Isothermal Nucleic Acid Amplification Methods and Compositions

• US 20090203085A1
• Filed: 02/12/2009
• Published: 08/13/2009
• Est. Priority Date: 02/12/2008
• Status: Active Grant

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: KURN, Nurith, Wang, Shenglong
• Granted Patent: US 8,034,568 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid

Abstract

Methods and compositions are provided related to the amplification of target polynucleotide sequences as well as total RNA and total DNA amplification. In some embodiments, the methods and compositions also allow for the immobilization and capture of target polynucleotides with defined 3′ and or 5′ sequences to solid surfaces. The polynucleotides attached to the solid surfaces can be amplified or eluted for downstream processing. In some cases, nucleotides attached to solid surfaces can be used for high throughput sequencing of nucleotide sequences related to target DNA or target RNA.

Claims

1-67. -67. (canceled)

68. A method for creating a partial RNA-DNA heteroduplex comprising.

a) providing a template nucleic acid in a reaction mixture;

b) adding an RNA-DNA chimeric primer;

c) adding a DNA polymerase; and

d) adding an exonuclease;

wherein said DNA polymerase comprises RNA-dependent polymerase activity.

69. A method for producing amplified nucleic acid comprising:

a) providing a template nucleic acid;

b) annealing a first primer or set of first DNA primers comprising a 3′

end whereby a portion of the 3′

end comprises a template annealing sequence and a 5′

tail sequence whereby a portion of the tail sequence comprises a sequence (A);

c) extending said first primer or set of first primers with a DNA polymerase to create a first primer extension product or first set of DNA polymerase products;

d) dissociating the primer extension product or products of said DNA polymerase from the template nucleic acid;

e) annealing a second primer comprising a 3′

DNA sequence and a 5′

RNA sequence, whereby a portion of the 3′

end comprises a randomized annealing sequence and a portion of the 5′

end comprises sequence (A) or sequence (B), to said primer extension product or products; and

f) extending said second primer with a DNA polymerase to create a second primer extension product or products to produce a double stranded DNA product or products comprising an A-A′

DNA duplex at one end.

70. The method of claim 69 wherein the template nucleic acid is selected from the group consisting of DNA and RNA.

71. The method of claim 69 wherein the template nucleic acid is RNA and the DNA polymerase of step (c) is an RNA dependent DNA polymerase.

72. The method of claim 71 wherein the dissociation step of step (d) is performed by degrading the RNA.

73. The method of claim 69 wherein the template nucleic acid is DNA and the DNA polymerase of step (c) is a DNA dependent DNA polymerase.

74. The method of claim 73 wherein the dissociation step of step (d) is performed by heat or chemical denaturation.

75. The method of claim 69 wherein the DNA polymerase does not exhibit substantial strand displacement activity

76. The method of claim 69 wherein each template annealing sequence of the set of primers is specific for a target or region of template nucleic acid.

77. The method of claim 69 wherein the template annealing sequence of the first primer comprises a random sequence.

78. The method of claim 69 wherein the members of the set of first primers each comprises a distinct 3′

DNA annealing sequence, each specific for a target or region of template nucleic acid.

79. The method of claim 69 wherein the first primer tail sequence is not complementary to the template nucleic acid.

80. The method of claim 69 wherein the 5′

tail sequence of the second primer comprises sequence (A).

81. The method of claim 69 wherein the 5′

tail sequence of the second primer does not comprise tail sequence (A).

82. The method of claim 69 further comprising step (g), degradation of single stranded 3′

end of said first primer extension product or products in the complex of first and second primer extension products with a single stranded DNA specific 3′

exonuclease.

83. The method of claim 82 further comprising step (h), the addition of a DNA polymerase with reverse transcriptase activity to create a double stranded nucleic acid with an A-A′

DNA duplex at one end and an B-B′

DNA-RNA heteroduplex at the other end.

84. The method of claim 83 further comprising SPIA with a reaction mixture comprising RNase H, a composite amplification primer comprising a 3′

DNA portion and a 5′

end whereby a portion of the 5′

end comprises RNA wherein the RNase H cleaves the RNA portion of the heteroduplex of step (h) to produce a single stranded end, and the amplification primer hybridizes to the single stranded end produced by RNase H, and a DNA polymerase with strand displacement activity.

85. The method of claim 69 wherein the first primer or set of primers comprises a ligand portion at the 5′

end.

