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 extending a nascent oligonucleotide-3′-OH in a template-dependent manner, the method comprising: contacting the nascent oligonucleotide-3′-OH with a nucleic acid template, a template-dependent polymerase and 4 N  oligonucleotide triphosphates each including N monomers, under conditions in which said nascent oligonucleotide-3′-OH hybridizes with said nucleic acid template and said template-dependent polymerase is active in incorporating said oligonucleotide triphosphates onto a growing 3′-OH group of the nascent oligonucleotide-3′-OH, thereby extending the nascent oligonucleotide-3′-OH in a template-dependent manner. 
     
     
         2 . A method of determining a sequence of a nucleic acid template, the method comprising the steps of—(a) contacting in one or more reaction vessels the nucleic acid template with a nascent oligonucleotide-3′-OH, a template-dependent polymerase, 4N oligonucleotide triphosphates each including N monomers, 4N oligonucleotide triphosphate analogs each including N monomers of which a 3′ monomer includes a chain terminator moiety, 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 oligonucleotide triphosphates and said oligonucleotide triphosphate analogs onto a growing Y—OH group of said nascent oligonucleotide-3′-OH, so as to obtain a population of nucleic acid chains each being terminated by a different oligonucleotide triphosphate analog of said 4N oligonucleotide triphosphate analogs; and (b) size separating said population of terminated nucleic acid chains, thereby determining the sequence of the nucleic acid template. 
     
     
         3 . The method of  claim 2 , wherein each of said 4N oligonucleotide triphosphate analogs is labeled by a unique label. 
     
     
         4 . The method of  claim 2 , wherein at least some of said unique labels is combinatorial labels. 
     
     
         5 . A method of engineering functional nucleic acid polymers and polypeptides by introduction of multiple point mutations in nucleic acid sequences, as a novel approach of directed evolution, the method comprising the steps of—(a) contacting in reaction vessels a nucleic acid template with a nascent oligonucleotide-3′-OH, a template-dependent polymerase, and 4N oligonucleotide triphosphates, each including N monomers, wherein N is an integer greater than 1, and wherein at least one of said oligonucleotide triphosphates has a mismatch as compared to the template sequence, under conditions in which the nascent oligonucleotide-3′-OH hybridizes with the nucleic acid template and the template-dependent polymerase is active in incorporating said oligonucleotide triphosphates and said at least one oligonucleotide triphosphate containing said mismatch onto a growing 3′-OH group of the nascent oligonucleotide-3′-OH, so as to obtain a population of nucleic acid chains each containing one or multiple mutations; and (b) amplifying said mutated population of nucleic acid chains and further shuffling, cloning and expressing them by methods known in the art to create pools of degenerate nucleic acid sequences and of degenerate polypeptides; and (c) screening said pools for individual clones with desired properties, and then using the selected clones as precursors for additional cycles of degeneration and selection, as described above, until the selected molecules are optimized for the desired function.