Parallel polymorphism scoring by amplification and error correction

Abstract

This invention provides a method of detecting polymorphisms, e.g., single nucleotide polymorphisms (SNPs), by amplification and error correction. The invention encompasses methods of performing amplification and error correction using an improved generation of nucleic acid polymerases, and methods of multiplexing the assay. The improvement to the polymerases is the joining of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the ability of the enzyme to bind and catalytically modify the nucleic acid.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application is a continuation of co-pending application Ser. No. 10 / 306,828 filed Nov. 27, 2002, which claims the benefit of U.S. provisional application No. 60 / 334,032, filed Nov. 28, 2001, which are incorporated by reference herein.FIELD OF THE INVENTION [0002] This invention provides a method of detecting polymorphisms, e.g., single nucleotide polymorphisms (SNPs) by amplification and error correction. The invention encompasses methods of performing amplification and error correction using an improved generation of nucleic acid polymerases, and methods of multiplexing the assay. The improvement to the polymerases is the joining of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the ability of the enzyme to bind and catalyticly modify the nucleic acid. BACKGROUND OF THE INVENTION [0003] The smallest possible difference between two DNA sequences is a change of a single base, a Single Nu...

AI Technical Summary

Benefits of technology

[0015] A fourth method comprises: (a) contacting a target nucleic acid comprising a query sequence with a probe oligonucleotide under conditions in which the probe specifically hybridizes to the target nucleic acid, wherein the 3′ nucleotide of the probe is a labeled query nucleotide; (b) providing an error-correcting polymerase comprising at least two heterologous domains, wherein a first domain that is a sequence-non-specific nucleic-acid-binding domain is joined to a second domain that is a polymerase domain, wherein the sequence non-specific nucleic-acid-binding domain: (i) binds to double-stranded nucleic acid, and (ii) enhances the processivity of the polymerase compared to an identical polymerase not having the sequence non-specific nucleic-acid-binding domain fused to it; (c) incubating the assay under conditions in which the probe is extended by the polymerase, wherein the labeled query nucleotide is cleaved from the probe when mismatched with the query sequence; and (d) detecting the amount of cleaved probe or cleaved label.

Problems solved by technology

All of these methods entail assays that are either difficult or expensive to develop, or difficult or expensive to perform.

There are a number of problems and deficiencies with this method, however.

First, known error-correcting polymerases, such as the Pyrococcus genus family B polymerases, are ill-suited to amplification of sequences directly from genomic DNA.

The processivity of the polymerases is too low to reliably complete a full-length copy of an amplicon in a single round.

This creates a problem, because it is preferable to use low amounts of genomic DNA in a PCR reaction in order to allow use of DNA that is not highly purified; and to reduce the amount of non-specific DNA, which can lead to side reactions, present in the reaction.

Second, the methods used for scoring whether error correction has occurred (and therefore what versions of an SNP are present in the original sample) are inadequate for low cost and high throughput.

Given the cost of reagents and disposables, and the amortized cost of equipment and space, it is exceedingly difficult to run a PCR for less than 10-20 US cents.

Yet, for many applications, SNP scoring is not economical unless it can be done for 1 US cent per locus.

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