Method for label-free detection of hybridized DNA targets

• US 6,579,680 B2
• Filed: 02/28/2001
• Issued: 06/17/2003
• Est. Priority Date: 02/28/2000
• Status: Expired due to Fees

• Current Assignee: Corning Incorporated
• Original Assignee: Corning Incorporated
• Inventors: Frutos, Anthony Glenn, Lahiri, Joydeep, Quesada, Mark A., Pal, Santona
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/795,158
• Publication Number:
  US 20020001844A1
• Time in Patent Office: 839 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/287.2, 536/22.1, 536/23.1, 536/24-- 2
• US Class Current: 435/6.1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis

Abstract

A method for the detection and analysis of genetic polymorphisms using arrays that do not require labeling of a target nucleic acid sequence. Hybridization of a perfectly complementary nucleic acid target sequence to an oligonucleotide probe sequence results in a displacement and complete removal of a hybridized probe sequence from the same oligonucleotide probe sequence by means of a thermo-kinetic reaction. The removal of the hybridized probe sequence, having a quencher element, increases the intensity of emission by an emitter element on the oligonucleotide probe sequence.

Claims

1. A method for detecting or identifying genetic polymorphisms, said method comprising:

providing (1) a set of first probes, each (i) having a nucleic acid sequence that is attached to a solid support, and (ii) differing from each other in at least one base location, and (2) a set of second probes, each having a nucleic acid sequence that incorporates at least one artificial mismatch site relative to a first probe, such that said first probe and second probe are bound to each other in a duplex;

providing each first probe with an emitter element, and each second probe with a quencher element;

hybridizing an unlabeled sample nucleic acid target to said duplex of first and second probes;

displacing said second probe from said duplex with said sample nucleic acid target, wherein said emitter element increases in emission intensity with such displacement.

2. The method according to claim 1, wherein each of said sample nucleic acid target is perfectly complementary to a number of said first probes.

3. The method according to claim 1, wherein each of said sample nucleic acid target is not perfectly complementary to a number of said first probes.

4. The method according to claim 1, wherein each nucleic acid sequence of said second probes has 1-5 artificial mismatch sites.

5. The method according to claim 4, wherein each artificial mismatch site is separated from one another by about 3-5 nucleotide positions.

6. The method according to claim 1, wherein said nucleic acid sequence of said first probes are attached to a solid support by covalent bonding.

7. The method according to claim 1, wherein said nucleic acid sequence of said first probes are attached to a solid support by non-covalent bonding.

8. The method according to claim 1, wherein said solid support is an article selected from a group consisting of porous substrates, non-porous substrates, three-dimensional surfaces, beads and planar surfaces.

9. The method according to claim 1, wherein said solid support is an article made from materials selected from a group consisting of glass, polymers, plastics, metals, and silicon.

10. The method according to claim 1, wherein said nucleic acid sequences of said first probes and said second probes each has a functional length of up to about 20-25 nucleotides.

11. The method according to claim 10, wherein said nucleic acid sequences of first probes and said second probes each has a functional length of about 14-19 nucleotides.

12. The method according to claim 1, wherein said first probe sequences are selected from the group consisting of DNA, PNA, RNA, and mixtures of DNA, PNA and RNA.

13. The method according to claim 1, wherein said sample nucleic acid target displaces said second probe by a thermo-kinetic mechanism wherein said sample nucleic acid target is favored to hybridize with a perfectly complementary first probe.

14. The method according to claim 13, wherein a first duplex containing at least one artificial mismatch site is formed when said first probe hybridizes with said second probe, and a second duplex is formed when said same first probe hybridizes with a perfectly complementary sample nucleic acid target, and said second duplex has a thermodynamic stability greater than a thermodynamic stability of said first duplex.

15. The method according to claim 13, wherein said second duplex has a melting temperature (T_m) greater than a melting temperature of said first duplex.

