WO2004003228A1

WO2004003228A1 - Genotyping method

Info

Publication number: WO2004003228A1
Application number: PCT/AU2003/000841
Authority: WO
Inventors: Garry Charles King; Daniel Di Giusto
Original assignee: Unisearch Limited
Priority date: 2002-07-01
Filing date: 2003-07-01
Publication date: 2004-01-08

Abstract

The present invention provides a method of incorporating at least one nucleotide or nucleotide analogue into a polynucleotide molecule in a primer-directed extension reaction, the method comprising hybridising a single-stranded polynucleotide primer with a single-stranded polynucleotide template molecule to generate a duplex molecule; and incorporating at least one nucleotide or nucleotide analogue into the duplex molecule at the 3’ end of the primer in the presence of a polymerase enzyme, wherein the 3’ terminus of the single-stranded primer is modified so as to maintain primer integrity. Also provided are methods for single nucleotide polymorphism genotyping, methods for detecting mutations at specific nucelotide positions and methods for diagnosing genetic disorders caused by mutations at specific nucleotide positions, as well as kits for carrying out the methods of the invention.

Description

Genotyping Method

Technical Field

The present invention relates generally to primer-directed base extension reactions. More particularly, the present invention relates to improved methods of single nucleotide incorporation in single-base extension (SBE) reactions. The present invention finds particular application in the genotyping of single nucleotide polymorphisms.

Background Art Genotyping of single nucleotide polymorphisms (SNPs) has attracted a considerable amount of interest in recent years. In particular this can be attributed to the use of SNPs as markers for the identification of disease susceptibility genes and to the promise of pharmacogenomics and personalized medicine. To date, the majority of SNP genotyping methods developed have been based upon one or more of four fundamental molecular biological processes: hybridisation, polymerisation (nucleotidyl transfer), ligation and nucleolysis.

Polymerisation is a particularly powerful tool, with applications ranging from full Sanger sequencing through limited pyrosequencing to single-base extension (SBE) or 'minisequencing' methods that identify a single allelic nucleotide immediately adjacent to a defined primer terminus. SBE has proven particularly attractive for its simplicity (there are only three major added components in the minimal implementation - primer, polymerase and triphosphate substrate) and for its adaptability to various detection formats. SBE offers about an order of magnitude better allele discrimination than allele-specific hybridisation (Pastinen et al., 1997).

With reference to its application to SNP genotyping, in a typical SBE reaction, a DNA polymerase catalyses the addition of a single nucleotide to the 3' end of a primer annealing to a template molecule immediately adjacent to the position of the SNP of interest, the single added nucleotide being complementary to the nucleotide at the site of the SNP. The identity of the nucleotide added defines the genotype. By appropriately labelling the nucleotide, its identity can be readily determined using a variety of detection methods, including mass spectrometry, fluorescence, chemiluminescence and electrochemistry, for example. One of the disadvantages of SBE as it has been applied to date resides in the potential for misincorporation of nucleotides by the DNA polymerase, that is the error rate for polymerase- directed base insertion. While the error rates for most polymerases are acceptably low in some applications, they can be significant and even remarkably high in others. For example, approximately 5% misincorporation of ddTTP as a sole substrate has been described for a homogeneous SBE assay (Haff & Smirnov, 1997). Anomalous allele frequencies that imply up to 30 % misincorporation have also been reported for homogeneous assays with the four ddNTPs (Bray et al., 2001) or four dye-labelled ddNTPs (Hsu et al., 2001) and in limited-base extensions containing three ddNTPs and one dNTP (Sun et al., 2000). Inhomogeneous assays such as arrayed primer extension (APEX) and bead minisequencing present additional complications due to

5 local concentration and steric effects, generally exhibiting significant variability and apparent misincorporation rates (Pastinen et al., 1997; Shapero et al., 2001 ; Lindroos et al., 2001).

In addition to these problems, established SBE methods often do not lend themselves to the determination of allele frequencies in pooled or markedly heterogeneous samples. Accordingly, there is a need for a method of SBE in which the accuracy of nucleotide incorporation is improved, ιo thereby enhancing the robustness of the assay for a variety of applications.

One solution has been provided by the primer-specific and mispair extension analysis (PSMEA) method (Hu et al., 1998; Hu et al., 2000), in which the proofreading (3'->5' exonuclease) activity of Pfu DNA polymerase has been employed to provide highly sensitive discrimination between genotypes. Proofreading is typically known to improve polymerase copy fidelity by one-to- i5 two orders of magnitude.

