Methods for increasing accuracy of nucleic acid sequencing

Abstract

The invention provides methods for improving the accuracy of a sequencing-by-synthesis reaction by sequencing at least a portion of a template and at least a portion of template complementary sequence.

Description

TECHNICAL FIELD OF THE INVENTION [0001] The invention generally relates to methods for increasing accuracy in nucleic acid synthesis reactions. BACKGROUND OF THE INVENTION [0002] In vitro nucleic acid synthesis is a foundation of many fundamental research and diagnostic tools, such as nucleic acid amplification and sequencing. In a template-dependent nucleic acid synthesis reaction, the sequential addition of nucleotides is catalyzed by a nucleic acid polymerase. Depending on the template and the nature of the reaction, the nucleic acid polymerase may be a DNA polymerase, an RNA polymerase, or a reverse transcriptase. [0003] The accuracy of template-dependent nucleic acid synthesis depends in part on the ability of the polymerase to discriminate between complementary and non-complementary nucleotides. Normally, the conformation of the polymerase enzyme favors incorporation of the complementary nucleotide. However, there is still an identifiable rate of misincorporation that depends ...

AI Technical Summary

Benefits of technology

The invention improves the accuracy of nucleic acid sequencing reactions by using a template-dependent sequencing-by-synthesis method. The method involves hybridizing a primer to a template and adding nucleotides to the end of the primer using a polymerase. The added nucleotides are detectable and the template is then removed, leaving the extended primer. The extended primer is then used as a template for further sequencing in the opposite direction. This process allows for resequencing in the 3' to 5' direction, increasing accuracy of the sequence information obtained from a given template. The invention also includes a method for removing the template from the duplex, resulting in a cleaner surface for further sequencing.

Problems solved by technology

However, there is still an identifiable rate of misincorporation that depends upon factors such as local sequence and the base to be incorporated.

Synthetic or modified nucleotides and analogs, such as labeled nucleotides, tend to be incorporated into a primer less efficiently than naturally-occurring nucleotides.

The reduced efficiency with which the unconventional nucleotides are incorporated by the polymerase can adversely affect the performance of sequencing techniques that depend upon faithful incorporation of such unconventional nucleotides.

Because single molecule techniques do not rely on ensemble averaging as do bulk techniques, errors due to misincorporation can have a significant deleterious effect on the sequencing results.

The incorporation of a nucleotide that is incorrectly paired, under standard Watson and Crick base-pairing, with a corresponding template nucleotide during primer extension may result in sequencing errors.

Furthermore, where the template being sequenced is present in only one or a few copies in the sample (a rare template), misincorporations can have a great impact on the sequence obtained because fewer sequences are obtained with which to compare to each other or with a reference sequence.

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