Real-time detection of rolling circle amplification products

• US 20040121338A1
• Filed: 12/19/2002
• Published: 06/24/2004
• Est. Priority Date: 12/19/2002
• Status: Abandoned Application

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Abarzua, Patricio, Alsmadi, Osama A.
• Application Number:
  US10/325,665
• Publication Number:
  US 20040121338A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

Disclosed are compositions and methods for real-time detection of rolling circle amplification products. Real-time detection is detection that takes place during the amplification reaction or operation. Real-time detection can be accomplished by, for example, using fluorescent change probes and/or primers during amplification. The fluorescent signals can be proportional to the amount of amplification product. The amplification can be multiply-primed rolling circle amplification in which replication of a circular template is primed at a plurality of sites on the circular template. Multiply-primed RCA increases the sensitivity of singly-primed rolling circle amplification. Multiply-primed RCA can be performed using a single primer (which hybridizes to multiple sites on the amplification target circle) or multiple primers (each of which can hybridize to a single site on the amplification target circle or multiple sites on the amplification target circle). Fluorescent change probes and primers are probes and primers that involve a change in fluorescence intensity or wavelength based on a change in the form or conformation of the probe or primer and nucleic acid to be detected, assayed or replicated.

Claims

1. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising

incubating a mixture comprising an amplification target circle, one or more rolling circle replication primers, and one or more fluorescent change probes, under conditions that promote rolling circle replication of the amplification target circle, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and

detecting, during the incubation, fluorescent change probes interacting with the tandem sequence DNA.

2. The method of claim 1 wherein the mixture contains a plurality of rolling circle replication primers.

3. The method of claim 1 wherein the mixture contains one rolling circle replication primer.

4. The method of claim 1 wherein detection of fluorescent change probes interacting with the tandem sequence DNA comprises measuring fluorescence from the fluorescent change probes continuously during the incubation.

5. The method of claim 4 wherein detection of fluorescent change probes interacting with the tandem sequence DNA further comprises measuring the rate of increase in fluorescence from the fluorescent change probes, wherein the rate of increase in fluorescence from the fluorescent change probes indicates the rate of amplification of the amplification target circle, wherein the rate of amplification of the amplification target circle indicates the amount of the amplification target circle present in the mixture.

6. The method of claim 5 wherein the amplification target circle is derived from a nucleic acid molecule in a nucleic acid sample, wherein the amount of the amplification target circle present in the mixture indicates the amount of the nucleic acid molecule from which the amplification target circle is derived that is present in the nucleic acid sample.

7. The method of claim 6 wherein the amplification target circle comprises a single stranded bacteriophage DNA, a double stranded DNA plasmid or other vector, a bacterial artificial chromosome vector, a yeast artificial chromosome vector, or a clone derived from such a vector.

8. The method of claim 7 wherein the amplification target circle is a sub-chromosomal fragment.

9. The method of claim 8 wherein the sub-chromosomal fragment is generated by restriction digestion chromosomal DNA and circularization of a chromosomal fragment.

10. The method of claim 6 wherein the amplification target circle comprises the nucleic acid molecule in the nucleic acid sample.

11. The method of claim 10 wherein the amplification target circle is a bacterial chromosome.

12. The method of claim 6 wherein the nucleic acid molecule is human DNA, yeast DNA, mitochondrial DNA, mRNA, cDNA, genomic DNA, viral DNA, viral RNA, bacteriophage DNA, bacteriophage RNA, or precursor RNA.

13. The method of claim 1 wherein the amplification target circle is derived from a nucleic acid molecule, wherein the nucleic acid molecule is human DNA, yeast DNA, mitochondrial DNA, mRNA, cDNA, genomic DNA, viral DNA, viral RNA, bacteriophage DNA, bacteriophage RNA, or precursor RNA.

14. The method of claim 1 wherein detection of fluorescent change probes interacting with the tandem sequence DNA comprises measuring fluorescence from the fluorescent change probes a plurality of times during the incubation.

15. The method of claim 1 wherein the rolling circle replication primers each comprise a complementary portion, wherein the amplification target circle comprises a plurality of primer complement portions, wherein the complementary portion of the rolling circle replication primers is complementary to one or more of the primer complement portions of the amplification target circle.

