Amplification method for polynucleotide detection assays

• US 5,397,698 A
• Filed: 11/02/1993
• Issued: 03/14/1995
• Est. Priority Date: 07/23/1987
• Status: Expired due to Term

• Current Assignee: Dade Behring Marburg GmbH (Siemens AG)
• Original Assignee: Syntec Incorporated (Atos SE)
• Inventors: Rose, Samuel, Ullman, Edwin F., Becker, Martin, Goodman, Thomas C.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/146,297
• Time in Patent Office: 497 Days
• Field of Search: 435/6, 435/91.1, 536/22.1
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

A method is disclosed for producing multiple copies of a primary polynucleotide sequence located at the 3'"'"' terminus of a polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a primary polynucleotide sequence hybridized with a template sequence of a single stranded pattern polynucleotide comprising two or more template sequences each containing one or more site specific cleavage sequences, (b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences, (c) dissociating said fragments, (d) hybridizing said fragments with single stranded pattern polynucleotide, and repeating steps (a)-(d). Steps (a)-(d) may be conducted simultaneously or wholly or partially sequentially. The method may be applied in the detection of a polynucleotide analyte in a sample suspected of containing such analyte to facilitate such detection. Also disclosed are compositions for conducting the method of the invention.

Claims

1. A method of determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises:

(a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a polynucleotide analyte having a 3'"'"' hydroxy nucleotide, a part thereof including said 3'"'"' hydroxy nucleotide being hybridized with a binding polynucleotide sequence of a single stranded pattern polynucleotide comprising said binding polynucleotide sequence connected at its 5'"'"' end to two or more template sequences each containing one or more site specific cleavage sequences,

(b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences,

(c) dissociating said fragments,

(d) hybridizing said fragments with said single stranded pattern polynucleotide,

(e) forming in the presence of said nucleoside triphosphates and said template dependent polynucleotide polymerase an extension of said fragments hybridized with said single stranded pattern polynucleotide,

(f) repeating steps (b)-(e) above wherein steps (b)-(e) are conducted simultaneously or wholly or partially sequentially, and

(g) detecting said fragments or fragments complementary thereto, the presence thereof indicating the presence of said polynucleotide analyte in said sample.

2. The method of claim 1 wherein said fragments are formed by a restriction enzyme and said single stranded pattern polynucleotide is DNA.

3. The method of claim 1 wherein said nucleotide triphosphates are selected from three members of the group consisting of dATP, dTTP, dGTP, and dCTP and derivatives thereof and said template sequences and the sequences connecting said template sequences consist of a continuous sequence of nucleotides selected from three members of the group consisting of A and dA, U and dT, C and dC, and G and dG and derivatives thereof that are complementary to said nucleoside triphosphates.

4. The method of claim 1 wherein prior to said forming said sample is incubated sequentially with (1) reagents for modifying the 3'"'"' end of any polynucleotide in said sample to prevent said 3'"'"' end from reacting with said template-dependent polynucleotide polymerase and (2) a restriction endonuclease capable of cleaving said polynucleotide analyte.

5. The method of claim 4 wherein said reagents comprise an enzyme capable of catalyzing a reaction of a polynucleotide 3'"'"'-hydroxyl group.

6. The method of claim 4 wherein said reagents are a ligase and an oligonucleotide terminated at the 3'"'"' end by a group that does not react to provide chain extension by said template-dependent polynucleotide polymerase.

7. The method of claim 4 wherein said reagents are a terminal transferase and a dideoxynucleoside triphosphate.

8. The method of claim 1 for determining the presence of a RNA analyte in a sample suspected of containing said RNA analyte, wherein said polynucleotide analyte terminating in a 3'"'"'-hydroxy nucleotide is provided by:

combining in an aqueous medium said sample, a single stranded DNA primer comprising a deoxynucleic acid sequence containing a restriction site and capable of hybridizing with said RNA sequence, and a restriction enzyme capable of cleaving said primer at said restriction site when said primer is hybridized with said RNA sequence, and

incubating said medium for a time sufficient to permit cleaving to occur.

9. The method of claim 8 wherein said primer is at least a portion of a cyclic polynucleotide or is terminated at its 3'"'"' end by a group incapable of reacting in a reaction catalyzed by said template-dependent polynucleotide polymerase.

