Methods and compositions for linear isothermal amplification of polynucleotide sequences using a RNA DNA composite primer
First Claim
Patent Images
1. :
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
(a) hybridizing a polynucleotide comprising a termination polynucleotide sequence to a DNA template-composite primer complex, wherein said complex comprises a composite primer hybridized to a single stranded DNA template comprising the target sequence, said composite primer comprising an RNA portion and a 3′
DNA portion, whereby said polynucleotide comprising a termination polynucleotide sequence is hybridized to a region of the template which is 5′
with respect to hybridization of the composite primer to the template;
(b) extending the composite primer in the DNA template-composite primer complex of step (a) with DNA polymerase;
(c) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced.
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Abstract
The present invention provides novel isothermal, single primer linear nucleic acid amplification methods. Methods for amplifying complementary DNA using a composite primer, primer extension, strand displacement, and optionally a termination sequence, are provided. Methods for amplifying sense RNA using a composite primer, primer extension, strand displacement, optionally template switching, a propromoter oligonucleotide and transcription are also provided. The invention further provides compositions and kits for practicing said methods, as well as methods which use the amplification products.
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Citations
49 Claims
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1. :
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
(a) hybridizing a polynucleotide comprising a termination polynucleotide sequence to a DNA template-composite primer complex, wherein said complex comprises a composite primer hybridized to a single stranded DNA template comprising the target sequence, said composite primer comprising an RNA portion and a 3′
DNA portion,whereby said polynucleotide comprising a termination polynucleotide sequence is hybridized to a region of the template which is 5′
with respect to hybridization of the composite primer to the template;
(b) extending the composite primer in the DNA template-composite primer complex of step (a) with DNA polymerase;
(c) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced. - View Dependent Claims (5, 8, 9, 11, 12, 22, 23)
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
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2. :
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
(a) extending a composite primer in a complex comprising (i) a single stranded DNA template comprising the target sequence; and
(ii) the composite primer, said composite primer comprising an RNA portion and a 3′
DNA portion, wherein the DNA template is hybridized to the composite primer;
(b) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced.
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
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3. :
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
(a) extending a composite primer in a complex comprising (i) a single stranded DNA template comprising the target sequence;
(ii) the composite primer, said composite primer comprising an RNA portion and a 3′
DNA portion, wherein the composite primer is hybridized to the DNA template; and
(iii) a polynucleotide comprising a termination polynucleotide sequence, wherein the polynucleotide comprising a termination polynucleotide sequence is hybridized to a region of the template which is 5′
with respect to hybridization of the composite primer to the template;
(b) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced.
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
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4. :
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
cleaving an RNA portion of a composite primer extension product in a complex comprising;
(a) a single stranded DNA template comprising the target sequence; and
(b) the composite primer extension product, wherein the composite primer of the composite primer extension product comprises an RNA portion and a 3′
DNA portion, wherein the composite primer extension product is hybridized to the DNA template;
wherein said cleaving is with an enzyme that cleaves RNA from an RNA/DNA hybrid, whereby another composite primer hybridizes to the target polynucleotide sequence and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced.
- A method for amplifying a polynucleotide sequence complementary to a target polynucleotide sequence comprising;
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6. :
- A method for amplifying a target polynucleotide sequence comprising;
hybridizing a primer extension product with a polynucleotide comprising a propromoter and a region which hybridizes to the primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the primer extension product, wherein the primer extension product is a displaced primer extension product generated by;
(a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′
DNA portion;
(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′
with respect to hybridization of the composite primer to the template;
(c) extending the composite primer with DNA polymerase;
(d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement to produce displaced primer extension product;
whereby multiple copies of the target sequence are produced.
- A method for amplifying a target polynucleotide sequence comprising;
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7. :
- A method for amplifying a target polynucleotide sequence comprising;
(a) hybridizing a first composite primer to a single stranded DNA template comprising the complementary sequence of the target polynucleotide sequence, said composite primer comprising an RNA portion and a 3′
DNA portion, wherein said single stranded DNA template is generated by a method comprising;
(i) hybridizing a polynucleotide comprising the target polynucleotide sequence with a second composite primer, said second composite primer comprising an RNA portion and a 3′
DNA portion;
(ii) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the polynucleotide comprising the target polynucleotide sequence which is 5′
with respect to hybridization of the second composite primer to said polynucleotide;
(iii) extending the second composite primer with DNA polymerase;
(iv) cleaving the RNA portion of the annealed second composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the polynucleotide comprising the target polynucleotide sequence and repeats primer extension by strand displacement, whereby multiple copies of a single stranded DNA template comprising the complementary sequence of the target polynucleotide sequence are generated;
(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′
with respect to hybridization of the first composite primer to the template;
(c) extending the first composite primer with DNA polymerase;
(d) cleaving the RNA portion of the annealed first composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement, whereby multiple copies of the target polynucleotide sequence are produced. - View Dependent Claims (10, 13, 24)
- A method for amplifying a target polynucleotide sequence comprising;
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14-21. -21. (canceled)
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25-47. -47. (canceled)
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48. :
- A method for amplifying a target polynucleotide sequence comprising;
(a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′
DNA portion;
(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′
with respect to hybridization of the composite primer to the template;
(c) extending the composite primer with DNA polymerase;
(d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement to produce displaced primer extension product;
(e) hybridizing a polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products, whereby at least 100 copies of RNA transcripts are produced from each displaced primer extension product. - View Dependent Claims (49)
- A method for amplifying a target polynucleotide sequence comprising;
Specification