Methods of identifying nucleic acid sequences using polycations

• US 6,436,646 B1
• Filed: 11/28/2000
• Issued: 08/20/2002
• Est. Priority Date: 05/21/1999
• Status: Expired due to Term

• Current Assignee: Caliper Holdings Incorporated
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Nikiforov, Theo T.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/727,532
• Time in Patent Office: 630 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/194, 435/183, 435/253.1, 435/255.1, 435/287.2, 436/164, 436/171, 436/172, 536/22.1, 536/23.1, 536/24-- 2, 204/155
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Methods, systems, kits for carrying out a wide variety of different assays that comprise providing a first reagent mixture which comprises a first reagent having a fluorescent label. A second reagent is introduced into the first reagent mixture to produce a second reagent mixture, where the second reagent reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent. A polyion is introduced into at least one of the first and second reagent mixtures, and the fluorescent polarization in the second reagent mixture relative to the first reagent mixture is determined, this fluorescent polarization being indicative of the rate or extent of the reaction.

Claims

1. A method of identifying a subsequence of nucleic acids in a target sequence, comprising:

providing a target nucleic acid sequence;

contacting the target nucleic acid sequence with a labeled probe that is complementary to a known nucleic acid sequence, wherein the probe is neutral or positively charged, to produce a first mixture;

contacting the first mixture with a polycation in a second mixture;

identifying when the labeled probe hybridizes with the target nucleic acid sequence by identifying an increase in a level of polycation that associates with the labeled probe in the second mixture.

2. The method of claim 1, wherein the labeled probe comprises a fluorescent label.

3. The method of claim 2, wherein the identifying step comprises monitoring a level of polarized fluorescence in the second mixture, an increase in the level of polarized fluorescence being indicative that the labeled probe hybridized with the target nucleic acid sequence.

4. The method of claim 1, wherein the labeled probe comprises a neutral or positively charged nucleic acid analog.

5. The method of claim 4, wherein the labeled probe comprises a peptide nucleic acid or a methyl phosphonate polymer.

6. The method of claim 1, wherein the subsequence of nucleic acids comprises a known polymorphic locus.

7. The method of claim 6, wherein the polymorphic locus comprises a single nucleotide polymorphism.

8. The method off claim 6, wherein the labeled probe is complementary to one variant of the polymorphic locus.

9. The method of claim 1, wherein the second mixture is provided in a well of a multiwell plate.

10. The method of claim 1, wherein the second mixture is provided in a microscale fluid channel.

11. A method of monitoring hybridization reaction, comprising:

providing a target nucleic acid sequence having a first subsequence of nucleic acids;

contacting the target nucleic acid sequence with a fluorescently labeled neutral or positively charged nucleic acid analog probe that is complementary to the subsequence of nucleic acids to form a fluorescently labeled hybrid in a first mixture;

contacting the fluorescently labeled hybrid with a polycation in a second mixture; and

comparing a level of polarized fluorescence in the second mixture to a level of polarized fluorescence of the fluorescently labeled nucleic acid probe that has not formed a fluorescently labeled hybrid, an increase in polarized fluorescence being indicative of the formation of the fluorescently labeled hybrid.

12. A method of monitoring a reaction, comprising:

providing first and second reactants, wherein the first reactant includes a fluorescent label;

reacting the first and second reactants in a first reaction mixture to produce a product, wherein the product includes either an increase in negative charge or a decrease in positive charge relative to the first reactant;

contacting the product with a polycation to the first reaction mixture; and

measuring a level of polarized fluorescence from the first mixture, an increase in polarized fluorescence in the first mixture relative to a level of polarized fluorescence of the first reactant alone, being indicative of an amount of the product produced in the first reaction mixture.