Methods for detecting and assaying nucleic acid sequences

• US 5,487,973 A
• Filed: 10/19/1992
• Issued: 01/30/1996
• Est. Priority Date: 09/10/1986
• Status: Expired due to Term

• Current Assignee: CF Partners Acquisition III, LLC
• Original Assignee: Polyprobe, Inc.
• Inventors: Nilsen, Thor W., Prensky, Wolf
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/963,107
• Time in Patent Office: 1,198 Days
• Field of Search: 435/6, 435/91, 435/291, 435/5, 435/91.2, 436/501, 436/811, 536/27-29, 536/22.1, 536/23.1, 536/24.1, 536/24.3-24.33, 536/25.3, 935/32, 935/78, 935/88
• US Class Current: 435/5
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/811 Test for named disease, body condition or organ function

Abstract

A novel class of reagents for assaying nucleic acid sequences comprise successive layers of polynucleotides of a specific structure, including a double-stranded waist and single-stranded, free arms at the molecule ends, formed by hybridization of the arms to adjacent molecule arms. The reagent is used to assay for specific nucleic acid sequences. The outer layer of polynucleotides are specific for the sequence to be assayed through their non-annealed, free, single-stranded arms. The reagents are useful in the assay of a wide variety of nucleic acid sequences including those associated with pathogens such as pathogenic bacteria and virus.

Claims

1. A method of determining the presence of a specific sequence of nucleotides in a nucleic acid target molecule in a sample by detecting the presence of a hybrid thereof, and not detecting the hybrid in the absence thereof which comprises;

(A) providing a reagent for the detection and assay of the sequence of nucleotides in the nucleic acid target molecule, which comprises;

(a) a plurality of molecules, each of which comprises a first partially double-stranded polynucleotide having a structure comprising a first molecule end, a second molecule end and a double-stranded body portion intermediate of the first and second ends thereof;

said first and second ends thereof each having at least one of first and second arms consisting of a single strand of polynucleotide;

said single strands being hybridizable with a pre-determined sequence of nucleotides in a nucleic acid;

the first and second arms of each of said first and second ends being non-hybridizable with each other;

(b) a plurality of molecules, each of which comprises a second partially double-stranded polynucleotide having a structure comprising a first molecule end, a second molecule end and a double-stranded body portion intermediate of the first and second ends thereof;

said first and second ends thereof each having at least one of first and second arms thereof consisting of a single strand of polynucleotide;

said single strands being hybridizable with a pre-determined sequence of nucleotides in a nucleic acid;

the first and second arms of each of said first and second ends being non-hybridizable with each other;

said plurality of molecules of the first polynucleotide and the second polynucleotide being joined through annealing of one or more arms thereof, to form a matrix; and

at least one non-annealed arm of said plurality of first and second polynucleotide molecules located on the surface of the matrix being free to hybridize and capable of hybridizing with the sequence of nucleotides in the nucleic acid target molecule, the presence of which is to be determined;

(B) contacting the reagent with the sample under hybridization conditions such that the at least one non-annealed arm of the matrix can hybridize with sequence of nucleotides in the nucleic acid target molecule, only if present in the sample to form the hybrid thereof; and

(C) detecting the presence of the hybrid, if present, as indicative of the presence of the specific sequence of nucleotides in a nucleic acid target molecule.

2. The method of claim 1 wherein the first and second polynucleotides are molecules of DNA.

3. The method of claim 1 wherein the reagent bears a detectable, signal generating marker.

4. The method of claim 1 wherein the nucleic acid target molecule is one associated with a bacterial or a viral pathogen.

