Transient electrical signal based methods and devices for characterizing molecular interaction and/or motion in a sample

• US 7,223,540 B2
• Filed: 01/15/2003
• Issued: 05/29/2007
• Est. Priority Date: 10/20/2000
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Pourmand, Nader, Hassibi, Arjang
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US10/345,653
• Publication Number:
  US 20030152985A1
• Time in Patent Office: 1,595 Days
• Field of Search: None
• US Class Current: 435/6.19
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

Devices for detecting a transient electrical signal in a sample are provided. Also provided are systems that include the subject devices. The subject devices and systems find use in a variety of applications, particularly in the characterization of a sample, and more particularly in the characterization of molecular entities in the sample.

Claims

1. A method of detecting an analyte through the occurrence of a polymerase mediated template dependent complementary primer extension reaction in a conducting medium, said method comprising:

(a) providing a system comprising an immobilized template nucleic acid and a sample;

(b) forming a template primer duplex nucleic acid made up of template and any complementary primer nucleic acids in the analyte in said sample in contact with said medium, wherein said medium comprises a polymerase and a plurality of nucleotides added to a second electrode in said medium;

(c) detecting a transient electrical signal indicative of whether or not a primer extension reaction occurs in the medium and produced in said medium by movement of charged nucleotides through the medium towards the immobilized template primer duplex and resultant covalent bonding of said at least one nucleotide to a terminus of said primer nucleic acid; and

(d) relating said detected transient electrical signal to the occurrence of a polymerase mediated template dependent primer extension reaction in said medium, said transient electrical signal being a signal dependent on complementary primer nucleic acids in the analyte.

2. The method according to claim 1, wherein said medium is an aqueous fluid medium.

3. The method according to claim 1, wherein said medium comprises only one type of nucleotide.

4. The method according to claim 1, wherein said medium comprises at least two different types of nucleotides.

5. The method according to claim 4, wherein said medium comprises dATP, dGTP, dCTP and dTTP.

6. The method according to claim 1, wherein said nucleic acid analyte comprises a SNP (single nucleotide polymorphism) site.

7. The method according to claim 1, wherein said nucleic acid analyte is a nucleic acid from a pathogenic organism.

8. The method according to claim 1, wherein said method is a method of gene expression profiling.

9. The method according to claim 1, wherein said method is a method of monitoring a PCR reaction as the reaction progresses.

12. The method of claim 1 wherein the medium is in a defined volume between about 10 μ

l and 0.05 ml.

13. The method of claim 1 further comprising amplifying the transient electrical signal with a differential amplifier having inputs from the two electrodes.

14. The method of claim 1 wherein said step of providing a system further comprises providing multiple immobilized primers, each having a separate electrode.

10. A method of sequencing a nucleic acid, said method comprising:

(a) providing a system comprising an immobilized template primer duplex nucleic acid made up of said nucleic acid hybridized to a primer nucleic acid, wherein said immobilized template primer duplex nucleic acid is immobilized on a surface of a working electrode and is in contact with a conducting medium comprising a polymerase;

(b) adding a plurality of molecules of only one type of nucleotide, at a time to the medium between the working electrode and a second electrode;

(c) detecting a transient electrical signal produced in said medium by movement of the molecules of one type of nucleotide and covalent bonding if said one type of nucleotide is complementary to a nucleotide at a terminus of said template nucleic acid; and

(d) determining said sequence of said nucleic acid using said detected transient electrical signal occurring upon movement and binding of a complementary charged nucleotide, said method further comprising the step of;

two or more iterations of (a) through (c) to produce a plurality of transient electrical signals from which said sequence of said nucleic acid is determined.

11. The method according to claim 10, wherein said medium alternatively comprises one of dGTP, dATP, dCTP and dTTP and said determining step comprises reading said sequence from a single detected transient electrical signal.

15. The method of claim 10 wherein the medium is in a defined volume between about 10 μ

l and 0.05 ml.

16. The method of claim 10 further comprising amplifying the transient electrical signal with a differential amplifier having inputs from the two electrodes.