Detection of nucleic acids

• US 20030022231A1
• Filed: 09/12/2002
• Published: 01/30/2003
• Est. Priority Date: 08/13/1999
• Status: Abandoned Application

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Wangh, Lawrence, Rice, John, Hartshorn, Cristina, Sanchez, J. Aquiles, Pierce, Kenneth
• Application Number:
  US10/242,395
• Publication Number:
  US 20030022231A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/537 Protease
• CPC Class Codes: C12Q 2547/107 Use of permeable barriers, e.g. waxes
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Disclosed are compositions, methods, and kits useful for the detection of the presence and/or quantity of one or more chromosomes from single cells, groups of cells, or subcellular compartments. Provided is a lysis buffer for the preparation of substantially accessible nucleic acid molecules from a single cell. Also provided are moderately-repeated highly-conserved nucleic acid sequences, and oligonucleotide primer and probe molecules which hybridize specifically thereto. Methods for the detection of the presence or quantity of one or more chromosomes from a single cell are included, as are methods for the assessment of the reliability of the results of the methods of the invention. Kits for the convenient practice of the invention are also included.

Claims

1. An isolated moderately-repeated highly-conserved nucleic acid sequence that is:

a) repeated 3-100 times within the genome of a cell or part thereof; and

b) sufficiently conserved such that at least two non-overlapping oligonucleotide primer molecules are able under stringent conditions to hybridize to and permit the amplification of the plurality of the copies of said nucleic acid sequence.

2. An oligonucleotide primer, comprising a nucleotide sequence that is sufficiently complementary to the moderately-repeated highly-conserved nucleic acid sequence of claim 1 to permit hybridization of the primer under stringent conditions to a plurality of the copies of said moderately-repeated highly-conserved nucleic acid sequence present in the nucleic acid molecules comprising the genomes of fewer than 10 cells or part thereof.

3. The primer of claim 2, wherein said primer is complementary to the moderately-repeated highly-conserved nucleic acid sequence only at the 5′

and 3′

ends of the primer molecule, such that hybridization of the primer to said moderately-repeated highly-conserved sequence results in the circularization of the primer molecule.

4. The primer of claim 2, wherein the nucleic acid molecules are isolated from a mammalian cell.

5. The primer of claim 4, wherein the mammalian cell is a human cell.

6. The primer of claim 5, wherein said primer is sufficiently complementary to a moderately-repeated highly-conserved nucleic acid sequence contained within human chromosome 17 to permit hybridization under stringent conditions to a plurality of the copies of said moderately-repeated highly-conserved nucleic acid sequence in human chromosome 17.

7. The primer of claim 6, wherein said primer comprises the nucleotide sequence of SEQ ID NO:

1.

8. The primer of claim 6, wherein said primer comprises the nucleotide sequence of SEQ ID NO:

2.

9. The primer of claim 5, wherein said primer is sufficiently complementary to a moderately-repeated highly-conserved nucleic acid sequence contained within the human Y chromosome to permit hybridization under stringent conditions to a plurality of the copies of said moderately-repeated highly-conserved nucleic acid sequence in the human Y chromosome.

10. The primer of claim 9, wherein said primer comprises the nucleotide sequence of SEQ ID NO:

4.

11. The primer of claim 9, wherein said primer comprises the nucleotide sequence of SEQ ID NO:

5.

12. The primer of claim 2, wherein said primer includes a detectable label.

13. The primer of claim 12, wherein the label is detectable only when said primer is hybridized to the moderately-repeated highly-conserved nucleic acid sequence or its specific amplicon.

14. The primer of claim 12, wherein the label is detectable only when said primer is not hybridized to the moderately-repeated highly-conserved nucleic acid sequence or its specific amplicon.

15. An oligonucleotide probe, comprising a nucleotide sequence that is sufficiently complementary to the moderately-repeated highly-conserved nucleic acid sequence of claim 1 to permit hybridization of the primer under stringent conditions to a plurality of the copies of said moderately-repeated highly-conserved nucleic acid sequence present in the nucleic acid molecules comprising the genomes of fewer than 10 cells or part thereof.