5. The method of claim 1 wherein the hybrid is bound to a water-insoluble support surface.

6. A method of detecting and assaying for the HIV-I virus in a sample by determining the presence in the sample of a sequence of nucleotides in a nucleic acid associated with HIV-I virus, by detecting a hybrid thereof, which comprises;

(A) providing a reagent, which comprises;

(a) a plurality of molecules, each of which comprises a first partially double-stranded polynucleotide having a structure comprising a first molecule end, a second molecule end and a double-stranded body portion intermediate of the first and second ends thereof;

said first and second ends each having at least one of first and second arms consisting of a single strand of polynucleotide;

said single strands being hybridizable with a pre-determined sequence of nucleotides in a nucleic acid;

the first and second arms of each of said first and second ends being non-hybridizable with each other;

(b) a plurality of molecules, each of which comprises a second partially double-stranded polynucleotide having a structure comprising a first molecule end, a second molecule end and a double-stranded body portion intermediate of the first and second ends thereof;

said first and second ends thereof each having at least one of first and second arms thereof consisting of a single strand of polynucleotide;

said single strands being hybridizable with a pre-determined sequence of nucleotides in a nucleic acid;

the first and second arms of each of said first and second ends being non-hybridizable with each other;

said plurality of molecules of the first polynucleotide and the second polynucleotide being joined together through annealing of one or more arms thereof, to form a matrix; and

at least one non-annealed arm of said plurality of first and second polynucleotide molecules located on the outer surface of the matrix being free to hybridize and capable Of hybridizing with the sequence of nucleotides in the nucleic acid associated with the HIV-I virus, the presence of which is to be determined;

(B) contacting the reagent with the sample under hybridization conditions such that the at least one non-annealed arm of the matrix can hybridize with the sequence of nucleotides in the nucleic acid associated with the HIV-I virus, only if present in the sample, to form a hybrid thereof; and

(C) detecting the presence of the hybrid, if present, as indicative of the presence in a sample of a sequence of nucleotides in a nucleic acid associated with HIF-I virus.

7. The method of claim 6 wherein the reagent bears a detectable, signal generating marker.

8. The method of claim 6 wherein the hybrid is bound to a water-insoluble support surface.

9. A method of detecting or quantitating a specific sequence of nucleotides in an analyte of interest by detecting the presence of or quantitating the amount present of a hybrid thereof and not detecting the presence or quantitating the amount of the hybrid in the absence thereof, which method comprises:

(a) forming a mixture of

(i) a sample which may contain said sequence of nucleotides in the analyte of interest, and

(ii) a composition containing a plurality of polynucleotides each having at at least three single stranded hybridization regions, wherein any one polynucleotide is bonded by hybridization or hybridization and covalent bonding to any other polynucleotide at least one such hybridization region, and when bonded to more than one such hybridization region of the same polynucleotides are not bonded, and wherein a plurality of the single stranded hybridization regions are not bonded to any other polynucleotides, at least one of the non-bonded single stranded hybridization regions of said composition being complementary to the sequence of nucleotides in the analyte of interest;

(b) allowing said composition and said sequence of nucleotides in the analyte of interest, only if present in the sample, to hybridize and form the hybrid; and

(c) detecting the presence of or quantitating the amount present of the hybrid, if present, as indicative of the presence or quantity, respectively, of a specific sequence of nucleotides in the analyte of interest.

10. A method according to claim 9, wherein the composition is bound to a non-nucleic acid support.

11. A method according to claim 9, wherein the composition further contains a label and said label is detected or quantitated in said hybrid.

12. A method according to claim 9, wherein the analyte of interest is a sequence of nucleotides of a pathogen.

13. A method according to claim 9, wherein the pathogen is a HIV-I virus.

14. A method of detecting or quantitating a specific sequence of nucleotides in an analyte of interest by detecting the presence of or quantitating the amount present of a hybrid complex thereof and not detecting the presence or quantitating the amount of the hybrid complex in the absence thereof, which method comprises:

(a) forming a first mixture of

(i) a simple which may contain said sequence of nucleotides in the analyte of interest and

