Methods and compositions for amplification of RNA sequences
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Abstract
The invention provides methods for linear and exponential amplification of RNA. They are particularly suitable for amplifying a plurality of RNA species in a sample. The methods are based on hybridization of polynucleotide comprising a propromoter sequence to a primer extension product to generate an intermediate polynucleotide capable of driving transcription, whereby multiple copies of RNA products comprising sequences complementary to an RNA sequence of interest are generated. 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.
140 Citations
51 Claims
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1-37. -37. (canceled)
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38. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising:
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(a) extending a first primer hybridized to a target RNA with an RNA-dependent DNA polymerase, whereby a complex comprising a first primer extension product and the target RNA is produced;
(b) cleaving RNA in the complex of step (b) with an enzyme that cleaves RNA from an RNA/DNA hybrid;
(c) extending a second primer hybridized to the first primer extension product with a DNA-dependent DNA polymerase whereby a complex comprising the first primer extension product and a second primer extension product is produced;
(d) denaturing the complex of step (c);
(e) hybridizing a composite primer to a single stranded second primer extension product resulting from step (d), wherein the composite primer comprises an RNA portion and a 3′
DNA portion;
(f) extending the composite primer with a DNA-dependent DNA polymerase, whereby a complex comprising a primer extension product and the second primer extension product is formed;
(g) cleaving RNA in the complex of step (f) with an enzyme that cleaves RNA from an RNA/DNA hybrid, such that another composite primer hybridizes to the second primer extension product and repeats primer extension by strand displacement, whereby multiple copies of the complement of the RNA sequence of interest are produced. - View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
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51-84. -84. (canceled)
Specification