16. The probe of claim 15, wherein the nucleic acid molecules are isolated from a mammalian cell.

17. The probe of claim 16, wherein the mammalian cell is a human cell.

18. The probe of claim 17, wherein said probe is sufficiently complementary to a moderately-repeated highly-conserved nucleic acid sequence contained within human chromosome 17 to permit hybridization under stringent conditions to a plurality of the copies of said moderately-repeated highly-conserved nucleic acid sequence in human chromosome 17.

19. The probe of claim 18, wherein said probe comprises the nucleotide sequence of SEQ ID NO:

3.

20. The probe of claim 17, wherein said probe is sufficiently complementary to a moderately-repeated highly-conserved nucleic acid sequence contained within the human Y chromosome to permit hybridization under stringent conditions to a plurality of the copies of said moderately-repeated highly-conserved nucleic acid sequence in the human Y chromosome.

21. The probe of claim 20, wherein said probe comprises the nucleotide sequence of SEQ ID NO:

6.

22. The probe of claim 15, wherein said probe includes a detectable label.

23. The probe of claim 22, wherein:

the detectable label comprises a fluor and a quencher such that in the absence of hybridization of said probe to the moderately-repeated highly-conserved nucleic acid sequence or its specific amplicon, said probe forms a hairpin loop structure that brings said fluor and quencher sufficiently proximate such that fluorescence is substantially quenched; and

wherein upon hybridization of said probe to said moderately-repeated highly-conserved nucleic acid sequence or its specific amplicon, said fluor and quencher are separated and a fluorescent signal is emitted.

24. The probe of claim 22, wherein the label is detectable only when the probe is not hybridized to the moderately-repeated highly-conserved nucleic acid sequence or its specific amplicon.

25. A method of detecting the presence or quantity of a nucleic acid sequence present in a sample of nucleic acid molecules comprising the genomes of fewer than 10 cells or part thereof, comprising:

a) contacting the cells or part thereof with a protease-based lysis buffer comprising;

i) an ionic detergent;

ii) a protease; and

iii) a buffering agent, to form a mixture;

b) incubating the mixture at a temperature at which the protease is active such that a sample of substantially accessible nucleic acid molecules is obtained;

c) incubating the sample at a temperature at which the protease is substantially inactivated;

d) contacting the sample with at least one nucleic acid primer complementary to a plurality of the copies of said nucleic acid sequence;

e) amplifying said nucleic acid sequence by an amplification reaction; and

f) detecting the amplicon of the nucleic acid sequence as indicative of the presence or quantity of said nucleic acid sequence in said sample.

26. The method of claim 25, wherein the nucleic acid sequence is a moderately-repeated highly-conserved nucleic acid sequence.

27. The method of claim 26, wherein the moderately-repeated highly-conserved sequence is contained within human chromosome 17.

28. The method of claim 27, wherein the amplification reaction is a cyclical amplification reaction and the sample is contacted with two primers comprising the nucleotide sequences of SEQ ID NO:

1 and SEQ ID NO;

2, respectively.

29. The method of claim 26, wherein the moderately-repeated highly-conserved sequence is contained within the human Y chromosome.

30. The method of claim 29, wherein the amplification reaction is a cyclical amplification reaction and the sample is contacted with two primers comprising the nucleotide sequences of SEQ ID NO:

4 and SEQ ID NO;

5, respectively.

31. The method of claim 25, wherein the sample is contacted with two primers complementary to opposite strands of the nucleic acid sequence and wherein the amplification reaction is a cyclical amplification reaction.

32. The method of claim 25, wherein the sample is contacted with at least two primer molecules comprising:

a) a first primer having both a 3′ and

5′

end complementary to nonoverlapping regions of the nucleic acid sequence such that upon hybridization, the primer forms a circular structure containing a gap; and

b) at least a second primer which is complementary to a sequence found between the 3′ and

5′

ends of the first primer molecule, such that the second primer is able to hybridize to the first primer and fill the gap between the ends of the first primer; and

wherein the amplification reaction is a rolling circle amplification reaction.

33. The method of claim 25, wherein at least one of the primers includes a detectable label.

34. The method of claim 33, wherein the label is detectable only when the primer is hybridized to the nucleic acid sequence or its specific amplicon.

