Modified stem-loop oligonucleotide mediated reverse transcription and base-spacing constrained quantitative PCR
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United States of America Patent
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Dec 26, 2017
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Jun 13, 2011
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Jun 14, 2010
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Abstract
There is provided a method for detecting a target RNA molecule in a sample comprising reverse transcription, amplification of the reverse transcription product, and detection of the amplification product, involving the use of (i) an RT oligonucleotide comprising a stem-loop portion containing one or more nucleotides modified or modifiable to block DNA polymerase extension and a target annealing portion that is complementary to a downstream portion of the target RNA, the target annealing portion located 3′ to the stem-loop portion, (ii) a first amplification primer that anneals to a downstream portion of a 3′ extended region of the reverse transcription product and (ii) a second amplification primer that anneals to an interface portion of a DNA strand complementary to the reverse transcription product, the interface portion comprising a region that is complementary to a 3′ portion of the RT oligonucleotide and a 5′ portion of the 3′ extended region in the reverse transcription product.
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