Method for detecting polynucleotides with immobilized polynucleotide probes identified based on T.sub.m

• US 5,639,612 A
• Filed: 01/26/1995
• Issued: 06/17/1997
• Est. Priority Date: 07/28/1992
• Status: Expired due to Term

• Current Assignee: Hitachi Chemical Company Limited (Resonac Holdings Corp.)
• Original Assignee: Hitachi Chemical Company Limited (Resonac Holdings Corp.)
• Inventors: Mitsuhashi, Masato, Cooper, Allan
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/379,078
• Time in Patent Office: 873 Days
• Field of Search: 435/6, 435/5, 435/810, 536/24.3, 536/24.31, 536/24.32, 935/78
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

A method for detecting the presence of a particular organism, infectious agent, or biological component of a cell or organism in a sample, based on sandwich hybridization in which first and second probes are used, and the specificity of the first probe is determined based on its melting temperature (T_m) with the target polynucleotide at a selected sodium and formamide concentration.

Claims

1. A method of identifying probes and thereafter detecting the presence of a particular organism, infectious agent, or biological component of a cell or organism in a sample derived from biological material that contains polynucleotides, by detecting an analyte polynucleotide in said sample that is indicative of the presence of said organism, infectious agent, or biological component, said method comprising the step of:

(a) identifying a first polynucleotide probe having a nucleotide sequence complementary to a first nucleotide sequence contained in an analyte polynucleotide specific to a particular organism, infectious agent, or biological component, said first polynucleotide probe having a melting temperature (T_m) within a preselected range with said analyte polynucleotide at a selected sodium and formamide concentration, by a method comprising;

(i) retrieving from a database a plurality of user-selected gene sequences including a gene sequence from said organism, infectious agent, or biological component, and inputting said sequences into a computer system for calculating T_m ;

(ii) calculating the T_m '"'"'s of a plurality of candidate probes having nucleotide sequences derived from the gene sequence from said organism, infectious agent, or biological component, at the selected sodium and formamide concentration with said gene sequence from said organism, infectious agent, or biological component, using said computer system;

(iii) calculating the T_m '"'"'s of said plurality of candidate probes having nucleotide sequences at the selected sodium and formamide concentration with each of said user-selected gene sequences other than that of said gene sequence from said organism, infectious agent, or biological component; and

(iv) identifying said first polynucleotide probe as the candidate probe which has a T_m with said analyte polynucleotide within the preselected range at the selected sodium and formamide concentration, and that has the lowest T_m overall with the other user-selected gene sequences;

(b) immobilizing said first polynucleotide probe to a solid support;

(c) hybridizing said analyte polynucleotide in said sample to said first nucleotide sequence of said first polynucleotide probe, if said analyte polynucleotide is present in said sample;

(d) identifying a second polynucleotide probe having a nucleotide sequence complementary to a second nucleotide sequence contained in said analyte polynucleotide, said second polynucleotide probe being different from said first nucleotide sequence, said second polynucleotide probe having a T_m with said analyte polynucleotide at a selected sodium and formamide concentration, said T_m being approximately the same or lower than the T_m of said first polynucleotide probe;

(e) hybridizing said second polynucleotide probe to said second sequence of said analyte polynucleotide hybridized to said first polynucleotide probe; and

(f) determining the presence of said particular organism, infectious agent, or biological component in said sample by detecting the presence of said second polynucleotide probe on said solid support.

2. A method according to claim 1, wherein said second nucleotide sequence is common to a plurality of different organisms, infectious agents, or biological components.

3. The method of claim 2, wherein said first polynucleotide probe has a T_m within the range of from approximately 48°

C. to approximately 60°

C.

4. The method of claim 2, wherein a label is attached to said second polynucleotide probe, and wherein the determining step comprises detecting the presence of said label.

5. The method of claim 4, wherein said label is selected from the group consisting of a radionuclide, an enzyme, an enzyme substrate, a specific binding moiety, an binding partner for a specific binding moiety, biotin, avidin, a nucleic acid stain, and a fluorescent material.

6. The method of claim 5, wherein said label is a nucleic acid stain selected from the group consisting of ethidium bromide, yoyo-1, and toro-1.

7. The method of claim 4, wherein said label can be measured by light emitted therefrom, and wherein step (f) comprises measuring the amount of light emitted by said label.