35. The method of claim 33, wherein the label is detectable only when the primer is not hybridized to the nucleic acid sequence or its specific amplicon.

36. The method of claims 25, wherein the step of detecting comprises:

a) contacting the sample with an oligonucleotide probe which specifically hybridizes to a plurality of the copies of the nucleic acid sequence or its specific amplicon; and

b) detecting said probe.

37. The method of claim 36, wherein the probe includes a detectable label.

38. The method of claim 37, wherein:

the detectable label comprises a fluor and a quencher such that in the absence of hybridization of said probe to the nucleic acid sequence or its specific amplicon, said probe forms a hairpin loop structure that brings said fluor and quencher sufficiently proximate such that fluorescence is substantially quenched; and

wherein upon hybridization of said probe to said nucleic acid sequence or its specific amplicon, said fluor and quencher are separated and a fluorescent signal is emitted.

39. The method of claim 37, wherein the label is detectable only when the probe is not hybridized to the nucleic acid sequence or its specific amplicon.

40. The method of claim 25, wherein the amplicon is detected or quantified in real time during the amplification reaction.

41. The method of claim 25, wherein at least one of the primers includes a detectable label and the amplicon is detected or quantified in real time during the amplification reaction by measuring the label associated with the primer hybridized to the amplicon.

42. The method of claim 36, wherein the probe contains a detectable label and the amplicon is detected or quantified in real time during the amplification reaction by measuring the label associated with the probe hybridized to the amplicon.

43. The method of claim 42, further comprising the step of comparing the quantity of amplicon detectable at a first selected time of amplification and the quantity of amplicon detectable at a later second selected time of amplification to predetermined quantity values for the first and second selected times of amplification as an indication of the presence or quantity of the amplification reaction.

44. The method of claim 42, further comprising the step of comparing the quantity of amplicon detectable at a first selected time of amplification and the quantity of amplicon detectable at a later second selected time of amplification to predetermined quantity values for the first and second selected times of amplification as an indication of the efficiency of the amplification reaction

45. The method of claim 25, wherein the sample of nucleic acid molecules is isolated from a single cell.

46. The method of claim 25, wherein the sample of nucleic acid molecules is isolated from one part of one cell.

47. The method of claim 25, wherein the protease is proteinase K.

48. The method of claim 25, wherein the temperature at which the protease is active is about 50°

C.

49. The method of claim 25, wherein the temperature at which the protease is substantially inactivated is about 95°

C.

50. The method of claim 25, wherein the buffering agent maintains the pH of the reaction at or near the optimal pH for the activity of the protease.

51. The method of claims 25, wherein the buffering agent is Tris.

52. The method of claim 25, wherein the buffering agent maintains the pH above 7.2 at the incubation temperature of the first incubation step.

53. The method of claim 25, wherein the ionic detergent is sodium dodecyl sulfate.

54. The method of claim 25, wherein the lysis buffer does not include chaotropic salts or Mg²⁺.

55. The method of claim 25, wherein the first incubation step lasts about one hour.

56. The method of claim 25, wherein the protease is proteinase K;

the buffering agent is Tris;

the ionic detergent is sodium dodecyl sulfate; and

the lysis buffer does not include chaotropic salts or Mg²⁺.

57. The method of claim 25, wherein:

a) the entirety of the method is conducted in a sealed reaction vessel; and

b) polymerase and Mg²⁺ molecules are added to the sample in a form such that they are made available for the amplification step only after the step wherein the protease is inactivated.

58. The method of claim 57, wherein the polymerase and Mg²⁺ molecules are encased in wax, and wherein the wax is melted and the polymerase and Mg²⁺ molecules are made available for the amplification step during the step wherein the protease is inactivated.

59. The method of claim 25, wherein the protease-based lysis buffer is replaced by an alkaline lysis buffer that does not contain DTT or any other reducing agent;

wherein the first incubation step lasts for a time sufficient to obtain substantially accessible nucleic acid molecules; and

wherein, prior to the amplification, the pH of the sample is neutralized by the addition of an acid and a buffering agent.

