Constant cluster seeding

Abstract

The invention provides methods for controlling the density of different molecular species on the surface of a solid support. A first mixture of different molecular species is attached to a solid support under conditions to attach each species at a desired density, thereby producing a derivatized support having attached capture molecules. The derivatized support is treated with a second mixture of different molecular species, wherein different molecular species in the second mixture bind specifically to the different capture molecules attached to the solid support. One or more of the capture molecules can be reversibly modified such that the capture molecules have a different activity before and after the second mixture of molecular species are attached. In particular embodiments, the different molecular species are nucleic acids that are reversibly modified to have different activity in an amplification reaction.

1 . A method of amplifying polynucleotides, comprising:
 (a) providing a plurality of oligonucleotides immobilised to a solid support, wherein the plurality of oligonucleotides comprises first oligonucleotide species comprising a capture sequence and second oligonucleotide species comprising an amplification sequence, wherein the first oligonucleotide species are immobilised on the solid support at a lower density than the second oligonucleotide species;   (b) applying a plurality of template polynucleotide molecules to the solid support under conditions wherein the template polynucleotide molecules hybridise to the capture sequence of the first oligonucleotide species but do not hybridise to the second oligonucleotide species;   (c) extending the first oligonucleotide species to generate polynucleotide complements of the template polynucleotide molecules, wherein the polynucleotide complements are immobilised on the solid support; and   (d) amplifying the polynucleotide complements under conditions wherein the polynucleotide complements hybridize to the amplification sequences of the second oligonucleotide species.   
   
   
       2 . The method of  claim 1 , wherein the plurality of oligonucleotides further comprises a third oligonucleotide species. 
   
   
       3 . The method of  claim 2 , wherein the third oligonucleotide species comprises a second amplification sequence. 
   
   
       4 . The method of  claim 3 , wherein the amplifying occurs under conditions wherein the template polynucleotide molecules hybridize to the second amplification sequences of the third oligonucleotide species. 
   
   
       5 . The method of  claim 1 , wherein the first oligonucleotide species is immobilised at a density of at least 1000 fold lower than the amplification sequences. 
   
   
       6 . The method of  claim 1 , wherein the oligonucleotide species is immobilised at a density of 10 6 -10 9  copies per cm 2 . 
   
   
       7 . The method of  claim 1 , wherein the capture sequence is longer than the amplification sequences. 
   
   
       8 . The method of  claim 1 , wherein the second oligonucleotide species comprises a reversible blocking group. 
   
   
       9 . The method of  claim 8 , wherein the reversible blocking group comprises a chemical species attached to the 3′ end of the second oligonucleotide species. 
   
   
       10 . The method of  claim 9 , wherein the chemical species is a phosphate group. 
   
   
       11 . The method of claim B, wherein the reversible blocking group comprises a self-complementary hairpin structure. 
   
   
       12 . The method of  claim 11 , wherein the second oligonucleotide species is cleaved after extension of the capture sequence, thereby removing the self-complementary hairpin structure from the amplification sequence on the solid support. 
   
   
       13 . The method of  claim 1 , wherein the amplification is isothermal. 
   
   
       14 . The method of  claim 13 , wherein the isothermal amplification comprises cycles of polymerase extension and chemical denaturation. 
   
   
       15 . The method of  claim 1 , wherein the template polynucleotide molecules that are applied to the solid support are single stranded.