However direct application of proofreading polymerases to conventional SBE is not possible because the 3' to 5' exonuclease activity of the polymerase causes extensive primer degradation (Syvanen et al., 1990; Haff & Smirnov, 1997).

The present invention is based on the finding that the above problems can be overcome o by providing a primer with a modified 3' terminus, such that the 3' terminus is protected from degradation by the exonuclease activity of the polymerase enzyme. In a particular application of the invention it has been found that carrying out an SBE reaction using both a polymerase with proofreading capabilities and a primer with a phosphorothioate or LNA modification at its 3' terminus dramatically reduces levels of nucleotide misincorporation without primer degradation,

2₅ thus providing a more robust assay than has previously been available.

Summary of the Invention

According to a first embodiment of the present invention there is provided a method of incorporating at least one nucleotide or nucleotide analogue into a polynucleotide molecule in a 3o primer-directed extension reaction, the method comprising the steps of:

(a) hybridising a single-stranded polynucleotide primer with a single-stranded polynucleotide template molecule to generate a primer-template duplex molecule; and

(b) incorporating at least one nucleotide or nucleotide analogue into the duplex molecule at the 3' end of the primer in the presence of a polymerase enzyme;

3₅ wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity. Preferably, the modification to the 3' terminus of the primer is one or more phosphorothioate linkages wherein the residue(s) at the 3' terminus of the primer contain at least one sulphur substitution at the scissile phosphate. More preferably, the phosphorothioate linkage is the Sp isomer. Still preferably, the phosphorothioate linkage is a phosphorodithioate linkage in which two sulphur substitutions are made at the scissile phosphate of the 3' terminal residue of the primer.

In an alternative embodiment, the modification to the 3' terminus of the primer may be provided by other non-native groups such as a methylphosphonates, phosphorothiolates, phosphoramidates, boron derivatives or other replacements. In a further alternative embodiment, the modification to the 3' terminus of the primer may be provided by one or more replacement sugar moieties located at the 3' terminal position, the 3' penultimate position, or any mixture of positions that includes one or both of these locations.

Preferably, the sugar moiety is a bicyclic "locked" nucleic acid analogue. Preferably, the locked nucleic acid analogue contains a methylene or ethylene linkage between the 2'-0 position and 4'-C position of the furanose ring of the nucleic acid.

Preferably the exonuclease activity is 3' -> 5' exonuclease activity provided by the polymerase enzyme, being a DNA polymerase with proofreading capabilities. More preferably, the

DNA polymerase is selected from the group consisting of: exo⁺ Klenow fragment of £ coli DNA polymerase I; T4 DNA polymerase; T7 DNA polymerase; Vent DNA polymerase; and Pfu DNA polymerase. Alternatively the exonuclease activity may be supplied by an additional 3' -> 5' exonuclease enzyme, such as exonuclease III.

Preferably the or each nucleotide or nucleotide analogue incorporated is a deoxynucleotide selected from the group consisting of: dATP, dTTP, dUTP, dCTP and dGTP. Also preferably, the or each nucleotide or nucleotide analogue incorporated is a dideoxynucleotide selected from the group consisting of: ddATP, ddTTP, ddUTP, ddCTP and ddGTP. Still preferably, the or each nucleotide or nucleotide analogue incorporated is an acyclonucleotide or acyclonucleotide analogue selected from the group consisting of: acyATP, acyUTP, acyTTP, acyCTP and acyGTP.

Preferably, the or each nucleotide or nucleotide analogue is labelled with a suitable label enabling detection. The label may be an electrochemically active agent, a fluorescent dye, a chromophore, a hapten, a chemiluminescent agent, a radioisotope, intrinsic or^'engineered mass, or the like.

For electrochemical detection, the electrochemically active agent may be selected from the group consisting of: metal complexes; metallocenes; and organic molecules exhibiting facile electron transfer behaviour. Preferably, the electrochemically active agent is a ferrocene derivative or a complex of a transition metal such as ruthenium or osmium.

The fluorescent dye may be selected from derivatives of the fluorescein, rhodamine, bodipy, cyanine or Alexa families, or other fluorescent compounds suitable for conjugation to a nucleotide. Dyes such as FAM, ROX, TAMRA, R110, R6G, Joe, HEX, TET, Alexa, Cy3 and Cy5 are particularly preferred.

In addition to the modification to the primer, the template molecule may also be suitably modified so as to provide protection from exonuclease activity. Preferably template modification is achieved by the addition of one or more phosphorothioate linkages to the 3' end of the template molecule. Also preferably, the template may be engineered to contain a 3' terminal nucleotide sequence that folds into a nuclease-resistant conformation.

