Enzymatic nucleic acid synthesis: methods for inhibiting pyrophosphorolysis during sequencing synthesis

Abstract

Nucleotide triphosphate probes containing a molecular and / or atomic tag on a γ and / or β phosphate group and / or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.

Description

RELATED APPLICATIONS [0001] The present invention claims priority of U.S. patent application Ser. No. 10 / 007,621 filed Dec. 3, 2001, which claims provisional priority U.S. Provisional Patent Application Ser. No. 60 / 250764 filed Dec. 1, 2000.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to compositions and methods for altering the fidelity of nucleic acid synthesis. [0004] More particularly, the present invention relates to the following general areas: (1) nucleotide triphosphate monomers having at least one molecular or atomic tag bonded to and / or chemically and / or physically associated with one or more of the phosphate groups of the triphosphate moiety of the monomers, the base moiety, and / or the sugar moiety in the case of a nucleoside analog; (2) methods for enzymatic DNA synthesis with altered fidelity; (3) methods of sequencing DNA, based on the detection of base incorporation using tags bonded to and / or chemically and / or phys...

AI Technical Summary

Benefits of technology

This approach significantly reduces pyrophosphorolysis, increases the fidelity of nucleotide incorporation, and enables rapid, accurate DNA sequencing without the need for electrophoresis, facilitating automation and real-time monitoring of sequencing reactions.

Problems solved by technology

The halogen quencher used in the assay is very inefficient producing only about a two fold decrease in fluorescent efficiency.

This problem is believed to be caused by pyrophosphorolysis of the primer extension product by a reverse nucleotide addition reaction promoted by the accumulation of pyrophosphates in the reaction mixture.

It has been recognized that pyrophosphorolysis, where an oligonucleotide is reduced in length, is detrimental to primer extension reactions.

A drop-out may not be readily detected by the operator, leading to errors in the interpretation of the data either by a human or computer-driven analyzer.

Since the electrophoresis step as well as the subsequent detection of the separated DNA fragments are cumbersome procedures, a great effort has been made to automate these steps.

However, despite the fact that automated electrophoresis units are commercially available, electrophoresis is not well suited for large-scale genome projects or clinical sequencing where relatively cost-effective units with high throughput are needed.

However, radioactive methods are not well suited for routine clinical applications and hence the development of a simple non-radioactive method for rapid DNA sequence analysis has also been of interest.

However, the PPi-based sequencing methods mentioned above are not without drawbacks.

This makes it difficult to sequence a template which is not bound to a solid support.

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