Compositions and methods for nucleic acid sequencing

Abstract

Provided herein are methods and compositions for improved sequencing techniques using, for example, polymeric particles and/or three-dimensional structures.

1 - 112 . (canceled) 
     
     
         113 . A particle comprising:
 a hydrogel polymer scaffold;   a polymerase complex comprising a polymerase bound to a double-stranded nucleic acid molecule; and   a polymer shell surrounding both the hydrogel polymer scaffold and polymerase complex, wherein said polymer shell is permeable to a polymerase.   
     
     
         114 . The particle of  claim 113 , wherein said double-stranded nucleic acid molecule is a polynucleotide primer hybridized to a target polynucleotide. 
     
     
         115 . The particle of  claim 114 , wherein said polynucleotide primer is non-covalently attached to the hydrogel polymer scaffold. 
     
     
         116 . The particle of  claim 114 , wherein said polynucleotide primer is covalently attached to the hydrogel polymer scaffold. 
     
     
         117 . The particle of  claim 113 , wherein said double-stranded nucleic acid molecule is retained within said particle. 
     
     
         118 . The particle of  claim 113 , wherein the hydrogel polymer scaffold is permeable to a polymerase. 
     
     
         119 . The particle of  claim 113 , wherein the hydrogel polymer scaffold comprises polyacrylamide, polyacrylate, polymethacrylate, polyethylene glycol, polyolefin, polystyrene, polycarbonate, polyurethane, polysiloxane, polyalkyloxide, polynorbornene, a polysaccharide polymer, or a copolymer thereof. 
     
     
         120 . The particle of  claim 113 , wherein the hydrogel polymer scaffold comprises polymerized units of acrylamide, poly(ethylene glycol) methyl ether methacrylate, glicydyl methacrylate, hydroxyethylmethacrylate, hydroxyethyl acrylate, or hydroxypropylmethacrylate. 
     
     
         121 . The particle of  claim 113 , wherein the hydrogel polymer scaffold and the polymer shell comprises one or more crosslinking agents. 
     
     
         122 . The particle of  claim 113 , wherein the polymer shell is formed by polymerized units of shell monomers, and the polymer shell is not covalently attached to the polynucleotide primer. 
     
     
         123 . The particle of  claim 113 , wherein the polymer shell is a hydrogel. 
     
     
         124 . The particle of  claim 113 , wherein the polymer shell comprises polymerized units of acrylamide, poly(ethylene glycol) methyl ether methacrylate, glicydyl methacrylate, hydroxyethylmethacrylate, hydroxyethylacrylate, or hydroxypropylmethacrylate. 
     
     
         125 . The particle of  claim 113 , wherein the diameter of said particle is greater than 1000 nm. 
     
     
         126 . The particle of  claim 113 , the hydrogel polymer scaffold has a core diameter, and the polymer shell surrounding the hydrogel polymer scaffold has a shell diameter, wherein the core diameter is about 80 to 99% of the shell diameter. 
     
     
         127 . A solid support comprising one or more particles of  claim 113 . 
     
     
         128 . The solid support of  claim 127 , further comprising 1 to 10 channels, wherein each channel comprises a plurality of particles. 
     
     
         129 . A method of amplifying a target polynucleotide, said method comprising:
 contacting a particle of  claim 114  with an amplification reagent comprising a plurality of nucleotides; and   amplifying the target polynucleotide, wherein amplifying comprises extending said primer along the target polynucleotide to form amplification products within the polymer particle.   
     
     
         130 . The method of  claim 129 , wherein amplifying comprises an isothermal amplification reaction. 
     
     
         131 . The method of  claim 129 , wherein amplifying comprises bridge-PCR, recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), strand displacement amplification, or RCA with exponential strand displacement amplification. 
     
     
         132 . The method of  claim 129 , wherein amplification comprises strand displacement amplification (SDA) or rolling circle amplification (RCA). 
     
     
         133 . The method of  claim 129 , further comprising sequencing said amplification products.