8. The method of claim 7, wherein the step of measuring the amount of light emitted by said label comprises:

recording the amount of light on film; and

measuring the exposure of the film using a densitometer.

9. The method of claim 7, wherein said label comprises alkaline phosphatase, and wherein step (f) of claim 1 comprises adding ATTOPHOS and measuring the fluorescense emitted using a fluorimeter.

10. The method of claim 2, wherein said solid support comprises a microtiter plate having a plurality of wells.

11. The method of claim 2, wherein said first polynucleotide probe comprises DNA.

12. The method of claim 2, wherein said first and second polynucleotide probes comprise DNA.

13. The method of claim 2, additionally comprising the step of washing said solid support after hybridizing said analyte polynucleotide in said sample to said first polynucleotide probe so that substantially all of said biological sample not annealed to said first polynucleotide probe is removed from said solid support.

14. The method of claim 2, additionally comprising the step of washing said solid support after hybridizing said second polynucleotide probe with said analyte polynucleotide in said sample which is hybridized to said first polynucleotide probe so that substantially all of said second polynucleotide probe not hybridized with said analyte polynucleotide is removed from said solid support.

15. The method of claim 2, wherein the polynucleotide hybridized to said first polynucleotide probe is selected from the group consisting of mRNA, rRNA, and genomic DNA.

16. The method of claim 1, wherein said melting temperature is determined by the formula;

space="preserve" listing-type="equation">Tm=81.5-16.6(log[Na])-0.63%(formamide)+0.41(%/(G+C))-600/N,

wherein Log[Na] is the log function of the sodium concentration, 0.63% (formamide) is the concentration of formamide, %(G+C) is the percentage of matched GC base pairs, and N is the probe length.

24. The method of claim 10, wherein each of said wells has a first polynucleotide probe covalently immobilized thereon.

28. The method of claim 1, wherein step (a) further comprises determining the hybridization strengths between said user-selected gene sequences and said candidate polynucleotide sequences by the calculated T_m '"'"'s in steps (a)(ii) and (iii), and identifying said first polynucleotide probe as the candidate probe in step (a)(iv) by numbers of hybridizations or false hybridizations to said user-selected gene sequences calculated by said hybridization strengths.

29. The method of claim 1, wherein said candidate probes in step (a) are selected at positions along said analyte polynucleotide, based on a nucleation threshold that places a minimum value on the number of base pairs at any nucleation site, selected by the user, in addition to said preselected T_m range.

30. The method of claim 1, wherein said preselected T_m range is specified by a minimum T_m with said analyte polynucleotide.

31. The method of claim 1, wherein said second polynucleotide probe is identified by a method comprising the steps of:

calculating the T_m '"'"'s of a plurality of candidate probes at the selected sodium and formamide concentration with said analyte polynucleotide at every possible hybridization point; and

identifying said second polynucleotide probe as the candidate probe which has a T_m within the preselected range with the nucleotide sequence complementary to the second nucleotide sequence of said analyte polynucleotide other than the first nucleotide sequence of said analyte polynucleotide and has the lowest T_m with the nucleotide sequence complementary to said first polynucleotide probe.

32. The method of claim 31, wherein said candidate probes are selected at positions along said analyte polynucleotide, based on a nucleation threshold that places a minimum value on the number of base pairs at any nucleation site, selected by the user, in addition to said preselected T_m range.

33. The method of claim 31, wherein a preselected T_m range for said second polynucleotide probe is specified by a minimum T_m with said analyte polynucleotide.

34. The method of claim 31, wherein said melting temperature is determined by the formula;

space="preserve" listing-type="equation">Tm=81.5-16.6(log[Na])-0.63%(formamide)+0.41(%(G+C))-600/N,

wherein Log[Na] is the log function of the sodium concentration, 0.63% (formamide) is the concentration of formamide, %(G+C) is the percentage of matched GC base pairs, and N is the probe length.

