Solid phase synthesis

Abstract

The present invention relates to a method for synthesis of a polynucleotide on a dendron-modified surface of a substrate, and a method for stably maintaining a polynucleotide immobilized on a solid surface.

1 . A method for synthesis of a polynucleotide on a dendron-modified surface of a substrate, wherein the dendron-modified surface is obtained by chemically modifying with a dendron in which a plurality of termini of the branched region of the dendron are bound to the surface and a terminus of the linear region of the dendron is functionalized.  
     
     
         2 . The method according to  claim 1 , wherein the polynucleotide is synthesized by reacting (a) at least one primer immobilized on the dendron, with (b) a solution comprising polymerase, dNTP or NTP, and template DNA or RNA.  
     
     
         3 . The method according to  claim 1 , wherein the polynucleotide is synthesized by reacting (a) template DNA or RNA immobilized on the dendron, with (b) a solution comprising polymerase, dNTP or NTP, and primers.  
     
     
         4 . The method according to  claim 1 , wherein the polynucleotide is synthesized by reacting (a) polymerase immobilized on the dendron, with (b) a solution comprising dNTP or NTP, primers, and template DNA or RNA.  
     
     
         5 . The method according to  claim 1 , wherein the dendrons are spaced at regular intervals between about 0.1 nm and about 100 nm among the linear functionalized groups.  
     
     
         6 . The method according to  claim 1 , wherein the terminus of the branched region is functionalized with —COZ, —NHR, —OR′, or —PR″3, wherein Z is a leaving group, R is an alkyl, R′ is alkyl, aryl, or ether, and R″ is H, alkyl, or alkoxy.  
     
     
         7 . The method according to  claim 1 , wherein the functional group is —NH2, —OH, PH3, —COOH, —CHO or —SH.  
     
     
         8 . The method according to  claim 1 , wherein the linear region comprises a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, ether, polyether, ester, or aminoalkyl group.  
     
     
         9 . The method according to  claim 1 , wherein the substrate is glass, semiconductor, metal, plastics, silicone, silicate, metal alloy, or synthetic organic metal.  
     
     
         10 . The method according to  claim 1 , wherein the substrate is in a form of a slide, a particle, a bead, a micro-well plate, or a porous material.  
     
     
         11 . The method according to  claim 1 , wherein the polynucleotide is DNA, RNA, oligonucleotide, cDNA, nucleotide analog, or a combination thereof.  
     
     
         12 . The method according to  claim 1 , wherein the dendron-modified surface is obtained by chemically modifying with the dendron after treating the substrate with a silane compound having a hydroxyl group.  
     
     
         13 . The method according to  claim 1 , wherein the polynucleotide is synthesized by a Klenow DNA polymerase I.  
     
     
         14 . The method according to  claim 1 , wherein the synthesis of polynucleotide is carried out by using RT-PCR or PCR method.  
     
     
         15 . The method according to  claim 14 , wherein the PCR method include a denaturing step, an annealing step, and an extension step with Taq polymerase or a polymerase derived from Taq polymerase.  
     
     
         16 . A method of stably maintaining a polynucleotide immobilized on a solid surface of a substrate under a thermal stress, wherein the substrate is chemically modified with a dendron in which a plurality of termini of the branched region of the dendron are bound to the surface and a terminus of the linear region of the dendron is functionalized.  
     
     
         17 . The method of stably maintaining a polynucleotide according to  claim 16 , wherein the thermal stress is a temperature of 60 to 100□.  
     
     
         18 . The method of stably maintaining a polynucleotide according to  claim 16 , wherein the thermal stress is long lasting.  
     
     
         19 . The method of stably maintaining a polynucleotide according to  claim 16 , wherein the thermal stress is put by repetitive heating and cooling.  
     
     
         20 . The method according to  claim 16 , wherein the substrate is chemically modified with the dendron, after treating the substrate with a silane compound having at least a hydroxyl group.