Polynucleotide determination with selectable cleavage sites

• US 4,775,619 A
• Filed: 10/16/1984
• Issued: 10/04/1988
• Est. Priority Date: 10/16/1984
• Status: Expired due to Term

• Current Assignee: Chiron Corporation (Bayer AG)
• Original Assignee: Chiron Corporation (Bayer AG)
• Inventors: Urdea, Mickey S.
• Primary Examiner(s): Warren, Charles F.
• Assistant Examiner(s): Jay, Jeremy M.
• Application Number:
  US06/661,508
• Time in Patent Office: 1,449 Days
• Field of Search: 435/6, 435/803, 435/91, 435/810, 935/78, 436/501, 436/518, 436/527, 436/532, 436/533, 436/824, 536/27
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Methods for diagnosis employing polynucleotides having oligonucleotide sequences substantially homologous to a sequence of interest, where the presence or absence of hybridization at a predetermined stringency provides for the release of a label from a support. Particularly, various techniques are employed for binding a label to a support, whereupon cleavage of either a single or double strand, a label may be released from a support, where the release of the label can be detected as indicative of the presence of a particular oligonucleotide sequence in a sample. The method finds use in diagnosis of disease, genetic monitoring, and analyzing nucleic acid mixtures.

Claims

1. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample,

said method comprising:

combining under hybridizing conditions said nucleic acid sample with a polynucelotide reagent, wherein one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynuceleotide reagent results in a label being bound to said support through a selectable cleavage site, wherein said selectable cleavage site is a restriction site resulting from the formation of a duplex at the oligonucleotide sequence of interest;

substantially freeing said support of label bound to said support other than through said selectable cleavage site;

cleaving said cleavage site with a restriction endonuclease which recognizes said site; and

detecting label free of said support.

2. A method according to claim 1, wherein said hybridization is effected through the binding of at least 4 complementary base pairs.

3. A method according to claim 1, wherein said polynucelotide reagent comprises a labeled polynucleotide bound to a support.

4. A method according to claim 1, wherein said polynucleotide reagent comprises a first polynucleotide bound to a support and a labeled second polynucleotide, wherein said first and second polynucleotides have oligonucleotide sequences complementary to sequences present in said analyte to form duplexes under said hybridizing conditions, at least one of said oligonucleotide sequence being a sequence of interest, wherein (1) either of said duplexes defines a restriction site, as said selectable cleavage site and said cleavage is with a restriction endonulease which recognizes said site, or (2) said label is linked to said polynucleotide through a selectable cleavage site and said cleavage is by cleaving said selectable cleavage site enzymatically or with light.

5. A method according to claim 1, wherein said nucleic acid sample is bound to said support and said polynucleotide reagent is labeled polynucleotide.

6. A method according to claim 1, wherein said label is a radionuclide.

7. A method according to claim 1, wherein said label is a fluorescer.

8. A method according to claim 1, wherein said label is an enzyme.

9. A method according to claim 1, wherein said label is an enzyme substrate or cofactor.

10. A method according to claim 1, wherein there is more than one label bound to said support through a plurality of selectable cleavage sites;

and

a plurality of cleavage sites are cleaved.

11. A method according to claim 1, wherein said hybridization is effected through the binding of at least 12 complementary base pairs.

13. A method according to claim 11, wherein said polynucleotide reagent comprises a labelled polynucleotide bound to a support.

14. A method according to claim 11, wherein said polynucleotide reagent comprises a first polynucleotide bound to a support and a labeled second polynucleotide, wherein said first and second polynucleotides have oligonucleotide sequences complementary to sequences present in said analyte to form duplexes, at least one of said oligonucleotide sequences being a sequence of interest, wherein (1) either of said duplexes defines a restriction site as said selectable cleavage site and said cleavage is with a restriction endonuclease which recognizes said site, or (2) said label is linked to said polynucleotide through a selectable cleavage site.

15. A method according to claim 14, wherein said nucleic acid sample is bound to said support and said polynucleotide reagent is labeled polynucleotide.

12. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample,

said method comprising:

combining under hybridizing conditions in an aqueous medium, said nucleic acid sample with a polynucleotide reagent, where one of said sample or a component of said reagent is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through a selectable cleavage site, wherein said selectable cleavage site is a restriction site resulting from the formation of a duplex at the oligonucleotide sequence of interest;

separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium;

washing said support with a medium of different hybridizing stringency from said aqueous medium to remove label bound to said support other than through said selectable cleavage site;

cleaving said cleavage site with a restriction endonuclease which recognizes said site; and

detecting label free of said support.

16. A method according to claim 12, wherein said hybridization is effected through the binding of at least 4 complementary base pairs.

17. A method according to claim 12, wherein said hybridization is effected through the binding of at least 12 complementary base pairs.

18. A reagent for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a polynucleotide sequence bound proximal to one end to a support and proximal to the opposite end to a label capable of providing, directly or indirectly, a detectable signal, said polynucleotide sequence having a sequence which when duplexed with said sequence of interest defines a selectable cleavage site cleavable by a restriction endonuclease which recognizes said site.

19. A reagent composition for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a first polynucleotide having a first oligonucleotide sequence complementary to a first portion of said sequence of interest;

and a labeled second polynucleotide, wherein said label provides, directly or indirectly a detectable signal, said second labeled polynucleotide having a second oligonucleotide sequence complementary to a second portion of said sequence of interest, said first and second portions comprising the entirety of said sequence of interest, wherein a continuous duplex may be formed, under hybridizing conditions, between said first oligonucleotide sequence and said first portion of said sequence of interest and between said second oligonucleotide sequence and said second portion of said sequence of interest, said continuous duplex containing a selectable cleavage site cleavage by a restriction endonuclease which recognizes said site.

20. A deoxyribonucleotide reagent comprising a deoxyribonucleotide sequence of at least four bases joined to a glass particle support through the reaction product of a semicarbazide and the dialdehyde reaction product of a ribonuceleotide and an oxidant, wherein said at least four bases are selected so as to form a duplex with a sequence of interest under hybridizing conditions, the duplex so formed containing a selectable cleavage site cleavable with a restriction endonuclease which recognizes said site.

21. A deoxyribonucleotide reagent comprising a deoxyribonucleotide sequence of at least four bases joined to a glass support through a phosphothiacetamide link, said link formed by reaction between a bromoacetamide substitutent on said glass support and a 5'"'"'-thophosphate moiety on said deoxyribonucleotide reagent, wherein said at least four bases are selected so as to form a duplex with a sequence of interest under hybridizing conditions, the duplex so formed containing a selectable cleavage site cleavable with a restriction endonuclease which recognizes said site.

22. The reagent composition of claim 21, wherein said duplex is formed from at least 12 complementary base pairs.

23. A reagent composition for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, comprising a first component which is a polynucleotide bound to a support and a second component which is a labeled second polynucleotide, wherein said label provides, directly or indirectly, a detectable signal, wherein said first and said second polynucleotides have oligonucleotide sequences complementary to sequences present in said analyte which form duplexes with said analyte under hybridizing conditions, at least one of said oligonucleotide sequences being complementary to a sequence of interest, wherein one of said duplexes defines a selectable cleavage site cleavable by a restriction endonuclease which recognizes said site.

24. The reagent composition of claim 23, wherein said duplexes are formed from at least 4 complementary base pairs.

25. The reagent composition of claim 23, wherein said duplexes are formed from at least 12 complementary base pairs.