16. The method of claim 1 wherein the rolling circle replication primers are random primers.

17. The method of claim 16 wherein the random primers comprise unmodified deoxyribonucleotides, unmodified ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, nucleotide analogs, one or a combination of oligonucleotide analogs, or a combination thereof.

18. The method of claim 17 wherein the random primers are chimeric.

19. The method of claim 1 wherein the rolling circle replication primers comprise unmodified deoxyribonucleotides, unmodified ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, nucleotide analogs, one or a combination of oligonucleotide analogs, or a combination thereof.

20. The method of claim 19 wherein the rolling circle replication primers are chimeric.

21. The method of claim 1 wherein the rolling circle replication primers comprise a mixture of random and specific primers.

22. The method of claim 1 wherein the rolling circle replication primers are within the range of 2 to 50 nucleotides in length.

23. The method of claim 1 wherein the rolling circle replication primers are within the range of 2 to 35 nucleotides in length.

24. The method of claim 1 wherein the rolling circle replication primers are within the range of 2 to 10 nucleotides in length.

25. The method of claim 1 wherein at least one of the rolling circle replication primers are hexamers.

26. The method of claim 1 wherein at least one of the rolling circle replication primers are octamers.

27. The method of claim 1 wherein at least one of the rolling circle replication primers comprises a non-complementary portion, wherein the non-complementary portion is not complementary to the amplification target circle, wherein the non-complementary portion is at the 5′

end of the rolling circle replication primer.

28. The method of claim 1 wherein the amplification target circle is a single stranded DNA circle.

29. The method of claim 1 wherein the amplification target circle is a duplex DNA circle having at least one nick.

30. The method of claim 1 wherein the amplification target circle is a duplex DNA circle having no nicks.

31. The method of claim 30 further comprising a denaturation step to separate the two strands of the duplex DNA circle.

32. The method of claim 1 wherein the amplification target circle is a supercoiled duplex DNA circle.

33. The method of claim 1 wherein the amplification target circle is derived from a nucleic acid sample, wherein the nucleic acid sample is derived from a biological sample.

34. The method of claim 33 wherein the biological sample comprises a bacterial colony, a bacterial cell, a bacteriophage plaque, a bacteriophage, a virus plaque, a virus, a yeast colony, a yeast cell, a baculovirus plaque, a baculovirus, a biological agent, an infectious biological agent, a biological threat agent, a eukaryotic cell culture, a eukaryotic cell, a culture of transiently transfected eukaryotic cells, or a transiently transfected eukaryotic cell.

35. The method of claim 33 wherein the biological sample comprises a blood sample, a urine sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a plasma sample, a serum sample, a pus sample, an amniotic fluid sample, a bodily fluid sample, a stool sample, a biopsy sample, a needle aspiration biopsy sample, a swab sample, a mouthwash sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a cell lysate sample, a crude cell lysate sample, a forensic sample, an environmental sample, an archeological sample, an infection sample, a nosocomial infection sample, a community-acquired infection sample, a biological threat sample, a production sample, a drug preparation sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, or a combination.

36. The method of claim 1 wherein the amplification target circle is derived from a nucleic acid molecule, wherein the nucleic acid molecule is derived from a biological sample.

37. The method of claim 36 wherein the biological sample comprises a blood sample, a urine sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a plasma sample, a serum sample, a pus sample, an amniotic fluid sample, a bodily fluid sample, a stool sample, a biopsy sample, a needle aspiration biopsy sample, a swab sample, a mouthwash sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a cell lysate sample, a crude cell lysate sample, a forensic sample, an environmental sample, an archeological sample, an infection sample, a nosocomial infection sample, a community-acquired infection sample, a biological threat sample, a production sample, a drug preparation sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, or a combination.

38. The method of claim 37 wherein the biological sample comprises a bacterial colony, a bacterial cell, a bacteriophage plaque, a bacteriophage, a virus plaque, a virus, a yeast colony, a yeast cell, a baculovirus plaque, a baculovirus, a biological agent, an infectious biological agent, a biological threat agent, a eukaryotic cell culture, a eukaryotic cell, a culture of transiently transfected eukaryotic cells, or a transiently transfected eukaryotic cell.

