Template-dependent nucleic acid polymerization using oligonucleotide triphosphates building blocks

Abstract

A novel use of a template-dependent polymerase. The novel use is effected by employing the template-dependent polymerase for incorporating at least one oligonucleotide triphosphate onto a nascent oligonucleotide-3′-OH in a template-dependent manner.

1 . A method of amplifying a nucleic acid template, the method comprising the step of contacting the nucleic acid template with a nascent oligonucleotide-3′-OH, a template-dependent polymerase and at least one oligonucleotide triphosphate under conditions in which said nascent oligonucleotide-3′-OH hybridizes with the nucleic acid template and said template-dependent polymerase is active in incorporating said at least one oligonucleotide triphosphate onto a growing 3′-OH group of said nascent oligonucleotide-3′-OH, thereby amplifying the nucleic acid template.  
     
     
         2 . A method of amplifying a nucleic acid template, the method comprising the step of contacting the nucleic acid template with a nascent oligonucleotide-3′-OH, a template-dependent polymerase and 4 N  oligonucleotide triphosphates each including N monomers, wherein N is an integer greater than 1, under conditions in which said nascent oligonucleotide-3′-OH hybridizes with the nucleic acid template and said template-dependent polymerase is active in incorporating said at least one oligonucleotide triphosphate onto a growing 3′-OH group of said nascent oligonucleotide-3′-OH, thereby amplifying the nucleic acid template.  
     
     
         3 . A method of exponentially amplifying a nucleic acid template, the method comprising the step of contacting the nucleic acid template with a pair of nascent oligonucleotides-3′-OH, said nascent oligonucleotides-3′-OH being hybridizable with opposite strands of the nucleic acid template, a template-dependent polymerase and 4 N  oligonucleotide triphosphates each including N monomers, wherein N is an integer greater than 1, under conditions in which said nascent oligonucleotides-3′-OH hybridize with said opposite strands of the nucleic acid template and said template-dependent polymerase is active in incorporating said at least one oligonucleotide triphosphate onto a growing 3′-OH group of each of said nascent oligonucleotides-3′-OH, thereby exponentially amplifying the nucleic acid template.  
     
     
         4 . A method of amplifying a nucleic acid template, the method comprising the step of contacting the nucleic acid template with a nascent oligonucleotide-3′-OH, a template-dependent polymerase, at least one oligonucleotide triphosphate and at least one nucleotide triphosphate, wherein said at least one oligonucleotide triphosphate and said at least one nucleotide triphosphate are selected such that at least one monomer of said at least one oligonucleotide triphosphate is absent from said at least one nucleotide triphosphate, under conditions in which said nascent oligonucleotide-3′-OH hybridizes with the nucleic acid template and said template-dependent polymerase is active in incorporating said at least one oligonucleotide triphosphate onto a growing 3′-OH group of said nascent oligonucleotide-3′-OH, thereby amplifying the nucleic acid template.