to Fees

• Current Assignee: Mds Sciex
• Original Assignee: Signature BioScience, Inc.
• Inventors: Hefti, John
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/839,860
• Publication Number:
  US 20010034030A1
• Time in Patent Office: 278 Days
• Field of Search: 435/6
• US Class Current: 435/6.11
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: H01J 49/04 Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components

Abstract

The present invention provides a variety of methods for conducting analyses of nucleic acids using a detection system which is sensitive to the unique dielectric properties of different hybridization complexes and which can distinguish between different hybridization complexes directly without the use of labels. Methods using the system to perform sequence checking, expression analysis, de novo sequencing and a variety of other nucleic acid analyses are provided.

Claims

1. A method for detecting nucleic acid hybridization complexes, comprising:

(1) acquiring a spectrum for a hybridization complex formed between (a) a nucleic acid probe that is electromagnetically coupled to a portion of a signal path wherein one or more said signal path and one or more said probe are present in an apparatus in which said method is carried out, each signal path having a corresponding nucleic acid probe, and wherein a plurality of probes and signal paths, if plurality is present, are optionally arranged as elements of an array, and (2) a nucleic acid target in a sample, by propagating a test signal along said signal path and detecting a response signal for the hybridization complex formed between the probe and target, wherein said propagating step comprises varying said test signal with time; and

(b) examining said spectrum for the presence of a signal which is characteristic for a hybridization complex between said probe and a particular target,

wherein said acquiring takes place without washing unbound nucleic acids from said probe before acquiring said spectrum.

2. The method of claim 1, wherein said method is being carried out to distinguish complementary from mismatch hybridization.

3. The method of claim 2, wherein:

(a) the target includes a known polymorphic site, which can include a first or a second base, and wherein the target forms a complementary hybridization complex with said probe if said target includes said first base and forms a mismatch hybridization complex if said target includes said second base; and

(b) examining comprises examining for the presence of a complementary signal and a mismatch signal, the presence of the complementary signal being indicative of said target including the first base at the polymorphic site and the presence of the mismatch signal being indicative of said target including said second base at said polymorphic site.

4. The method of claim 1, wherein said probe and said target are unlabeled.

5. The method of claim 1, wherein said varying comprises varying the frequency of the test signal that is propagated along the signal path.

6. The method of claim 5, wherein said test signal is a microwave signal.

7. The method of claim 1, wherein said signal path comprises a transmission line.

8. The method of claim 7, wherein said probe is directly attached to said transmission line.

9. The method of claim 1, wherein:

(a) said sample potentially comprises a target of known sequence;

(b) said probe has a sequence which is complementary to said target of known sequence; and

(c) said response signal is indicative of said sample containing said target of known sequence.

10. The method of claim 1, wherein said target is present in a sample containing nucleic acids amplified from genes expressed in a particular cell.

11. The method of claim 1, wherein said examining comprises measuring changes in signal amplitude or frequency at a plurality of different time points to obtain a plurality of measured values.

12. The method of claim 11, wherein said measured values are used to evaluate the kinetics of hybridization between said probe and target.

13. The method of claim 1, wherein a plurality of probes of the same sequence are electromagnetically coupled to the transmission line and wherein said examining is used to quantitate the number of hybridization complexes formed.

14. The method of claim 1, wherein said test signal has a frequency or range of frequencies in a range from 10 MHz to 1000 GHz.

15. The method of claim 1, wherein said test signal frequency is a resonant frequency f_res and said complementary signal and said mismatch signal have different f_res values from each other.

16. The method of claim 1, wherein a plurality of said signal paths and corresponding probes are arranged as elements in an array.

17. The method of claim 16, wherein:

(a) said sample potentially comprises a first target of known sequence and a second target of a different known sequence;

(b) said plurality of probes comprises a first probe complementary to said first target and a second probe complementary to said second target, said first and second probe being located at a first and second element of said array, respectively; and

(c) said detecting comprises monitoring said first and second element for a first and second response signal, respectively, wherein said first response signal indicates the presence of said first target in said sample and wherein said second response signal indicates the presence of said second target in said sample.

18. The method of claim 17, wherein said test signal has a frequency or range of frequencies in a range from 10 MHz to 1000 GHz.