39. The method of claim 38 wherein the nucleic acid molecule is human DNA, yeast DNA, mitochondrial DNA, mRNA, cDNA, genomic DNA, viral DNA, viral RNA, bacteriophage DNA, bacteriophage RNA, or precursor RNA.

40. The method of claim 33 wherein the biological sample is lysed.

41. The method of claim 40 wherein lysis is achieved by treatment of the biological sample with heat, an enzyme, an organic solvent, or a combination of these.

42. The method of claim 41 wherein lysis is achieved by treatment of the biological sample with an enzyme, wherein the enzyme is lysozyme, glucylase, xymolyase, or a combination of these.

43. The method of claim 1 wherein the amplification target circle is a single stranded RNA circle.

44. The method of claim 1 wherein the amplification target circle comprises no more than about 10,000 nucleotides.

45. The method of claim 1 wherein the amplification target circle comprises more than 10,000 nucleotides.

46. The method of claim 1 wherein the amplification target circle comprises no more than about 1,000 nucleotides.

47. The method of claim 1 wherein the amplification target circle comprise no more than about 100 nucleotides.

48. The method of claim 1 wherein the amplification target circle comprises a single stranded bacteriophage DNA, a double stranded DNA plasmid or vector, a bacterial artificial chromosome vector, a yeast artificial chromosome vector, or a clone derived from such a vector.

49. The method of claim 1 wherein the amplification target circle comprises a nucleic acid molecule in a nucleic acid sample.

50. The method of claim 1 wherein the amplification target circle is of unknown sequence composition.

51. The method of claim 1 wherein the fluorescent change probes each comprise a complementary portion, wherein the amplification target circle comprises at least one detection tag portion, wherein the complementary portion of the fluorescent change probes matches the sequence of at least one of the detection tag portions of the amplification target circle.

52. The method of claim 1 wherein the mixture comprises a plurality of amplification target circles.

53. The method of claim 52 wherein the fluorescent change probes each comprise a complementary portion, wherein the amplification target circles each comprise at least one detection tag portion, wherein the complementary portion of each of the fluorescent change probes matches the sequence of one or more of the detection tag portions of the amplification target circles.

54. The method of claim 53 wherein the mixture comprises a plurality of fluorescent change probes, wherein the complementary portion of each fluorescent change probe matches the sequence of one or more of the detection tag portions of a different one of the amplification target circles.

55. The method of claim 53 wherein the mixture comprises a plurality of fluorescent change probes, wherein the complementary portion of each fluorescent change probe matches the sequence of one or more of the detection tag portions of one or more of the amplification target circles.

56. The method of claim 53 wherein the mixture comprises a plurality of fluorescent change probes, wherein the complementary portion of each fluorescent change probe matches the sequence of one of the detection tag portions of a different one of the amplification target circles.

57. The method of claim 53 wherein the mixture comprises a plurality of fluorescent change probes, wherein the complementary portion of each fluorescent change probe matches the sequence of a plurality of the detection tag portions of a different one of the amplification target circles.

58. The method of claim 53 wherein the mixture comprises a plurality of fluorescent change probes, wherein the complementary portion of each fluorescent change probe matches the sequence of a plurality of the detection tag portions of one of the amplification target circles.

59. The method of claim 53 wherein the mixture comprises a plurality of fluorescent change probes, wherein the complementary portion of each fluorescent change probe matches the sequence of a plurality of the detection tag portions of a plurality of the amplification target circles.

60. The method of claim 53 wherein the mixture comprises a plurality of fluorescent change probes, wherein the complementary portion of each fluorescent change probe matches the sequence of one of the detection tag portions of a plurality of the amplification target circles.

61. The method of claim 53 wherein the mixture comprises a plurality of fluorescent change probes, wherein the complementary portion of each fluorescent change probe matches the sequence of one of the detection tag portions of one of the amplification target circles.

62. The method of claim 53 wherein detection of fluorescent change probes interacting with the tandem sequence DNA comprises measuring fluorescence from the fluorescent change probes continuously during the incubation.