10. A method for detecting the presence of a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte which comprises:

combining either simultaneously or wholly or partially sequentially (1) said sample, (2) means for causing said polynucleotide analyte to be terminated in a 3'"'"'--OH group when said analyte is not terminated in a 3'"'"'--OH group, (3) a single stranded pattern polynucleotide comprising a binding polynucleotide sequence hybridizable with said polynucleotide analyte and connected to the 3'"'"' end of a strand of two or more template sequences connected in tandem and containing site specific cleavage sequences, (4) nucleoside triphosphates, (5) template-dependent polynucleotide polymerase, and (6) means for specifically cleaving cleavable polynucleotide sequences complementary to said site specific cleavage sequences when said cleavable polynucleotide sequences are hybridized with said site specific cleavage sequences and

incubating said combination under conditions for either simultaneously or wholly or partially sequentially (a) causing said polynucleotide analyte to be terminated in a 3'"'"'--OH group when said analyte is not terminated in a 3'"'"'--OH group, (b) hybridizing said polynucleotide analyte with said single stranded pattern polynucleotide, (c) forming an extension of said polynucleotide analyte comprising a sequence complementary to said template sequence and to said site specific cleavage sequences, (d) cleaving said extension into fragments at said cleavable polynucleotide sequences, (e) dissociating said fragments, (f) hybridizing said dissociated fragments with single stranded pattern polynucleotide, and (g) forming an extension of said hybridized fragments along said pattern polynucleotide, and repeating steps (d)-(g), and

determining the presence of said fragments, or fragments complementary thereto, the presence thereof indicating the presence of said polynucleotide analyte in said sample.

11. The method of claim 10 wherein said site specific cleavage sequences are cleaved.

12. The method of claim 11 wherein said fragments include those of the single stranded pattern polynucleotide.

13. The method of claim 10 wherein said polynucleotide analyte is DNA.

14. The method of claim 10 wherein said single stranded pattern polynucleotide is DNA.

15. The method of claim 14 wherein said single stranded pattern polynucleotide comprises at least three of said template sequences.

16. The method of claim 15 wherein said site specific cleavage sequences contained in said template sequences are all identical.

17. The method of claim 14 wherein said single stranded pattern polynucleotide is terminated at the 3'"'"' end by a group that is incapable of reacting in a reaction catalyzed by said template dependent polynucleotide polymerase.

18. The method of claim 10 wherein said template sequence is 8 to 100 nucleotides in length.

19. The method of claim 10 wherein said site specific cleavage sequence is a restriction endonuclease site and said means for specifically cleaving said cleavable polynucleotide sequences comprises a restriction endonuclease.

20. The method of claim 10 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are ATP, GTP, CTP, and TTP.

21. The method of claim 10 wherein said incubation is carried out at a substantially constant temperature.

22. The method of claim 10 wherein the number of said fragments formed for each of said extensions is increased at least by a factor of three.

23. The method of claim 10 wherein said polynucleotide analyte is RNA.

24. The method of claim 10 wherein said single stranded pattern polynucleotide is at least part of a cyclic polynucleotide.

25. A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte, which method comprises:

combining in an aqueous medium either simultaneously or wholly or partially sequentially (1) said sample (2) means for obtaining from said polynucleotide analyte a target sequence and for terminating said sequence in a 3'"'"'--OH group when said sequence is not terminated in a 3'"'"'--OH group, (3) single stranded pattern polydeoxynucleotide comprised of a binding polynucleotide sequence complementary to said target sequence joined at its 5'"'"' end to a multiply repeated sequence of at least 10 nucleotides, said repeated sequence, when hybridized to its complementary sequence and incubated with a restriction endonuclease, promoting cleavage of said complementary sequence to form restriction fragments, (4) deoxynucleoside triphosphates, (5) a DNA dependent DNA polymerase for extending said target sequence to provide said complementary sequence, and (6) a restriction endonuclease for forming said restriction fragments,

said combining being carried out under conditions for either simultaneously or wholly or partially sequentially (a) obtaining said target sequence from said polynucleotide analyte, (b) hybridizing said target sequence to said single stranded pattern oligodeoxynucleotide, (c) extending said target sequence to form said complementary sequence, (d) cleaving said complementary sequence, (e) denaturing the duplex of said complementary sequence and said repeated sequence, (f) hybridizing said complementary sequence with single stranded pattern polydeoxynucleotide, (g) extending said complementary sequence, and repeating steps (d)-(g) above; and

detecting said cleaved complementary sequence, the presence thereof indicating the presence of said polynucleotide analyte in said sample.