60. The method of claim 59, wherein the alkaline lysis buffer contains potassium hydroxide.

103. A method for selecting best-possible primers, comprising:

a) performing the methods of either of claims 25 or 59, wherein at least one pair of primers is used to contact the sample; and

b) determining whether the primers are best-possible primers, wherein best possible primers are those that, when used in the method herein, have at least one of the following properties;

i) lower C_T value;

ii) smaller C_T value variance;

iii) higher fluorescence 4-6 cycles beyond the C_T value;

iv) smaller variance of the fluorescence 4-6 cycles beyond the C_T value;

v) a greater rate of signal increase; and

vi) fewer non-specific amplicons

than other primer pairs specific for the same nucleic acid sequence.

104. A method for selecting best-possible primers, comprising:

a) performing the methods of either of claims 25 or 59, wherein;

i) at least one pair of primers is utilized to contact the sample;

ii) the sample of nucleic acid molecules comprising the genomes of fewer than 10 cells or part thereof is replaced by a nucleic acid sample comprising the genomes 10 or more cells or part thereof; and

iii) wherein the nucleic acid sample is derived from gene-deleted DNA; and

b) determining whether the primers are best-possible primers, wherein best possible primers are those that, when used in the method herein, result in the fewest non-specific amplicons, as compared to other primers specific for the same nucleic acid sequence.

61. A method for preparing a sample of accessible nucleic acid molecules from fewer than 10 cells or parts thereof for an amplification reaction comprising:

a) contacting the cells or part thereof with an alkaline lysis buffer that does not contain DTT or any other reducing agent, to form a mixture;

b) incubating the mixture for an amount of time sufficient to obtain substantially accessible nucleic acid molecules;

c) neutralizing the pH mixture by adding an acid and a buffering agent,

such that substantially accessible nucleic acid molecules are obtained.

62. The method of claim 61, wherein the alkaline lysis buffer contains potassium hydroxide.

63. A method for preparing a sample of substantially accessible nucleic acid molecules from fewer than 10 cells or parts thereof for an amplification reaction, comprising:

a) contacting the cells or parts thereof with a protease-based lysis buffer comprising;

i) an ionic detergent

ii) a protease; and

iii) a buffering agent,

to form a mixture;

b) incubating the mixture at a temperature at which the protease is active;

such that substantially accessible nucleic acid molecules are obtained.

64. The method of claim 63, wherein the protease is proteinase K.

65. The method of claim 63, wherein the temperature is about 50°

C.

66. The method of claim 63, wherein the buffering agent maintains the pH of the reaction at or near the optimal pH for the activity of the protease.

67. The method of claim 63, wherein the buffering agent is Tris.

68. The method of claim 63, wherein the buffering agent maintains the pH above 7.2 at the incubation temperature.

69. The method of claim 63, wherein the ionic detergent is sodium dodecyl sulfate.

70. The method of claim 63, wherein the lysis buffer does not include chaotropic salts or Mg²⁺.

71. The method of claim 63, wherein the step of incubating lasts about one hour.

72. The method of claim 63, further comprising inactivating the protease prior to the amplification step.

73. The method of claim 63, wherein the protease is proteinase K;

the buffering agent is about Tris;

the ionic detergent is sodium dodecyl sulfate; and

the lysis buffer does not include chaotropic salts or Mg²⁺.

74. A protease-based lysis buffer comprising:

a) an ionic detergent;

b) a protease; and

c) a buffering agent sufficient to achieve and maintain a pH of about 7.2 or above at the incubation temperature of a method in which the lysis buffer is utilized, and wherein chaotropic salts and Mg²⁺ are not included in the lysis buffer.

75. The protease-based lysis buffer of claim 74, wherein the ionic detergent is sodium dodecyl sulfate, the protease is proteinase K, and the buffering agent is Tris.

76. A kit for the preparation of substantially accessible nucleic acid molecules from the nucleic acid molecules comprising the genomes of fewer than 10 cells or part thereof comprising:

a protease-based lysis buffer comprising an ionic detergent, a protease, and a buffering agent;

in at least a first container.