63. The method of claim 62 wherein the amplification target circle, the detection tag portion of which matches the sequence of the complementary portion of a fluorescent change probe, corresponds to the fluorescent change probe, wherein detection of fluorescent change probes interacting with the tandem sequence DNA further comprises measuring the rate of increase in fluorescence from one of the fluorescent change probes, wherein the rate of increase in fluorescence from the fluorescent change probe indicates the rate of amplification of the amplification target circle corresponding to the fluorescent change probe, wherein the rate of amplification of the amplification target circle indicates the amount of the amplification target circle present in the mixture.

64. The method of claim 62 wherein the amplification target circle, the detection tag portion of which matches the sequence of the complementary portion of a fluorescent change probe, corresponds to that fluorescent change probe, wherein detection of fluorescent change probes interacting with the tandem sequence DNA further comprises measuring the rate of increase in fluorescence from the fluorescent change probes, wherein the rate of increase in fluorescence from each of the fluorescent change probes indicates the rate of amplification of the amplification target circle corresponding to the fluorescent change probe, wherein the rate of amplification of the amplification target circle indicates the amount of the amplification target circle present in the mixture.

65. The method of claim 64 wherein the amplification target circles are derived from nucleic acid molecules, wherein each amplification target circle is derived from a different nucleic acid molecule.

66. The method of claim 65 wherein each nucleic acid molecule is derived from a different nucleic acid sample, wherein the amount of each amplification target circle present in the mixture indicates the amount of the nucleic acid molecule from which the amplification target circle is derived that is present in the nucleic acid sample from which the nucleic acid molecule is derived.

67. The method of claim 52 wherein replication of each amplification target circle results in formation of different tandem sequence DNAs.

68. The method of claim 67 wherein the fluorescent change probes each comprise a complementary portion, wherein the tandem sequence DNAs each comprise different probe complement portions, wherein the complementary portion of each of the fluorescent change probes is complementary to the sequence of a different one of the probe complement portions.

69. The method of claim 52 wherein at least one amplification target circle is a plasmid, wherein at least one amplification target circle is a bacterial chromosome.

70. The method of claim 69 wherein at least one of the amplification target circles is eukaryotic chromosomal DNA.

71. The method of claim 70 wherein the eukaryotic chromosomal DNA is human chromosomal DNA.

72. The method of claim 69 wherein the detection results in detection of the genotype of one or more of the amplification target circles and antibiotic resistance phenotype of one or more of the amplification target circles.

73. The method of claim 1 wherein one or more of the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.

82. The method of claim 1 wherein one or more of the fluorescent change probes are triplex hairpin quenched probes.

83. The method of claim 1 wherein one or more of the fluorescent change probes are triplex FRET probes.

74. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising

incubating a mixture comprising an amplification target circle and one or more rolling circle replication primers under conditions that promote rolling circle replication of the amplification target circle, wherein one or more of the rolling circle replication primers comprise a fluorescent change primer, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and

detecting, during the incubation, fluorescent change primers incorporated into the tandem sequence DNA.

75. The method of claim 74 wherein the fluorescent change primer is a hairpin quenched primer.

76. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising

incubating a mixture comprising an amplification target circle, one or more rolling circle replication primers, and one or more DNA strand displacement primers under conditions that promote rolling circle replication of the amplification target circle, wherein one or more of the DNA strand displacement primers comprise a fluorescent change primer, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and

detecting, during the incubation, fluorescent change primers incorporated into the tandem sequence DNA.

77. The method of claim 76 wherein the fluorescent change primer is a hairpin quenched primer.

78. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising

incubating a mixture comprising an amplification target circle, one or more rolling circle replication primers, and one or more fluorescent change probes, under conditions that promote rolling circle replication of the amplification target circle, wherein one or more of the rolling circle replication primers comprise a fluorescent change primer, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and

detecting, during the incubation, fluorescent change probes interacting with the tandem sequence DNA, fluorescent change primers incorporated into the tandem sequence DNA, or both.

79. The method of claim 78 wherein the mixture further comprises one or more DNA strand displacement primers, wherein one or more of the DNA strand displacement primers comprise a fluorescent change primer.

80. The method of claim 78 wherein one or more of the fluorescent change probes are hairpin quenched probes, cleavage quenched probes, cleavage activated probes, fluorescent activated probes, or a combination.

81. The method of claim 78 wherein the fluorescent change primer is a hairpin quenched primer.