Global amplification using random priming by a composite primer
First Claim
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1. A method for amplification of a template DNA, comprising:
- (a) incubating a reaction mixture, said reaction mixture comprising;
(i) a template DNA;
(ii) a first primer, wherein the first primer is hybridizable to a multiplicity of template sites, wherein the first primer is a population of different composite primers each comprising an RNA portion and a 3′
DNA portion, wherein each composite primer comprises a 3′
random sequence, and wherein each composite primer is a tailed primer that comprises a 5′
portion that is not hybridizable to the template DNA tinder conditions in which the first primer hybridizes to the template DNA;
(iii) a DNA-dependent DNA polymerase; and
(iv) an RNA-dependent DNA polymerase;
wherein the incubation is under conditions that permit generation of a complex comprising an RNA/DNA heteroduplex, wherein said complex comprising an RNA/DNA heteroduplex is produced by hybridization of the first primer to the template DNA, primer extension to generate a first primer extension product, hybridization of a second primer to the first primer extension product, and extension of the second primer to generate a second primer extension product; and
(b) incubating a reaction mixture, said reaction mixture comprising(i) at least a portion of the reaction products generated according to step (a);
(ii) an amplification primer, wherein said amplification primer is a composite primer comprising an RNA portion and a 3′
DNA portion, wherein the amplification primer comprises some of the sequence of the first primer, and wherein the first primer and the amplification primer are different primers;
(iii) a DNA-dependent DNA polymerase; and
(iv) an agent that cleaves RNA from an RNA/DNA heteroduplex;
wherein the incubation is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the first primer extension product when its RNA is cleaved and another amplification primer binds to the second primer extension product and is extended, such that primer extension and strand displacement are repeated, whereby multiple copies of a an amplification product are generated.
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Abstract
The invention relates to the field of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplification of (i.e., making multiple copies of) a multiplicity of different polynucleotide template sequences using a randomly primed RNA/DNA composite primer.
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Citations
74 Claims
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1. A method for amplification of a template DNA, comprising:
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(a) incubating a reaction mixture, said reaction mixture comprising; (i) a template DNA; (ii) a first primer, wherein the first primer is hybridizable to a multiplicity of template sites, wherein the first primer is a population of different composite primers each comprising an RNA portion and a 3′
DNA portion, wherein each composite primer comprises a 3′
random sequence, and wherein each composite primer is a tailed primer that comprises a 5′
portion that is not hybridizable to the template DNA tinder conditions in which the first primer hybridizes to the template DNA;(iii) a DNA-dependent DNA polymerase; and (iv) an RNA-dependent DNA polymerase; wherein the incubation is under conditions that permit generation of a complex comprising an RNA/DNA heteroduplex, wherein said complex comprising an RNA/DNA heteroduplex is produced by hybridization of the first primer to the template DNA, primer extension to generate a first primer extension product, hybridization of a second primer to the first primer extension product, and extension of the second primer to generate a second primer extension product; and (b) incubating a reaction mixture, said reaction mixture comprising (i) at least a portion of the reaction products generated according to step (a); (ii) an amplification primer, wherein said amplification primer is a composite primer comprising an RNA portion and a 3′
DNA portion, wherein the amplification primer comprises some of the sequence of the first primer, and wherein the first primer and the amplification primer are different primers;(iii) a DNA-dependent DNA polymerase; and (iv) an agent that cleaves RNA from an RNA/DNA heteroduplex; wherein the incubation is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the first primer extension product when its RNA is cleaved and another amplification primer binds to the second primer extension product and is extended, such that primer extension and strand displacement are repeated, whereby multiple copies of a an amplification product are generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 73, 74)
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20. A method for amplification of a template DNA, comprising:
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incubating a reaction mixture, said reaction mixture comprising; (a) a complex comprising a RNA/DNA partial heteroduplex, wherein the complex is generated by incubating a first reaction mixture, said first reaction mixture comprising; (i) a template DNA; (ii) a first primer;
wherein the first primer is hybridizable to a multiplicity of template sites, wherein the first primer is a population of different composite primers each comprising an RNA portion and a 3′
DNA portion, wherein the composite primer comprises a 3′
