Mispriming prevention reagents

Abstract

Provided herein are mispriming prevention reagents, compositions and kits comprising such reagents and methods of use thereof.

1 - 162 . (canceled) 
     
     
         163 . A method of creating a cDNA comprising:
 (a) forming a reaction mixture comprising:
 (i) RNA; 
 (iv) a reverse transcriptase; 
 (v) dNTPs; and 
 (vi) a mispriming prevention reagent comprising a nucleic acid molecule comprising, in 5′ to 3′ order:
 (1) a first condition-dependent stem region comprising a 5′ terminal covalently linked moiety and a first stem nucleic acid sequence, wherein the first stem nucleic acid sequence is at least 6 nucleotides in length and wherein the 5′ terminal covalently linked moiety comprises a cyclic or polycyclic planar moiety that does not have a bulky portion; 
 (2) a condition-dependent loop region comprising a loop nucleic acid sequence of at least 3 nucleotides in length; and 
 (3) a second condition-dependent stem region comprising a second stem nucleic acid sequence and a 3′ terminal covalently linked moiety, wherein the second stem nucleic acid sequence is at least 6 nucleotides in length and is complementary to the first stem nucleic acid sequence, wherein the 3′ terminal covalently linked moiety comprises a cyclic or polycyclic planar moiety that does not have a bulky portion, wherein the 3′ terminal covalently linked moiety is non-identical to the 5′ terminal covalently linked moiety, wherein the 3′ terminus of the second condition-dependent stem region is non-extendable by the reverse transcriptase or by a thermostable DNA polymerase, wherein the second condition-dependent stem region hybridizes to the first condition-dependent stem region with a stem melting temperature that is no greater than the first primer melting temperature and the second primer melting temperature, and wherein hybridization of the second condition-dependent stem region to the first condition-dependent stem region causes the reagent to acquire a stem-loop hairpin conformation; and 
 
   (b) incubating the reaction mixture under conditions such the RNA is reverse transcribed by the reverse transcriptase to form cDNA.   
     
     
         164 . The method of  claim 163 , wherein the loop nucleic acid sequence is a single nucleotide repeat sequence. 
     
     
         165 . The method of  claim 164 , wherein the single nucleotide repeat sequence is a poly-cytosine sequence. 
     
     
         166 . The method of  claim 163 , wherein the loop nucleic acid sequence is between 25 and 40 nucleotides in length. 
     
     
         167 . The method of  claim 166 , wherein the loop nucleic acid sequence is 28 nucleotides in length. 
     
     
         168 . The method of  claim 163 , wherein the first stem nucleic acid sequence and the second stem nucleic acid sequence are no more than 14 nucleotides in length. 
     
     
         169 . The method of  claim 163 , wherein the first stem nucleic acid sequence and the second stem nucleic acid sequence are each at least 8 nucleotides in length. 
     
     
         170 . The method of  claim 169 , wherein the first stem nucleic acid sequence and the second stem nucleic acid sequence are each 11 nucleotides in length. 
     
     
         171 . The method of  claim 163 , wherein the stem-loop hairpin conformation comprises a 5′ or 3′ overhang. 
     
     
         172 . The method of  claim 163 , wherein the stem-loop hairpin conformation comprises a blunt end. 
     
     
         173 . The method of  claim 163 , wherein:
 (a) the most 3′ nucleic acid of the first stem nucleic acid sequence is cytosine and the most 5′ nucleic acid of the second stem nucleic acid sequence is guanine; or   (b) the most 3′ nucleic acid of the first stem nucleic acid sequence is guanine and the most 5′ nucleic acid of the second stem nucleic acid sequence is a cytosine.   
     
     
         174 . The method of  claim 163 , wherein:
 (a) the most 5′ nucleic acid of the first stem nucleic acid sequence is cytosine and the most 3′ nucleic acid of the second stem nucleic acid sequence is guanine; or   (b) the most 5′ nucleic acid of the first stem nucleic acid sequence is guanine and the most 3′ nucleic acid of the second stem nucleic acid sequence is a cytosine.   
     
     
         175 . The method of  claim 163 , wherein the 5′ terminal covalently linked moiety comprises a dabcyl moiety. 
     
     
         176 . The method of  claim 163 , wherein the 3′ terminal covalently linked moiety comprises a coumarin moiety. 
     
     
         177 . The method of  claim 176 , wherein the coumarin moiety is selected from the group consisting of Courmarin 39, Courmarin 47 and Biosearch Blue. 
     
     
         178 . The method of  claim 177 , wherein the coumarin moiety is Biosearch Blue. 
     
     
         179 . The method of  claim 163 , wherein the 3′ terminal covalently linked moiety comprises a dabcyl moiety. 
     
     
         180 . The method of  claim 163 , wherein the 5′ terminal covalently linked moiety comprises a coumarin moiety. 
     
     
         181 . The method of  claim 180 , wherein the coumarin moiety is selected from the group consisting of Courmarin 39, Courmarin 47 and Biosearch Blue. 
     
     
         182 . The method of  claim 163 , wherein the mispriming prevention reagent does not hybridize to the target nucleic acid molecule with a melting temperature of greater than 32° C.