26. The method of claim 25 wherein said repeated sequence is cleaved by said restriction endonuclease.

27. The method of claim 26 wherein detecting said cleaved complementary sequence includes detecting said cleaved repeated sequence.

28. The method of claim 25 wherein said analyte is DNA and said means for obtaining a target sequence includes a restriction enzyme.

29. The method of claim 25 wherein said analyte is RNA and said means for obtaining a target sequence includes (1) a single stranded DNA primer comprising a deoxynucleic acid sequence capable of hybridizing with said target RNA sequence and containing a restriction site, said primer being terminated at its 3'"'"' end by a group that prevents chain extension by DNA-dependent DNA polymerase and (2) a restriction endonuclease capable of cleaving said primer at said restriction site when said primer is hybridized with said RNA sequence and said aqueous medium is incubated for a time sufficient to permit said RNA analyte to hybridize with said DNA primer and to permit said restriction endonuclease to cleave the hybridized RNA analyte-DNA primer.

30. The method of claim 25 wherein said repeated sequence contains 12 to 100 nucleotides.

31. The method of claim 25 wherein said deoxynucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.

32. The method of claim 25 wherein said conditions include substantially constant temperature.

33. The method of claim 25 wherein said deoxynucleoside triphosphates are selected from three members of the group consisting of dATP, dTTP, dGTP, and dCTP and derivatives thereof.

34. The method of claim 33 wherein said template sequences and polydeoxynucleotide sequences joining said templates sequences consist of a continuous sequence of nucleotides selected from three members of the group consisting of dA, dT, dC, dG and derivatives thereof.

35. The method of claim 25 wherein said cleaved complementary sequence is determined by a method selected from the group consisting of nucleic acid probe hybridization, spectroscopic detection and chromatographic detection.

36. A method for determining the presence of a target sequence of nucleotides in a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte, which method comprises:

combining either simultaneously or wholly or partially sequentially said sample and (1) means capable of causing said target sequence to be terminated in a 3'"'"'--OH group when said target sequence is not already terminated in a 3'"'"'--OH group, (2) single stranded pattern polydeoxynucleotide comprised of a sequence complementary to said target sequence, said sequence bonded at its 5'"'"' end to a multiply repeating sequence of at least 10 nucleotides which, when hybridized to a complementary sequence, promotes cleavage of said complementary sequence to form fragments (3) deoxynucleoside triphosphates, (4) DNA dependent DNA polymerase and (5) a restriction endonuclease for cleaving said complementary sequence when hybridized to said multiply repeating sequence;

incubating the individual components and mixtures formed therefrom under conditions which promote either wholly or partially sequential or simultaneous (a) denaturation of said target sequence when said target sequence is double stranded, (b) hybridization of said target sequence with said single stranded pattern polydeoxynucleotide, (c) extension of said target sequence by DNA dependent DNA polymerase to produce a duplex containing said complementary sequence, (d) cleavage of said complementary sequence in said duplex, (e) melting of said duplex, (f) hybridization of said cleaved complementary sequence, (g) extension of said complementary sequence by DNA dependent DNA polymerase to produce a duplex containing said complementary sequence, and repeating steps (d)-(g) above; and

detecting said complementary sequence or fragments of said pattern polydeoxynucleotide produced during cleavage of said complementary sequence, the presence thereof indicating the presence of said analyte in said sample.

37. The method of claim 36 wherein said polynucleotide is DNA and said means capable of causing said target sequence to terminate in a 3'"'"'--OH group includes a restriction enzyme.

38. The method of claim 36 wherein said complementary sequence contains 10 to 100 nucleotides.

39. The method of claim 36 wherein said deoxynucleoside triphosphates are dATP, dGTP, dCTP and dTTP.

40. The method of claim 36 wherein said conditions include substantially constant temperature.

41. The method of claim 36 wherein said deoxynucleoside triphosphates are selected from three members of the group consisting of dATP, dTTP, dGTP, and dCTP and derivatives thereof.

42. The method of claim 41 wherein said template sequences and polydeoxynucleotide sequences joining said template sequences consist of a continuous sequence of nucleotides selected from three members of the group consisting of dA, dT, dC, and dG, and derivatives thereof.