86. The method of claim 83 further comprising:

(i) cleavage of the RNA from the DNA-RNA heteroduplex with RNase H;

(j) annealing a chimeric oligonucleotide comprising a 5′

end whereby the 5′

end comprises sequence (C) and a 3′

end whereby the 3′

end comprises the DNA sequence (B) to the double stranded nucleic acid product;

(k) extending the double stranded nucleic acid product with a DNA polymerase to produce a C-C′

DNA-RNA heteroduplex; and

(l) amplifying the second primer extension product with a reaction mixture comprising RNase H, an amplification primer comprising a 3′

DNA portion and a 5′

end whereby a portion of the 5′

end comprises RNA that hybridizes to a portion of the single stranded end produced by RNase H, and a DNA polymerase with strand displacement activity to produce an amplification product comprising sequence (B) near its 3′

end and sequence (A′

) at the 5′

end.

87. A method for producing amplified nucleic acid comprising:

a) providing a template nucleic acid;

b) annealing a first DNA primer or set of first DNA primers comprising a 3′

end whereby a portion of the 3′

end comprises a template annealing sequence and a 5′

end whereby a portion of the 5′

end comprises a DNA tail sequence (A);

c) extending said first primer or set of first primers with a DNA polymerase to create a primer extension product or set of primer extension products;

d) dissociation of the primer extension product or products from the template nucleic acid;

e) annealing a second primer comprising a 3′

DNA end, whereby a portion of the 3′

end comprises a random template annealing sequence, and a 5′

RNA end, whereby a portion of the 5′

end comprises the tail sequence (A), to said primer extension product or products or to said template nucleic acid;

f) extending said second primer with a DNA polymerase to create a second primer extension product or set of second primer extension products; and

g) denaturing and annealing the second primer extension product or set of second primer extension products to form a stem loop structure comprising an A-A′

RNA-DNA heteroduplex.

88. The method of claim 87 further comprising the addition of a 3′

single stranded DNA specific exonuclease and single strand specific RNase to degrade unincorporated primers and template nucleic acid.

89. The method of claim 88 further comprising amplification with a reaction mixture comprising RNase H, an amplification primer comprising sequence (A′

), and a DNA polymerase with strand displacement activity, wherein the amplification primer comprises a 3′

-DNA sequence and a 5′

-RNA sequence.

90. The method of claim 87 wherein said template nucleic acid is selected from the group consisting of RNA and DNA.

91. The method of claim 87 wherein the template nucleic acid is RNA and the DNA polymerase of step (c) is an RNA dependent DNA polymerase.

92. The method of claim 87 wherein the template nucleic acid is DNA and the DNA polymerase of step (c) is a DNA dependent DNA polymerase.

93. The method of claim 91 wherein the dissociation step of step (d) is performed by degradation of the RNA.

94. The method of claim 92 wherein the dissociation step of step (d) is performed by chemical or heat denaturation.

95. The method of claim 87 wherein the DNA polymerase does not exhibit substantial strand displacement activity.

96. The method of claim 87 wherein each DNA annealing sequence in the set of first DNA primers is specific for a target or region of the template nucleic acid.

97. The method of claim 87 wherein a portion of the 5′

tail sequence of the first primer is sequence (A) and a portion of the 5′

tail sequence of the second primer is sequence (A).

98. The method of claim 87 wherein the tail sequence is not complimentary to the template nucleic acid:

99. A method of capturing and amplifying a target sequence or sequences comprising:

(a) providing a surface wherein a first primer or set of first primers comprising a 5′

tail sequence comprising sequence (A) and a 3′

template annealing sequence comprising sequence (P) are immobilized on said surface;

(b) annealing a target nucleic acid to said first primer or set of primers;

(c) extending the first primer or set of primers with a DNA polymerase to produce an immobilized primer extension product or set of immobilized primer extension products complimentary to and hybridized with the target nucleic acid to form a complex or set of complexes;

(d) dissociating the target nucleic acid-primer extension product complex or set of primer extension product complexes;

(e) annealing a second primer comprising a 3′

DNA template annealing sequence (P) or set of template annealing sequences and a 5′

RNA tail sequence comprising sequence (A) or sequence (B) to the immobilized primer extension product or products; and

(f) extending said second primer with a DNA polymerase to create a second primer extension product or set of primer extension products comprising a double stranded nucleic acid comprising a 3′

DNA-DNA duplex (A)-(A′

).

