Dumbbell PCR: A Method To Quantify Specific Small RNA Variants With A Single Nucleotide Resolution At Terminal Sequences

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.

1 - 14 . (canceled) 
     
     
         15 . A method of quantifying small RNAs with single nucleotide resolution at a 3′ terminal sequence, the method comprising:
 a) the following adapters and primers:
 (i) a 3′-db-adapter with a nucleotide sequence that has a stem-loop structure and having a nucleotide sequence that is complementary to a 3′-terminal sequence of target RNAs; 
 (iii) an RT primer; 
 (iv) a reverse primer; 
 (v) a forward primer; and 
 (vi) a probe targeting the boundary of the target RNAs and the 3′-db-adapter, wherein the probe contains a 5′fluorophore and a 3′ quencher; 
 
 b) hybridizing the 3′-db-adapter to the 3′-end of the target RNAs: 
 c) ligating the 3′db-adapter with the target RNAs by Rn12 ligation to form ligation products; and 
 d) amplifying and quantifying the ligation products by RT-PCR assay using a DNA polymerase with 5′ exonuclease activity, wherein the probe is complementary the boundary of the 3′-db-adapter and target RNAs to quantify ligated target RNAs. 
 
     
     
         16 . The method of  claim 15 , wherein the forward primer is complementary to a 5′-sequence of the target RNAs. 
     
     
         17 . The method of  claim 15 , wherein the nucleotide sequence of the 3′-db adapter is SEQ ID NO: 18. 
     
     
         18 . The method of  claim 15 , wherein the nucleotide sequence of the RT primer is SEQ ID NO: 19. 
     
     
         19 . The method of  claim 15 , wherein the nucleotide sequence of the forward primer is SEQ ID NO: 5. 
     
     
         20 . The method of  claim 15 , wherein the target RNA is miR-16. 
     
     
         21 . The method of  claim 15 , wherein the target RNAs are quantified in human cancer cells. 
     
     
         22 . The method of  claim 21 , wherein the human cancer cells are breast cancer cells. 
     
     
         23 . The method of  claim 21 , wherein the human cancer cells are prostate cancer cells.