Most preferably, according to method of the first embodiment, a single nucleotide or nucleotide analogue is incorporated into the duplex molecule in step (b).

According to a second embodiment of the present invention there is provided a method of determining the identity of at least one nucleotide or nucleotide analogue incorporated into a polynucleotide molecule in a primer-directed extension reaction, the method comprising the steps of:

(a) hybridising a single-stranded polynucleotide primer with a single-stranded polynucleotide template molecule to generate a primer-template duplex molecule; (b) incorporating at least one nucleotide or nucleotide analogue into the duplex molecule at the 3' end of the primer in the presence of a polymerase enzyme; and

(c) detecting of the identity of the at least one incorporated nucleotide or nucleotide analogue, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity. Preferably in the method of the second embodiment a single nucleotide or nucleotide analogue is incorporated into the duplex molecule in step (b).

According to a third embodiment of the present invention there is provided a method of genotyping by primer-directed base extension, the method comprising the steps of:

(a) hybridising a single-stranded polynucleotide primer with a single-stranded polynucleotide template molecule to generate a duplex primer-template molecule;

(b) incorporating at least one nucleotide or nucleotide analogue into the duplex molecule at the 3' end of the primer in the presence of a polymerase enzyme; and

(c) detecting the identity of the at least one incorporated nucleotide or nucleotide analogue, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity. Preferably in the method of the third embodiment a single nucleotide or nucleotide analogue is incorporated into the polynucleotide molecule.

According to a fourth embodiment of the present invention there is provided a method of single nucleotide polymorphism typing in a target polynucleotide molecule, the method comprising the steps of:

(a) hybridising a single-stranded polynucleotide primer with the target polynucleotide molecule in single-stranded form such that a duplex primer-target molecule is generated with the 3' end of the primer immediately adjacent the single nucleotide polymorphism in the target polynucleotide molecule; (b) incorporating one nucleotide or nucleotide analogue into the duplex molecule at the

3' end of the primer in the presence of a polymerase enzyme; and

(c) detecting the identity of the incorporated nucleotide or nucleotide analogue to thereby determine the identity of the single nucleotide polymorphism in the target polynucleotide molecule, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity. According to a fifth embodiment of the present invention there is provided a method for detecting a mutation at a specific nucleotide position in a target polynucleotide molecule, the method comprising the steps of:

(a) hybridising a single-stranded polynucleotide primer with the target polynucleotide molecule in single-stranded form such that a duplex primer-target molecule is generated with the 3' end of the primer immediately adjacent the specific nucleotide position in the target polynucleotide molecule;

(b) incorporating one nucleotide or nucleotide analogue into the duplex molecule at the 3' end of the primer in the presence of a polymerase enzyme; and (c) detecting the identity of the incorporated nucleotide or nucleotide analogue to thereby detect a mutation at the specific nucleotide position in the target polynucleotide molecule, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity.

According to a sixth embodiment of the present invention there is provided a method for diagnosing a genetic disorder or predisposition to the genetic disorder in an individual wherein said genetic disorder is caused by mutation of a single nucleotide, the method comprising the steps of:

(a) isolating a polynucleotide molecule containing the single nucleotide from the individual;

(b) hybridising a single-stranded polynucleotide primer with the polynucleotide molecule in single-stranded form such that a duplex molecule is generated with the 3' end of the primer immediately adjacent the single nucleotide in the polynucleotide molecule; (c) incorporating one nucleotide or nucleotide analogue into the duplex molecule at the 3' end of the primer in the presence of a polymerase enzyme; and

(d) detecting the identity of the incorporated nucleotide or nucleotide analogue to thereby detect a mutation at the specific nucleotide in the target polynucleotide molecule, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity.

In performing the methods defined in any one of the first to the sixth embodiments, the modified single-stranded primer may be obtained either by direct synthesis, or by re-use of a primer previously extended in a primer-directed extension reaction. In the latter case, the modified primer may be prepared by nucleolysis of the 3'-terminal nucleotide residue or residues from the previously extended primer to obtain the original 3'-modified primer. This nucleolysis may be achieved by the 3'-5' exonuclease activity of a proofreading polymerase or by a separate 3'-5' exonuclease.

According to a seventh embodiment of the present invention there is provided a kit for incorporating at least one nucleotide or nucleotide analogue into a polynucleotide molecule in a primer-directed extension reaction comprising:

(a) a single-stranded oligonucleotide primer;

(b) a mixture of nucleotides or nucleotide analogues for incorporation; and

(c) a polymerase enzyme; wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity. Typically, for the purposes of the above embodiments, the maintenance of primer integrity refers to modifications which protect the 3' terminus of the primer from exonuclease activity. Additionally or alternatively the modifications may prevent relative destabilization of the primer terminus.

