Methods and devices for amplification of nucleic acid

Abstract

The present invention relates to methods and devices for amplifying nucleic acid, and, in particular, amplifying so as to generate products on a surface without the use of emulsions. In a preferred embodiment, a plurality of groups of amplified product are generated on the surface, each group positioned in different (typically predetermined) locations on said surface so as to create an array.

1 .- 31 . (canceled) 
     
     
         32 . A method of amplifying and sequencing nucleic acid, comprising:
 a) providing:
 i) a population of different nucleic acid template molecules, 
 ii) a plurality of single stranded oligonucleotides immobilized on a bead, 
 iii) amplification reagents, 
 iv) sequencing reagents, and 
 v) a plurality of first and second sequencing primers; 
   b) hybridizing at least a portion of said population of nucleic acid template molecules to said plurality of single stranded oligonucleotides immobilized on said bead;   c) amplifying said nucleic acid template molecules so as to create a plurality of forward strands;   d) sequencing said forward oligonucleotide strands with said first sequencing primers;   e) amplifying said nucleic acid template molecules so as to create a plurality of reverse single stranded strands; and   f) sequencing said reverse oligonucleotide strands with said second sequencing primers.   
     
     
         33 . The method of  claim 32 , wherein said amplification reagents comprise polymerase and dNTPs. 
     
     
         34 . The method of  claim 32 , wherein said sequencing reagents comprise reagents for sequencing by synthesis. 
     
     
         35 . The method of  claim 32 , wherein the bead is enclosed in a chamber. 
     
     
         36 . The method of  claim 35 , wherein the chamber is formed between i) a first surface having a plurality of indentations and ii) a second surface. 
     
     
         37 . The method of  claim 35 , wherein the bead is located within a single indentation of the plurality of indentations. 
     
     
         38 . The method of  claim 36 , wherein each indentation of the plurality of indentation comprises a bead. 
     
     
         39 . The method of  claim 36 , wherein said chamber is sealed by an adhesive gasket between the first and second surfaces. 
     
     
         40 . The method of  claim 32 , wherein said plurality of single stranded oligonucleotides are immobilized to said bead through a linker. 
     
     
         41 . The method of  claim 40 , wherein said linker elevates said oligonucleotides away from said surface. 
     
     
         42 . The method of  claim 32 , wherein when the first sequencing primers are used in step d), said second sequencing primers are not active. 
     
     
         43 . A method of sequencing nucleic acid, comprising:
 a) providing i) a plurality of different amplicons immobilized on a bead enclosed in a chamber, and ii) sequencing reagents;   b) introducing said sequencing reagents into said chamber; and   c) performing sequencing by synthesis.   
     
     
         44 . The method of  claim 43 , wherein the chamber is formed between i) a first surface having a plurality of indentations and ii) a second surface. 
     
     
         45 . The method of  claim 43 , wherein said chamber is sealed by an adhesive gasket, between the first and second surfaces. 
     
     
         46 . The method of  claim 45 , wherein said adhesive gasket is sandwiched between said first and second surfaces. 
     
     
         47 . The method of  claim 43 , wherein said sequencing reagents comprise polymerase and a plurality of sequencing primers. 
     
     
         48 . The method of  claim 43 , wherein said amplicons are attached to the bead through extension of the primers which were attached to the bead prior to amplification. 
     
     
         49 . The method of  claim 48 , wherein said amplification performed without the use of emulsions. 
     
     
         50 . The method of  claim 44 , wherein the first surface comprises silicon. 
     
     
         51 . The method of  claim 50 , where the first surface is a microchip.