Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences
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United States of America Patent
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Dec 27, 2022
Grant Date -
Sep 6, 2018
app pub date -
Aug 22, 2016
filing date -
Aug 21, 2015
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Abstract
A method for specifically and efficiently quantifying the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5′-Dbs-adapter to the 5′-end of target RNAs, wherein the 5′-Dbs-adapter has a stem-loop structure whose protruding 5′-end base-pairs with the 5′-end of target RNAs, and wherein the loop region of 5′-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3′Db-adapter to the 3′-end of target RNAs, wherein the 3′-Db-adapter has a stem-loop structure whose protruding 3′-end base-pairs with the 3′-end of target RNAs; ligating both adapters with target RNAs by RN12 ligation to form a “dumbbell-like” structure; and, amplifying and quantifying the ligation product by RT-PCR.
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