(ii) a first composition containing a plurality of polynucleotides each having at least three single stranded hybridization regions, wherein any one polynucleotide is bonded by hybridization or hybridization and covalent bonding to any other polynucleotide at least one such hybridization region, and when bonded to more than one such hybridization region of the same polynucleotide there is an intermediate region where the two polynucleotides are not bonded, and wherein a plurality of the single stranded hybridization regions are not bonded to any other polynucleotides;

at least one of the non-bonded single stranded hybridization regions can be capable of hybridizing with the sequence or a portion of the sequence of nucleotides in the analyte of interest;

at least one of the non-bonded single-stranded hybridization regions can be capable of hybridizing with non-bonded single stranded hybridization regions of a second composition; and

at least one of the non-bonded single stranded hybridization regions is capable of hybridizing either to the sequence or portion of the sequence of nucleotides in the analyte of interest or to the non-bonded single stranded hybridization regions of the second composition;

(b) optionally allowing said first composition and said sequence of nucleotides in the analyte of interest to hybridize and form a first hybrid complex only if said sequence of nucleotides in the analyte of interest is present in the sample and if the at least one non-bonded single stranded hybridization regions of the first composition is capable of hybridizing with the sequence or a portion of the sequence of nucleotides in the analyte of interest;

(c) forming a second mixture of

(i) said first mixture and

(ii) the second composition, said second composition containing a plurality of polynucleotides each having at least three single stranded hybridization regions, wherein any one polynucleotide is bonded by hybridization or hybridization and covalent bonding to any other polynucleotide at at least one such hybridization region, and when bonded to more than one such hybridization region of the same polynucleotide there is an intermediate region where the two polynucleotides are not bonded, and wherein a plurality of the single stranded hybridization regions are not bonded to any other polynucleotides, at least one of the non-bonded single stranded hybridization regions of said second composition being complementary to at least one of the non-bonded single stranded hybridization regions of said first composition or to at least a portion of the sequence of nucleotides in the analyte of interest; and

(d) subjecting said second mixture to hybridization conditions to form a second hybrid complex only if the sequence of nucleotides in the analyte of interest is present, wherein said second hybrid complex comprises;

(i) the first and second compositions each hybridization bonded to the analyte of interest, (ii) the first and second compositions hybridization bonded to each other and the second composition hybridization bonded to the analyte of interest, or (iii) the first and second compositions hybridization bonded to each other and the first composition hybridization bonded to the analyte of interest; and

(e) detecting the presence of or quantitating the amount present of said second hybrid complex and thus of the specific sequence of nucleotides in the analyte of interest.

15. A method according to claim 14, wherein at least one of the first and second compositions is bound to a non-nucleic acid support.

16. A method according to claim 14, wherein at least one of the first and second compositions contains a label and said label is detected in said second hybrid complex.

17. A method according to claim 14, wherein the analyte of interest is a sequence of nucleotides of a pathogen.

18. A method according to claim 17, wherein the pathogen is a HIV-I virus.

19. A method of detecting or quantifying a specific sequence of nucleotides in an analyte of interest by detecting the presence of or quantitating the amount present of a hybrid complex thereof and not detecting the presence or quantitating amount of the hybrid complex in the absence thereof, which method comprises:

(a) forming a mixture of

(i) a sample which may contain said sequence of nucleotides in the analyte of interest and

(ii) first and second compositions containing a plurality of polynucleotides each having at least three single stranded hybridization regions, wherein any one polynucleotide is bonded by hybridization or hybridization and covalent bonding to any other polynucleotide at at least one such hybridization region, and when bonded to more than one such hybridization region of the same polynucleotide there is an intermediate region where the two polynucleotides are not bonded, and wherein a plurality of the single stranded hybridization regions are not bonded to any other polynucleotides, at least one of the non-bonded single stranded hybridization regions of said first and of said second compositions being complementary to different parts respectively of the sequence of nucleotides in the analyte of interest and to non-bonded single stranded hybridization regions of a third composition;

(b) allowing said first and second compositions and said sequence of nucleotides in the analyte of interest to hybridize forming a first hybrid complex only if said sequence of nucleotides in the analyte of interest is present in the sample;