16. The method according to claim 1, wherein said method is used in an analysis of gene expression.

17. A method for detecting genetic polymorphisms, said method comprising:

providing a plurality of DNA duplexes, each having a first nucleic acid sequence that is immobilized to a solid support and labeled with a fluorophore, and an attached second nucleic acid sequence that incorporates at least one mismatch site and is labeled with a fluorescence quencher;

exposing said first nucleic acid sequence to an unlabeled sample DNA sequence that is perfectly complementary to said first nucleic acid sequence;

dislodging said second nucleic acid sequence from said first nucleic acid sequence, thereby removing said quencher.

18. The method according to claim 17, wherein said exposure of said solid support to said sample DNA target occurs in solution.

19. The method according to claim 17, wherein said sample DNA target hybridizes with said first nucleic acid sequence at a temperature in the range from about 20°

C. to 100°

C.

20. The method according to claim 19, wherein said temperature ranges from about 25°

C. to 55°

C.

21. A method for detecting genetic polymorphisms, said method comprising:

immobilizing on a solid support a plurality of first oligonucleotide probes, each having a first nucleic acid sequence to which an emitting element is attached;

providing (1) a second probe having a second nucleic acid sequence, and (2) an unlabeled sample nucleic acid target;

hybridizing under competitive conditions said second probe and said sample nucleic acid target to said first oligonucleotide, wherein said second probe binds to said first oligonucleotide probe and said quenching element reduces an emission from said emitting element when said sample nucleic acid target is not complementary.

22. The method according to claim 21, wherein each of said sample nucleic acid target is perfectly complementary to a number of said first oligonucleotide probes.

23. The method according to claim 21, wherein each of said sample nucleic acid target is not perfectly complementary to a number of said first oligonucleotide probes.

24. The method according to claim 21, wherein each nucleic acid sequence of said second oligonucleotide probes has 1-5 artificial mismatch sites.

25. The method according to claim 24, wherein each artificial mismatch site is separated from one another by about 3-5 nucleotide positions.

26. The method according to claim 21, wherein said nucleic acid sequence of said first oligonucleotide probes are attached to solid support by covalent bonding.

27. The method according to claim 21, wherein said nucleic acid sequence of said first oligonucleotide probes are attached to a solid support by non-covalent bonding.

28. The method according to claim 21, wherein said solid support is an article selected from a group consisting or porous substrates, non-porous substrates, three-dimensional surfaces, beads and planar surfaces.

29. The method according to claim 21, wherein said solid support is an article made from materials selected from a group consisting of glass, polymers, plastics, metals, and silicon.

30. The method according to claim 21, wherein said nucleic acid sequences of said first oligonucleotide probes and said second oligonucleotide probes each has a functional length of up to about 20-25 nucleotides.

31. The method according to claim 30, wherein said nucleic acid sequences of first oligonucleotide probes and said second oligonucleotide probes each has a functional length of about 14-19 nucleotides.

32. The method according to claim 21, wherein said first oligonucleotide probe sequences are selected from the group consisting of DNA, PNA, RNA, and mixtures of DNA, PNA and RNA.

33. The method according to claim 21, wherein said sample nucleic acid target displaces said second oligonucleotide probe by a thermo-kinetic mechanism wherein said sample nucleic acid target is favored to hybridize with a perfectly complementary first oligonucleotide probe.

34. The method according to claim 33, wherein a first duplex containing at least one artificial mismatch site is formed when said first oligonucleotide probe hybridizes with said second oligonucleotide probe, and a second duplex is formed when said same first oligonucleotide probe hybridizes with a perfectly complementary sample nucleic acid target, and said second duplex has a thermodynamic stability greater than a thermodynamic stability of said first duplex.

35. The method according to claim 33, wherein said second duplex has a melting temperature (T_m) greater than a melting temperature of said first duplex.

36. The method according to claim 21, wherein said method as used in an analysis of gene expression.