35. The method of claim 10, wherein said first probe is immobilized to said microtiter plate via its 5'"'"' end.

36. The method of claim 10, wherein step (a) further comprises identifying in the same way at least one different first polynucleotide probe, each different first polynucleotide probe having a nucleotide sequence complementary to a first nucleotide sequence contained in a different analyte polynucleotide, specific to a particular organism, infectious agent, or biological component, each different first polynucleotide probe having a T_m with said analyte polynucleotide at a selected sodium and formamide concentration, said T_m being about the same as that of any other first polynucleotide probe so that each first polynucleotide probe can be hybridized to the corresponding analyte polynucleotide under the same conditions;

step (b) further comprises immobilizing each first polynucleotide probe to a different well of said microtiter plate having a plurality of wells;

in step (c), hybridization is conducted in such a way that each analyte polynucleotide in said sample is hybridized with each first polynucleotide probe, if said analyte polynucleotide is present in said sample, under the same conditions, whereby plural types of organisms, infectious agents, or biological components are simultaneously detected.

17. A kit for identifying the presence of an organism, infectious agent, or biological component of a cell or organism in a biological sample, comprising:

a microtiter plate having a plurality of wells, to which at least two different polynucleotide probes are immobilized, each polynucleotide probe being immobilized to a different well, each polynucleotide probe being complementary to or homologous to a first nucleotide sequence in an analyte polynucleotide specific to a different organism, infectious agent, or biological component to be detected, each polynucleotide probe having approximately the same T_m within the range of from approximately 48°

C. to approximately 60°

C., each polynucleotide probe being identified by a method comprising;

(i) retrieving from a database a plurality of user-selected gene sequences including a gene sequence from said organism, infectious agent, or biological component, and inputting said sequences into a computer system for calculating T_m ;

(ii) calculating the T_m '"'"'s of a plurality of candidate probes having nucleotide sequences derived from the gene sequence from said organism, infectious agent, or biological component, at the selected sodium and formamide concentration with said gene sequence from said organism, infectious agent, or biological component, using said computer system;

(iii) calculating the T_m '"'"'s of said plurality of candidate probes having nucleotide sequences at the selected sodium and formamide concentration with each of said user-selected gene sequences other than that of said gene sequence from said organism, infectious agent, or biological component; and

(iv) identifying each of said polynucleotide probes as the candidate probe which has a T_m with said analyte polynucleotide within the preselected range at the selected sodium and formamide concentration, and that has the lowest T_m overall with the other user-selected gene sequences; and

a common polynucleotide probe complementary to or homologous to a second nucleotide sequence in said analyte polynucleotide of said organism, infectious agent, or biological component, said common polynucleotide probe being complementary to polynucleotide contained in a plurality of different organisms, infectious agents, or biological components, said common polynucleotide probe having about the same or lower T_m than each specific polynucleotide probe.

18. The kit of claim 17, additionally comprising at least one of the following:

dNTP'"'"'s, a reverse transcriptase, a polymerase, and a buffer appropriate for addition of dNTP'"'"'s to a primer using a reverse transcriptase or polymerase.

19. The kit of claim 17, additionally comprising a buffer appropriate for the hybridization of said probes and polynucleotides, said polynucleotides being selected from the group consisting of mRNA, rRNA, and genomic DNA.

20. The kit of claim 17, wherein said second polynucleotide probe bears a label.

21. The kit of claim 20, wherein said label is selected from the group consisting of a radionuclide, an enzyme, an enzyme substrate, a specific binding moiety, an binding partner for a specific binding moiety, biotin, avidin, a nucleic acid stain, and a flouresecent material.

22. The kit of claim 17, wherein said specific polynucleotide probe comprises a first specific primer which is complementary or homologous to a sequence specific to a particular organism, infectious agent, or biological component, said kit additionally comprising a second specific polynucleotide primer which is complementary or homologous to a different sequence specific to said organism, infectious agent, or biological component.

23. The kit of claim 17, including a DNA polymerase that has significant polymerase activity at temperatures above 50°

C.

25. A method of identifying probes and thereafter detecting the presence of an organism, infectious agent, or biological component of a cell or organism in a sample derived from biological material that contains polynucleotides, by detecting an analyte polynucleotide in said sample that is indicative of the presence of said organism, infectious agent, or biological component, said method comprising the steps of:

(a) immobilizing a first polynucleotide probe to a solid support, said first polynucleotide probe having a nucleotide sequence complementary to a first nucleotide sequence contained in an analyte polynucleotide, common to a plurality of different organisms, infectious agents, or biological components, said first polynucleotide probe having a T_m within a preselected range between approximately 48°