77. The kit of claim 76, wherein the lysis buffer does not include chaotropic salts or Mg²⁺.

78. The kit of claim 76, wherein the protease is proteinase K, the ionic detergent is sodium dodecyl sulfate, and the buffering agent is Tris.

79. A kit for detecting the presence or quantity of a nucleic acid sequence present in a sample of nucleic acid molecules comprising the genomes of fewer than 10 cells or part thereof, comprising:

a) a protease-based lysis buffer comprising an ionic detergent, a protease, and a buffering agent, in at least a first container; and

b) at least one oligonucleotide primer which specifically hybridizes to a plurality of the copies of said nucleic acid sequence, in at least a second container.

80. The kit of claim 79, wherein the nucleic acid sequence is a moderately-repeated highly-conserved nucleic acid sequence.

81. The kit of claim 80, wherein the moderately-repeated highly-conserved nucleic acid sequence is contained in the human Y chromosome.

82. The kit of claim 80, wherein the moderately-repeated highly-conserved nucleic acid sequence is contained in human chromosome 17.

83. The kit of claim 79, wherein at least one of the primers includes a detectable label.

84. The kit of claim 83, wherein the label is detectable only when the primer is hybridized to the nucleic acid sequence or its specific amplicon.

85. The kit of claim 83, wherein the label is detectable only when the primer is not hybridized to the nucleic acid sequence.

86. The kit of claim 79, further comprising at least a third container containing an oligonucleotide probe which specifically hybridizes to a plurality of the copies of the nucleic acid sequence or its specific amplicon.

87. The kit of claim 86, wherein the probe includes a detectable label.

88. The kit of claim 87, wherein:

the detectable label comprises a fluor and a quencher such that in the absence of hybridization of said probe to the nucleic acid sequence or its specific amplicon, said probe forms a hairpin loop structure that brings said fluor and quencher sufficiently proximate such that fluorescence is substantially quenched; and

wherein upon hybridization of said probe to said nucleic acid sequence or its specific amplicon, said fluor and quencher are separated and a fluorescent signal is emitted.

89. The kit of claim 87, wherein the label is detectable only when the probe is not hybridized to the nucleic acid sequence or its specific amplicon.

90. The kit of claim 79, further comprising an amplification reagent comprising:

a) a polymerase;

b) a buffering agent;

c) one or more salts; and

d) deoxynucleotide triphosphate molecules.

91. The kit of claim 90, wherein the polymerase is provided encased in wax.

92. The kit of claim 91, further comprising Mg²⁺ molecules encased in wax.

93. The kit of claim 79, further comprising at least one enhancer of a molecular beacon probe.

94. The kit of claim 79, further comprising a plurality of primers which specifically hybridize to two or more nucleic acid sequences or their specific amplicons.

95. The kit of claim 79, further comprising a plurality of probes which specifically hybridize to two or more nucleic acid sequences or their specific amplicons.

96. The kit of claim 79, further comprising a plurality of enhancers of molecular beacon probes.

97. A method of preparing gene-deleted DNA for use in an amplification reaction comprising contacting a sample of complete-genome DNA with a sequence specific replication inhibitor, such that at least the first replication event of a specific nucleic acid sequence in the amplification reaction is prevented or delayed.

98. A sample of gene-deleted DNA prepared by the method of claim 97.

99. The method of claim 97, wherein the sequence specific replication inhibitor is a protein having an enzymatic activity.

100. The method of claim 97, where in the sequence specific replication inhibitor is an oligonucleotide.

101. The method of claim 97, wherein the sequence specific replication inhibitor is a non-enzymatic protein.

102. The method of claim 97, wherein the sequence specific replication inhibitor is at least one small molecule.

105. An enhancer of a probe, wherein said enhancer keeps an amplicon in a single-stranded or unhybridized state in the region where said probe hybridizes to its target sequence.

106. The enhancer of claim 105, wherein said enhancer is an oligonucleotide.

107. The enhancer of claim 105, wherein said enhancer is a protein having an enzymatic activity.

108. The enhancer of claim 105, wherein said enhancer is a non-enzymatic protein.

109. The enhancer of claim 105, wherein the probe is a molecular beacon.