Synthesis of 2', 3'-dideoxynucleosides for automated DNA synthesis and pyrophosphorolysis activated polymerization

Abstract

Methods for preparation of 2′,3′-dideoxynucleotides support structures, such as 2′,3′-dideoxyguanosine, 2′,3′-dideoxyadenosine, and 3′-deoxythymidine support structures are disclosed. Various methods of using such structures are also provided, such as their use for automated DNA synthesis and pyrophosphorolysis activated polymerization.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. patent application Ser. No. 13 / 117,029, filed May 26, 2011 which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Nos. 61 / 349,791, filed May 28, 2010 and 61 / 352,197, filed Jun. 7, 2010, which are herein incorporated by reference in their entirety.FIELD[0002]This invention relates generally to the field of nucleic acid polymerization and amplification. More specifically, this invention relates to methods of synthesis of various 2′,3′-dideoxynucleoside supports, such as 2′,3′-dideoxyguanosine, 2′,3′-dideoxyadenosine, 3′-deoxythymidine and 2′,3′-dideoxyuridine supports, and to their use.BACKGROUND[0003]Pyrophosphorolysis-activated polymerization (PAP) is a technique useful for nucleic acid polymerization and amplification. PAP is an allele-specific amplification method, the essence of which is that 3′-dideoxynucleotide-terminated primers (also d...

AI Technical Summary

Benefits of technology

[0009]Accordingly, better methods are needed for preparing oligonucleotides P* suitable for the PAP process. Such methods would allow for easy synthesis of the oligonucleotides and labeled primers, without resorting to expensive 5′-amidite synthesis or to 5′ to 3′ reverse synthesis. The primers so produced would have all the beneficial properties of the primers obtainable by the above-described earlier methods, such as selectivity of PAP in allele-specific PCR. The instant specification describes such synthetic methods.

Problems solved by technology

While PAP is undoubtedly a very attractive technique, it has certain deficiencies and drawbacks.

One difficulty lies in the process of obtaining a suitable oligonucleotide P. For instance, such products as ddA, ddG, and dT are not available commercially and have to be synthesized.

All such synthetic methods are labor intensive and costly.

Likewise, no adequate method for making a DNA containing dT has been described, other than expensive 5′-to-3′ reverse synthesis.

This is likely due to the fact that there is a lack of convenient exocyclic functional group for covalent linkage on the thymine base.

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