C. and approximately 60°

C. with said analyte polynucleotide at a selected sodium and formamide concentration;

(b) identifying a second polynucleotide probe having a label thereon, wherein the nucleotide sequence of said second polynucleotide probe is complementary to a second nucleotide sequence contained in said analyte polynucleotide, specific to an organism, infectious agent, or biological component, and has approximately the same T_m as said first polynucleotide probe so that said first and second polynucleotide probes can hybridize to said analyte polynucleotide under the same conditions, said identification being conducted, by a method comprising;

(i) retrieving from a database a plurality of user-selected gene sequences including the gene sequence of said organism, infectious agent, or biological component, and inputting said sequences into a computer system for calculating T_m ;

(ii) calculating the T_m '"'"'s of a plurality of candidate probes having nucleotide sequences derived from the gene sequence from said organism, infectious agent, or biological component, at the selected sodium and formamide concentration with said gene sequence from said organism, infectious agent, or biological component, using said computer system;

(iii) calculating the T_m '"'"'s of said plurality of candidate probes having nucleotide sequences at the selected sodium and formamide concentration with each of said user-selected gene sequences other than that of said gene sequence from said organism, infectious agent, or biological component; and

(iv) identifying said second polynucleotide probe as the candidate probe which has a T_m within the preselected range with said analyte polynucleotide and has the lowest T_m overall with the other user-selected gene sequences;

(c) contacting the polynucleotides present in said sample with said first polynucleotide probe and said second polynucleotide probe having a label thereon with said polynucleotides present in said sample, whereby said first nucleotide sequence contained in said analyte polynucleotide hybridizes to said first polynucleotide probe, and said second polynucleotide probe hybridizes to said second nucleotide sequence contained in said analyte polynucleotide, if said analyte polynucleotide is present in said sample; and

(d) determining the presence of said organism, infectious agent, or biological component in said sample by detecting the presence of said label on said second polynucleotide probe if hybridized to said analyte polynucleotide.

26. The method of claim 25, wherein said solid support is a microtiter plate having a plurality of wells, each of said wells having a specific polynucleotide probe covalently immobilized thereon.

27. The method of claim 25, additionally comprising the step of:

washing said solid support after hybridizing said first and second polynucleotide probes so that substantially all of said second polynucleotide probe not annealed to said analyte polynucleotide is removed from said solid support.

37. A method of identifying probes and thereafter detecting the presence of a particular polynucleotide in a sample, said method comprising the step of:

(a) identifying a first polynucleotide probe having a nucleotide sequence complementary to a first nucleotide sequence contained in said particular polynucleotide, said first polynucleotide probe having a melting temperature (T_m) within a preselected range with said particular polynucleotide at a selected sodium and formamide concentration, by a method comprising;

(i) retrieving from a database a plurality of user-selected gene sequences including the gene sequence of said organism, infectious agent, or biological component, and inputting said sequences into a computer system for calculating T_m ;

(ii) calculating the T_m '"'"'s of a plurality of candidate probes having nucleotide sequences derived from the gene sequence from said organism, infectious agent, or biological component, at the selected sodium and formamide concentration with said gene sequence form said organism, infectious agent, or biological component, using said computer system;

(iii) calculating the T_m '"'"'s of said plurality of candidate probes having nucleotide sequences at the selected sodium and formamide concentration with each of said user-selected gene sequence other than that of said gene sequence from said organism, infectious agent, or biological component; and

(iv) identifying said first polynucleotide probe as the candidate probe which has a T_m within the preselected range with said particular polynucleotide indicative of the presence of said organism, infectious agent, or biological component, and that has the lowest T_m overall with the other user-selected gene sequences;

(b) immobilizing said first polynucleotide probe to a solid support;

(c) hybridizing said particular polynucleotide in said sample to said first nucleotide sequence of said first polynucleotide probe, if said particular polynucleotide is present in said sample;

(d) identifying a second polynucleotide probe having a nucleotide sequence complementary to a second nucleotide sequence contained in said particular polynucleotide, different from said first nucleotide sequence, and having a T_m with said particular polynucleotide at a selected sodium and formamide concentration, said T_m being approximately the same or lower than the T_m of said first polynucleotide probe;

(e) hybridizing said second polynucleotide probe to said second sequence of said particular polynucleotide hybridized to said first polynucleotide probe; and

(f) determining the presence of said particular polynucleotide in said sample by detecting the presence of said second polynucleotide probe on said solid support.