(c) forming a mixture of

(i) said first hybrid complex and

(ii) the third composition, said third composition containing a plurality of polynucleotides each having at least three single stranded hybridization regions, wherein any one polynucleotide is bonded by hybridization or hybridization and covalent bonding to any other polynucleotide at at least one such hybridization region, and when bonded to more than one such hybridization region of the same polynucleotide there is an intermediate region where the two polynucleotides are not bonded, and wherein a plurality of the single stranded hybridization regions are not bonded to any other polynucleotides, at least one of the non-bonded single stranded hybridization regions of said third composition being complementary to at least one of the non-bonded single stranded hybridization regions of at least one of said first and second compositions;

(d) allowing said first hybrid complex and said third composition to hybridize forming a second hybrid complex; and

(e) detecting the presence of or quantitating the amount present of said second hybrid complex and thus of the specific sequence of the nucleotides in the analyte of interest.

20. A method according to claim 19, wherein at least one of the first, second and third compositions is bound to a non-nucleic acid support.

21. A method according to claim 19, wherein at least one of the first, second and third compositions contains a label and said label is detected in said second hybrid complex.

22. A method according to claim 19, wherein the analyte of interest is a sequence of nucleotides of a pathogen.

23. A method according to claim 22, wherein the pathogen is a HIV-I virus.

24. A method of determining the concentration of a specific sequence of nucleotides in an analyte of interest in a composition by reference to sizes of compositions in hybrid complexes thereof and not determining the concentration of the sequence of nucleotides in the analyte of interest in the composition in the absence of hybrid complexes thereof, which method comprises:

(a) contacting

(i) a plurality of compositions each containing a plurality of polynucleotides each having at least three single stranded hybridization regions, wherein any one polynucleotide is bonded by hybridization or hybridization and covalent bonding to any other polynucleotide at at least one such hybridization region, and when bonded to more than one such hybridization region of the same polynucleotide there is an intermediate region where the two polynucleotides are not bonded, and wherein a plurality of the single stranded hybridization regions are not bonded to any other polynucleotides, at least one of the non-bonded single stranded hybridization regions of each of said compositions being complementary to the sequence of nucleotides in the analyte of interest, and each said composition being a different size, and

(ii) a solid support containing bound sequence of nucleotides in the analyte of interest, under conditions conducive to hybridization of said compositions and said analyte of interest to form hybrid complexes on the solid support, only if said sequence of nucleotides in the analyte of interest is present in the sample;

(b) washing the solid support to remove unhybridized compositions;

(c) detecting said hybrid complexes if present, on the solid support; and

(d) determining the concentration of said analyte of interest by reference to the sizes of the compositions of said hybrid complexes on the solid support.

25. A method of detecting or quantitating a specific sequence of nucleotides in an analyte of interest by detecting the presence of or quantitating the amount of hybrid thereof and not detecting the presence or quantitating amount of the hybrid in the absence thereof, which method comprises

(a) contacting

(i) a single which may contain said sequence of nucleotides in the analyte of interest and

(ii) a composition containing a plurality of polynucleotides each having at least three single stranded hybridization regions, wherein any one polynucleotide is bonded by hybridization or hybridization and covalent bonding to any other polynucleotide at at least one such hybridization region, and when bonded to more than one such hybridization region of the same polynucleotide there is an intermediate region where the two polynucleotides are not bonded, said composition bound to a solid support, and, at least one of said single stranded hybridization regions capable of hybridizing under conditions conducive to hybridization with said sequence of nucleotides, only if present in the analyte of interest to form a hybrid;

and

(b) detecting the presence of or quantitating the amount of the hybrid, if present, as indicative the presence of quantity, respectively, of the specific sequence of nucleotides in the analyte of interest.

26. A method according to claim 25, wherein the analyte of interest is a sequence of nucleotides of a pathogen.

27. A method according to claim 25, wherein the pathogen is a HIV-I virus.