random sequence, and wherein the composite primer is a tailed primer that comprises a 5′
portion that is not hybridizable to the template DNA under conditions in which the first primer hybridizes to the template DNA;(iii) a DNA-dependent DNA polymerase; and (iv) an RNA-dependent DNA polymerase; wherein the incubation is under conditions that permit hybridization of the first primer to the template DNA, primer extension to generate a first primer extension product, hybridization of a second primer to the first primer extension product, and extension of the second primer to generate a second primer extension product, whereby a complex comprising an RNA/DNA partial heteroduplex is generated; (b) an amplification primer, wherein the amplification primer is a composite primer comprising an RNA portion and a 3′
DNA portion, wherein the amplification primer comprises some of the sequence of the first primer, and wherein the first primer and the amplification primer are different primers;(c) a DNA-dependent DNA polymerase; and (d) an agent that cleaves RNA from an RNA/DNA heteroduplex; wherein the incubation is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the first primer extension product when its RNA is cleaved and another amplification primer binds to the second primer extension product and is extended, such that primer extension and strand displacement are repeated, whereby multiple copies of an amplification product are generated. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 65, 66, 67, 68)
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34. A method for amplification of a template DNA, comprising:
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incubating a reaction mixture, said reaction mixture comprising; (a) a complex of a first primer extension product and a second primer extension product, wherein the first primer extension product is generated by extension of a first primer hybridized to the template DNA with a DNA polymerase, wherein the first primer is a hybridizable to a multiplicity of template polynucleotide sites, wherein the first primer is a population of different composite primers each comprising an RNA portion and a 3′
DNA portion, wherein each composite primer comprises a 3′
random sequence, and wherein each composite primer is a tailed primer that comprises a 5′
portion that is not hybridizable to the template DNA under conditions in which the first primer hybridizes to the template DNA, and wherein the second primer extension product is generated by extension of a second primer hybridized to the first primer extension product;(b) an amplification primer, wherein the amplification is a composite primer comprising an RNA portion and a 3′
DNA portion, wherein the amplification primer comprises some of the sequence of the first primer, wherein the first primer and the amplification primer are different primers, and wherein the amplification primer is hybridizable to the second primer extension product;(c) a DNA-dependent DNA polymerase; and (d) an agent that cleaves RNA from an RNA/DNA heteroduplex; wherein the incubation is under conditions that permit RNA cleavage, primer hybridization, primer extension, and displacement of the first primer extension product from the second primer extension product when its RNA is cleaved and another amplification primer binds and is extended, such that primer extension and strand displacement are repeated, whereby multiple copies of an amplification product are generated. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 69, 70, 71, 72)
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48. A method for amplification of a template DNA, comprising:
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(a) randomly priming a template DNA with a first primer, wherein said first primer is hybridizable to a multiplicity of template polynucleotide sites, wherein the first primer is a population of different composite primers each comprising an RNA portion and a 3′
DNA portion, and wherein each composite primer is a tailed primer that comprises a 5′
portion that is not hybridizable to the template DNA under conditions in which the first primer hybridizes to the template DNA;(b) extending said first primer with a DNA polymerase to generate a first primer extension product; (c) hybridizing a second primer to the first primer extension product; (d) extending said second primer with a DNA-dependent DNA polymerase and an RNA-dependent polymerase to generate a second primer extension product, whereby a complex comprising an RNA/DNA heteroduplex is generated; (e) cleaving RNA from the first primer with an agent that cleaves RNA from a RNA/DNA heteroduplex; (f) hybridizing an amplification primer to the second primer extension product, wherein said amplification primer is a composite primer comprising a RNA portion and a 3′
DNA portion, wherein the amplification primer comprises some of the sequence of the first primer, and wherein the first primer and the amplification primer are different primers;(g) extending the hybridized amplification primer by strand displacement DNA synthesis; (h) cleaving RNA from the amplification primer with an agent that cleaves RNA from a RNA/DNA heteroduplex, such that another amplification primer hybridizes and is extended, whereby multiple copies of an amplification product are generated. - View Dependent Claims (49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)
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Specification