100. The method of claim 99 wherein said template nucleic acid is selected from the group consisting of RNA and DNA.

101. The method of claim 99 wherein said first primer comprises a spacer element between the immobilizing surface and the remaining portion of the primer, a 3′

DNA end comprising a 3′

DNA annealing sequence (P), and a 5′

comprising a common 5′

DNA sequence tail (A) or sequence tail (B) between said spacer element and annealing sequence.

102. The method of claim 99 wherein the set of first primers comprises:

(a) a spacer element between the immobilizing surface and the remaining portion of the primer;

(b) a 3′

DNA end wherein members of the set of first primers comprise unique target specific 3′

DNA annealing sequences.); and

(c) a 5′

DNA end comprising a common 5′

DNA sequence tail (A) or DNA sequence tail (B) between said spacer element and annealing sequence.

103. The method of claim 99 wherein said annealing sequences (P) of said set of first primers are each specific to a target or region of the template nucleic acid.

104. The method of claim 99 wherein the 5′

tail sequence of the first primer is sequence (A) and the 5′

tail sequence of the second primer is sequence (B).

105. The method of claim 99 wherein the 5′

tail sequence of the first primer is sequence (A) and the 5′

tail sequence of the second primer is sequence (A).

106. The method of claim 99 wherein said DNA polymerase does not exhibit strand displacement activity.

107. The method of claim 99 further comprising step (g) dissociation of the second primer extension product by heat or chemical denaturation.

108. The method of claim 99 wherein the surface is selected from the group consisting of a bead, a magnetic particle, a microarray, a gene chip, and an array.

109. The method of amplifying the eluted DNA polymerase product of claim 107 further comprising:

(h) annealing an amplification primer comprising a 5′

RNA sequence and a 3′

DNA sequence;

(i) extending said annealed primer with a DNA polymerase; and

(j) amplifying the eluted DNA polymerase product with a reaction mixture comprising RNase H, an amplification primer comprising a DNA portion and a 5′

RNA portion, and a DNA polymerase with strand displacement activity to produce an amplified DNA product or products having a defined 5′

sequence (A′

) or (B′

).

110. The method of claim 99 further comprising:

(g) degradation of the single stranded 3′

end of said first primer extension product and single stranded immobilized first primers with a single stranded DNA specific 3′

exonuclease;

(h) extending the 3′

end of the first primer extension product with an RNA-dependent DNA polymerase to generate an immobilized double stranded product with an RNA-DNA heteroduplex at one end.

(i) cleavage of spacer element with light or chemical cleavage to release the immobilized double stranded nucleic acid from the surface; and

(j) amplifying the released double stranded nucleic acid in solution with a reaction mixture comprising RNase H, an amplification primer comprising a DNA portion and a 5′

RNA portion, and a DNA polymerase with strand displacement activity to produce an amplified DNA product or products having a defined 3′

sequence comprising sequence (A′

).

111. The method of claim 99 further comprising:

(g) degrading the single stranded 3′

end of said first primer extension product or products and unextended immobilized first primers with a single stranded DNA specific 3′

exonuclease;

(h) extending said first primer extension product with a DNA polymerase; and

(i) degrading RNA in the heteroduplex from the first primer extension product and the RNA portion of the second primer with RNAse H.

(j) annealing an amplification primer to the single stranded portion of the second primer extension product wherein the amplification primer has a DNA portion and a 5′

RNA portion;

(k) extending the amplification primer with a DNA polymerase having strand displacement activity to produce an amplified product hybridized to the second primer extension product; and

(l) repeating steps (i)-(k) to produce multiple copies of amplified product or products having a defined 3′

sequence comprising sequence (A′

).

112. The method of claim 105 further comprising:

(g) separating the second primer extension product or products from the first primer extension product or products by heat or chemical denaturation;

(h) annealing the second primer extension product in solution to create a stem loop structure with a DNA sequence (A′

)-RNA sequence (A) heteroduplex end;

(i) degrading RNA in the heteroduplex with RNase H;

(j) annealing an amplification primer to the single stranded portion of the second primer extension product wherein the amplification primer has a DNA portion and a 5′

RNA portion, wherein the RNA portion of the amplification primer forms a heteroduplex with a DNA portion of the second primer extension product;

(k) extending the amplification primer with a DNA polymerase having strand displacement activity to produce an amplified product hybridized to the second primer extension product to form a double stranded nucleic acid, said amplified product forming an RNA-DNA heteroduplex at one end of the double stranded nucleic acid; and

(l) repeating steps (i)-(k) to produce amplified DNA.

113. The method of claim 112 wherein between step (f) and step (g), a single stranded DNA specific 3′

exonuclease is added to degrade unincorporated primers.