Nucleic Acid Amplification

• US 20140295439A1
• Filed: 03/15/2014
• Published: 10/02/2014
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Labrador Diagnostics LLC (SoftBank Group Corp.)
• Original Assignee: Theranos Incorporated
• Inventors: Patel, Pranav
• Granted Patent: US 9,416,387 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

Methods and compositions for the amplification of nucleic acids are disclosed. Amplification methods provided herein may be performed under isothermal conditions. Methods and compositions may include reagents such as restriction enzymes, polymerases, ligases, primers, and polynucleotide adaptors.

Claims

1. (canceled)

2. A method for amplifying a linear double-stranded nucleic acid template comprising two separate complementary strands, comprising:

(A) treating the linear double-stranded nucleic acid template with two polynucleotide adaptors under conditions such that a circular strand comprising the two adaptors and the two complementary strands is formed, wherein each adaptor comprises a single nucleic acid strand which contains a single strand component of a restriction enzyme recognition sequence and which is configured to, under certain conditions, adopt a stem-loop structure;

(B) treating the circular strand of step (A) with a first oligonucleotide primer and a polymerase, under conditions such that an extension product of the first primer is synthesized;

(C) treating the extension product of the first primer of step (B) with a second oligonucleotide primer and a polymerase, under conditions such that an extension product of the second primer is synthesized which is complementary to the extension product of the first primer of step (B), to produce a new double-stranded nucleic acid comprising at least a portion of the extension product of the first primer and at least a portion of the extension product of the second primer; and

(D) treating the new double-stranded nucleic acid from step (C) with a restriction enzyme that recognizes a full double-stranded restriction enzyme recognition sequence corresponding to a single strand component of a restriction enzyme sequence of step (A), under conditions such that the new double-stranded nucleic acid from step (C) is cleaved to form two or more shorter double-stranded nucleic acids, at least two of which comprise at least a portion of a copy of the linear double-stranded nucleic acid template of step (A), thereby amplifying the linear double-stranded nucleic acid template.

4. The method of claim 2, further comprising repeating steps (A)-(D) for one or more additional cycles, using a shorter double-stranded nucleic acid of step (D) of a first cycle as the linear double-stranded nucleic acid template of step (A) of a second cycle.

5. The method of claim 2, wherein at least two of the shorter double-stranded nucleic acids of step (D) contain a complete copy of the linear double-stranded nucleic acid template of step (A).

15. The method of claim 2, wherein the two polynucleotide adaptors contain single strand components of a restriction enzyme recognition sequence corresponding to the same restriction enzyme.

19. The method of claim 2, wherein at least one of the adaptors contains a nucleotide sequence comprising a 5′

region, a middle region, and a 3′

region, wherein the 5′

region and 3′

region of the sequence are complementary to each other such that under certain conditions they anneal to each other and form the stem of a stem-loop structure, and wherein the stem contains a blunt end.

27. The method of claim 2, wherein all steps of the method are performed at a temperature of no greater than 70 C.

32. The method of claim 2, wherein the linear double-stranded nucleic acid template is amplified at least 10-fold within 60 minutes of initiation of the method.

3. (canceled)

6-14. -14. (canceled)

16-18. -18. (canceled)

20-26. -26. (canceled)

28-31. -31. (canceled)

33-55. -55. (canceled)

56. A vessel, comprising in fluidic communication therein:

(A) an isolated nucleic acid ligase,

(B) an isolated nucleic acid polymerase,

(C) an isolated restriction enzyme, and

(D) a polynucleotide adaptor, wherein the adaptor comprises a single nucleic acid strand which contains the single strand component of a restriction enzyme recognition sequence and which is configured to, under certain conditions, adopt a stem-loop structure.

57. The vessel of claim 56, further comprising a first oligonucleotide primer and a second oligonucleotide primer.

58. The vessel of claim 57, further comprising a linear double-stranded nucleic acid template comprising two complementary strands.

59. The vessel of claim 58, wherein the first oligonucleotide primer is complementary to a complementary strand of the linear double-stranded nucleic acid template.

65. The vessel of claim 56, wherein the isolated restriction enzyme recognizes a full double-stranded restriction enzyme recognition sequence corresponding to the single strand component of a restriction enzyme sequence of the adaptor.

71. The vessel of claim 56, wherein the adaptor contains a nucleotide sequence comprising a 5′

region, a middle region, and a 3′

region, wherein the 5′

region and 3′

region of the sequence are complementary to each other such that under certain conditions they anneal to each other and form the stem of a stem-loop structure, and wherein the stem contains a blunt end.

72. The vessel of claim 56, wherein the adaptor contains a nucleotide sequence comprising a 5′

region, a middle region, and a 3′

region, wherein the 5′

region and 3′

region of the sequence are complementary to each other such that under certain conditions they anneal to each other and form the stem of a stem-loop structure, and wherein the outermost part of the stem contains a sticky end.

60-64. -64. (canceled)

66-70. -70. (canceled)

73-76. -76. (canceled)

77. A kit for detecting a target nucleic acid of interest, the kit comprising two or more fluidically isolated containers, the containers collectively comprising:

(A) an isolated nucleic acid ligase,

(B) an isolated nucleic acid polymerase,

(C) an isolated restriction enzyme, and

(D) a polynucleotide adaptor, wherein the adaptor comprises a single nucleic acid strand which contains the single strand component of a restriction enzyme recognition sequence and which is configured to, under certain conditions, adopt a stem-loop structure.

78. The kit of claim 77, further comprising a first oligonucleotide primer and a second oligonucleotide primer.

79. The kit of claim 78, further comprising a linear double-stranded nucleic acid template comprising two complementary strands.

80. The kit of claim 79, wherein the first oligonucleotide primer is complementary to a complementary strand of the linear double-stranded nucleic acid template.

86. The kit of claim 77, wherein the isolated restriction enzyme recognizes a full double-stranded restriction enzyme recognition sequence corresponding to the single strand component of a restriction enzyme sequence of the adaptor.

92. The kit of claim 77, wherein the adaptor contains a nucleotide sequence comprising a 5′

region, a middle region, and a 3′

region, wherein the 5′

region and 3′

region of the sequence are complementary to each other such that under certain conditions they anneal to each other and form the stem of a stem-loop structure, and wherein the stem contains a blunt end.

81-85. -85. (canceled)

87-91. -91. (canceled)

93-97. -97. (canceled)