Methods and compositions for cluster generation by bridge amplification

Abstract

The present disclosure is concerned with compositions and methods for reducing the steps used in the generation of monoclonal clusters by combining the enzymes used for linearization and removal of unused surface primers.

1 - 7 . (canceled) 
     
     
         8 . A method of preparing nucleic acids for a sequencing reaction, the method comprising:
 (a) providing an array comprising a plurality of amplification sites, wherein amplification sites comprise
 (i) a plurality of capture nucleic acids attached to the amplification sites,
 wherein a first population of the plurality of capture nucleic acids comprises a cleavage site, and 
 
 (ii) a plurality of clonal double-stranded modified target nucleic acids,
 wherein both strands of each double-stranded target nucleic acid are attached at their 5′ ends to a capture nucleic acid, 
 wherein one strand is attached to a capture nucleic acid that comprises the cleavage site, and 
 wherein the cleavage site is positioned in a double-stranded region of each double-stranded molecule; 
 
   (b) contacting the array with a composition comprising at least one enzyme to produce an abasic site at the cleavage site and an exonuclease comprising a 3′ to 5′ single-stranded DNA exonuclease activity,
 wherein cleavage occurs at the cleavage site, 
 wherein cleavage converts one strand of double-stranded target nucleic acids into a first strand attached to the amplification site and a second strand that is not attached to the amplification site; and 
 wherein single-stranded capture nucleic acids comprising a free 3′ end are reduced in length by the exonuclease. 
   
     
     
         9 . The method of  claim 8 , wherein the at least one enzyme to produce an abasic site at the cleavage site comprises uracil DNA glycosylase and an endonuclease. 
     
     
         10 . The method of  claim 8 , wherein the endonuclease is DNA glycosylase-lyase Endonuclease VIII. 
     
     
         11 . The method of  claim 8 , further comprising removal of the at least one enzyme to produce an abasic site at the cleavage site and the exonuclease from the array. 
     
     
         12 . The method of  claim 8 , further comprising subjecting the cleaved double-stranded target nucleic acids to conditions that remove the second strand that is not attached to the amplification site. 
     
     
         13 . The method of  claim 12 , wherein the conditions that remove the second strand comprise a denaturant, wherein the denaturant results in immobilized single-stranded nucleic acids comprising a target nucleic acid covalently attached to a second population of capture nucleic acid, wherein the second population of capture nucleic acids is attached to the amplification sites. 
     
     
         14 . The method of  claim 11 , wherein the denaturant comprises formamide. 
     
     
         15 . The method of  claim 11 , further comprising re-annealing the immobilized single-stranded nucleic acid to a member of the first population of capture nucleic acids to generate an immobilized partially single-stranded nucleic acids. 
     
     
         16 . The method of  claim 8 , wherein the cleavage site is positioned in the capture nucleic acid region of the double-stranded region of each double-stranded target nucleic acid. 
     
     
         17 . The method of  claim 8 , wherein the cleavage site comprises a uracil, wherein an abasic site is generated by the uracil DNA glycosylase, and wherein the abasic site is cleaved by the endonuclease. 
     
     
         18 . The method of  claim 8 , wherein the exonuclease is Exonuclease I. 
     
     
         19 . The method of  claim 13 , further comprising hybridizing a sequencing primer to the immobilized single-stranded nucleic acids of  claim 13  or a single stranded region of the immobilized partially single-stranded nucleic acids of  claim 15 , thereby preparing single-stranded nucleic acids for a sequencing reaction. 
     
     
         20 . The method of  claim 19 , further comprising performing a sequencing reaction to determine the sequence of at least one region of the immobilized single-stranded nucleic acids or the immobilized partially single-stranded nucleic acids. 
     
     
         21 . The method of  claim 19 , wherein the sequencing reaction comprises sequencing-by-synthesis. 
     
     
         22 . The method of  claim 8 , wherein the array is produced by amplifying a plurality of target nucleic acids using the capture nucleic acids as amplification primers. 
     
     
         23 . The method of  claim 22 , wherein amplifying comprises exclusion amplification.