Methods and Compositions Employing Blocked Primers

Abstract

The present disclosure provides methods, compositions, and systems employing blocked primers. Aspects of the disclosure include providing a blocked primer reaction mixture that includes a blocked primer and a template nucleic acid component from a single cell; unblocking the blocked primer to produce an active primer reaction mixture and subjecting the activated primer reaction mixture to primer extension conditions, such as nucleic acid implication conditions.

1 - 15 . (canceled) 
     
     
         16 . A method comprising:
 (a) combining a blocked primer comprising a light labile blocking moiety with a template nucleic acid component to produce a blocked primer reaction mixture;   (b) unblocking the blocked primer of the blocked primer reaction mixture by exposing the blocked primer reaction mixture to light sufficient to remove the blocking moiety to produce an activated primer reaction mixture; and   (c) subjecting the activated primer reaction mixture to template dependent primer extension reaction conditions.   
     
     
         17 . The method according to  claim 16 , wherein the blocked primer is a member of a pair of blocked amplifications primers. 
     
     
         18 . The method according to  claim 16 , wherein the template dependent primer extension reaction conditions comprise nucleic acid amplification conditions. 
     
     
         19 . The method according to  claim 16 , wherein the template nucleic acid component comprises ribonucleic acids or deoxyribonucleic acids. 
     
     
         20 . The method according to  claim 16 , wherein the template nucleic acid component is produced by a polymerase mediated template dependent primer extension reaction. 
     
     
         21 . The method according to  claim 20 , wherein the polymerase mediated template dependent primer extension reaction comprises a reverse transcriptase mediated template dependent primer extension reaction. 
     
     
         22 . The method according to  claim 16 , wherein the template nucleic acid component is produced by obtaining a single cell and lysing the obtained single cell. 
     
     
         23 . The method according to  claim 16 , wherein the method comprises producing the blocked primer reaction mixture by introducing a single cell into a vessel comprising the blocked primer and lysing the introduced single cell. 
     
     
         24 . The method according to  claim 16 , wherein the method further comprises performing a second template mediated primer extension reaction. 
     
     
         25 . The method according to  claim 24 , wherein the second template mediated primer extension reaction is performed with an activated primer produced by unblocking a second blocked primer that is differentially blocked from the first blocked primer. 
     
     
         26 . The method according to  claim 25 , wherein the second blocked primer is a member of a second pair of blocked amplification primers and the method comprises unblocking the second pair of blocked amplification primers and then using the unblocked second pair of amplification primers in a second nucleic acid amplification. 
     
     
         27 . The method according to  claim 16 , wherein the light labile blocking moiety is attached to the 3′ end of the blocked primer. 
     
     
         28 . The method according to  claim 16 , wherein the blocked primer comprises non-template nucleotide sequence. 
     
     
         29 . The method according to  claim 16 , wherein the method is used in preparing a library selected from an immune cell receptor repertoire library, a T-cell receptor (TCR) repertoire library, a B-cell receptor (BCR) repertoire library, an expression library, a sequencing library, and a next-generation sequencing (NGS) library. 
     
     
         30 . A method comprising:
 (a) combining a blocked primer comprising a thermally labile blocking moiety with a template nucleic acid component to produce a blocked primer reaction mixture;   (b) unblocking the blocked primer of the blocked primer reaction mixture by heating the blocked primer reaction mixture to a temperature sufficient to remove the thermally labile blocking moiety to produce an activated primer reaction mixture; and   (c) subjecting the activated primer reaction mixture to template dependent primer extension reaction conditions.   
     
     
         31 . The method according to  claim 30 , wherein the blocked primer is a member of a pair of blocked amplifications primers. 
     
     
         32 . The method according to  claim 30 , wherein the template nucleic acid component comprises ribonucleic acids or deoxyribonucleic acids. 
     
     
         33 . The method according to  claim 30 , wherein the template nucleic acid component is produced by a reverse transcriptase mediated template dependent primer extension reaction. 
     
     
         34 . The method according to  claim 30 , wherein the template nucleic acid component is produced by obtaining a single cell and lysing the obtained single cell. 
     
     
         35 . The method according to  claim 30 , wherein the blocked primer comprises non-template nucleotide sequence. 
     
     
         36 . The method according to  claim 30 , wherein the method is used in preparing a library selected from an immune cell receptor repertoire library, a T-cell receptor (TCR) repertoire library, a B-cell receptor (BCR) repertoire library, an expression library, a sequencing library, and a next-generation sequencing (NGS) library. 
     
     
         37 . A method comprising:
 (a) combining a blocked primer comprising an enzymatically labile blocking moiety with a template nucleic acid component to produce a blocked primer reaction mixture;   (b) adding an enzyme to the blocked primer reaction mixture to enzymatically remove the enzymatically labile blocking moiety of the blocked primer, thereby unblocking the blocked primer of the blocked primer reaction mixture to produce an activated primer reaction mixture; and   (c) subjecting the activated primer reaction mixture to template dependent primer extension reaction conditions.   
     
     
         38 . The method according to  claim 37 , wherein the blocked primer is a member of a pair of blocked amplifications primers. 
     
     
         39 . The method according to  claim 37 , wherein the template nucleic acid component comprises ribonucleic acids or deoxyribonucleic acids. 
     
     
         40 . The method according to  claim 37 , wherein the template nucleic acid component is produced by a reverse transcriptase mediated template dependent primer extension reaction. 
     
     
         41 . The method according to  claim 37 , wherein the template nucleic acid component is produced by obtaining a single cell and lysing the obtained single cell. 
     
     
         42 . The method according to  claim 37 , wherein the blocked primer comprises non-template nucleotide sequence. 
     
     
         43 . The method according to  claim 37 , wherein the method is used in preparing a library selected from an immune cell receptor repertoire library, a T-cell receptor (TCR) repertoire library, a B-cell receptor (BCR) repertoire library, an expression library, a sequencing library, and a next-generation sequencing (NGS) library. 
     
     
         44 - 47 . (canceled)