Methods and kits for amplification of RNA sequences using composite primers
First Claim
1. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, said method comprising the steps of:
- (a) extending a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;
(b) cleaving RNA in the complex of step (a) with at least one enzyme that cleaves RNA from an RNA/DNA hybrid;
(c) extending a second primer hybridized to the first primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity and at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the second primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a second primer extension product is produced to form a complex of first and second primer extension products;
(d) cleaving RNA from the first primer in the complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid such that a composite amplification primer hybridizes to the second primer extension product, wherein the composite amplification primer comprises an RNA portion and a 3′
DNA portion;
(e) extending the composite amplification primer hybridized to the second primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity;
whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated, and whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.
4 Assignments
0 Petitions
Accused Products
Abstract
The invention provides methods for isothermal amplification of RNA. The methods are particularly suitable for amplifying a plurality of RNA species in a sample. The methods employ a composite primer, a second primer and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In another aspect, the methods employ a single primer (which is a composite primer) and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In some embodiments, a transcription step is included to generate multiple copies of sense RNA of an RNA sequence of interest. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.
-
Citations
63 Claims
-
1. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, said method comprising the steps of:
- (a) extending a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;
(b) cleaving RNA in the complex of step (a) with at least one enzyme that cleaves RNA from an RNA/DNA hybrid;
(c) extending a second primer hybridized to the first primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity and at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the second primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a second primer extension product is produced to form a complex of first and second primer extension products;
(d) cleaving RNA from the first primer in the complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid such that a composite amplification primer hybridizes to the second primer extension product, wherein the composite amplification primer comprises an RNA portion and a 3′
DNA portion;
(e) extending the composite amplification primer hybridized to the second primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity;
whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated, and whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53)
- (a) extending a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′
-
24. A method of determining presence or absence of a sequence of interest in a target RNA, said method comprising (i) amplifying the target RNA, said amplifying comprising extending a composite amplification primer hybridized to a polynucleotide complex generated from the target RNA comprising a 3′
- single stranded region corresponding to the sequence of interest, wherein the RNA portion of the composite amplification primer is known to hybridize to a reference sequence comprising the sequence of interest, and wherein the polynucleotide complex is generated by a method comprising;
(a) extending a first primer hybridized to a target RNA, wherein the first primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a complex comprising a first primer extension product and the target RINA is produced;
(b) extending a second primer hybridized to the first prime extension product, wherein the second primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby a second primer extension product is produced to form a complex of first and second primer extension products; and
(c) cleaving RNA in the complex of first and second primer extension products, whereby the polynucleotide complex comprising the 3′
single stranded region is generated; and
(ii) comparing the amplification products if any from step (i) with the amount of amplification products from a polynucleotide complex generated from a reference template comprising a 3′
single stranded region comprising the reference sequence, wherein the 3′
single stranded region of the polynucleotide complex of the reference template corresponds to the sequence of interest, wherein production of detectably fewer amplification products from the polynucleotide complex from the target RNA as compared to the amount of amplification products from the polynucleotide complex of the reference template indicates that the sequence of interest is absent from the target RNA and the target RNA may comprise a sequence variant with respect to the reference sequence hybridizable to the RNA portion of the composite primer. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31)
- single stranded region corresponding to the sequence of interest, wherein the RNA portion of the composite amplification primer is known to hybridize to a reference sequence comprising the sequence of interest, and wherein the polynucleotide complex is generated by a method comprising;
-
54. A method for generating multiple copies of a polynucleotide sequence complementary to an RNA sequence, said method comprising the steps of:
- (a) synthesizing a polynucleotide strand by extending a first primer hybridized to a target RNA, wherein the first primer is a composite primer comprising an RNA portion and a 3′
DNA portion, and whereby a complex comprising the first polynucleotide strand and the target RNA is produced;
(b) synthesizing a second polynucleotide strand complementary to the first polynucleotide strand by extending a second primer hybridized to a target RNA, wherein the second primer is a composite primer comprising an RNA portion and a 3′
DNA portion, whereby the second polynucleotide strand forms a complex with the first polynucleotide strand;
(c) cleaving RNA from the first composite primer in the complex of first polynucleotide strand and the second polynucleotide strand such that a composite amplification primer hybridizes to the second polynucleotide strand, wherein the composite amplification primer comprises an RNA portion and a 3′
DNA portion;
(d) extending the composite amplification primer hybridized to the second polynucleotide strand, such that said first polynucleotide strand is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated, whereby multiple copies of a polynucleotide sequence complementary to the target RNA are generated. - View Dependent Claims (55, 56, 57, 58, 59, 60, 61, 62, 63)
- (a) synthesizing a polynucleotide strand by extending a first primer hybridized to a target RNA, wherein the first primer is a composite primer comprising an RNA portion and a 3′
Specification