Brief Description of the Drawings

The present invention will now be described, by way of example only, with reference to the following drawings.

Fig. 1. Primer and template sets. Corresponding phosphodiester, phosphorothioate and LNA groups of primers are indicated by "o", "s" and underline respectively. Template residues which direct primer extension are indicated in bold.

Fig.2. Primer degradation by a proofreading polymerase with strong 3'-5' exonuclease activity, (a-c): P1 primer incubated with T4 DNA polymerase for 0, 0.17 and 1.0 h. (d-f): SP1 primer incubated with T4 DNA polymerase for 0, 1.0 and 16.0 h. Fig. 3. Timecourse of SP1 primer incubation with exo⁺ Klenow fragment of E. coli DNA polymerase I (filled triangles) and T4 DNA polymerase (open circles). Percentage intact primer is reported for triplicate samples relative to a separate internal standard oligonucleotide added post-reaction. Fig. 4. Primer extension with deoxynucleotide triphosphates. All spectra contain one additional equivalent of primer as a post-reaction standard.

(a-d): Incorporation of dGTP for the following primer/template/polymerase combinations (KF = Klenow fragment of £ coli DNA polymerase I).

(a) P1/LTT/exo- KF. (b) SP1/LTT/exo- KF. (c) SP1/LTT/exo⁺ KF. (d) SP1/LTC/exo⁺ KF. (e-h): Incorporation of dATP for primer/template/polymerase combinations. (e) P1/LTT/exo- KF. (f) SP1/LTT/exo- KF. (g) SP1/LTT/exo⁺ KF. (h) SP1/LTT/T4 DNA polymerase.

All spectra include a signal from primer added post-reaction as an internal standard.

Fig. 5. Extension of SP1 primer by proofreading T7 DNA polymerase. All spectra contain one additional equivalent of primer as a post-reaction internal standard.

(a-d): Correct primer-substrate pairs. (a) LTT/dATP. (b) LTA/dTTP. (c) LTG/dCTP. (d) LTC/dGTP.

(e-h): Incorrect primer-substrate pairs.

(e) LTG/dATP. (f) LTC/dTTP. (g) LTA/dCTP. (h) LTA/dGTP.

Fig. 6. Extrinsic proofreading of exo- Klenow fragment of £ coli DNA polymerase l-catalysed reactions by exonuclease III. (a) Misincorporation of dGTP opposite LTT template.

(b) Double addition of dGTP opposite LTC template.

(c) Proofreading of (a) by 5 U exo III.

(d) Proofreading of (b) by 5 U exo III.

Fig. 7. Primer extension under limiting substrate concentrations. LTG template with dCTP - 2.5 μM ( ), 10 μM (o), 40 μM (■) and 160 μM (Δ). (a) 1 U Sequenase 2.0. (b) 1 U exo⁺ T7 DNAP. (c) 0.25 U Sequenase 2.0. (d) 0.25 U exo⁺ T7 DNAP.

Fig.8. Extension and proofreading of LNA-modified primers. Primer and template sequences are as shown in Fig. 1. Lane (1): Untreated PAI-C3 (3'-terminal LNA) primer; (2): Untreated PAI-C5 (3'- penultimate LNA) primer; (3): PAI-T2g template; (4): PAI-C3 + T2g-long; (5): PAI-C5 + T2g-long; (6): #1 + exo III; (7): #2 + exo III; (8): #3 + exo 111; (9): #4 + exo III; (10): #5 + exo III; (11): PAI-C3 + T2g, pol-extended; (12): PAI-C5 + T2g, pol-extended; (13): #11 + exo III; (14): #12 + exo III. Fig. 9. Proofreading of fluorescent-labelled terminators on alternative primer constructs. Exo⁺- Vent DNAP degradation of R6G-ddCTP-extended PAI-C1 (normal DNA, diamonds), PAI-C3 (3'- LNA, squares) and PAI-C4 (3'-phosphorothioate, triangles). Fig. 10. Proofreading incorporation of electochemically-labelled nucleotides. (a) OSWV trace of Fc1-dUTP extended EP-2A primer captured by an immobilized 2A antitag oligonucleotide. (b) OSWV trace of VF1-dUTP extended EP-UT primer captured by the UT antitag.

Definitions

In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

The term "phosphorothioate modification" as used herein includes a modification to at least one nucleotide or nucleotide analogue in which the scissile phosphate group of the nucleotide or nucleotide analogue includes at least one sulphur substitution. For example, a phosphoromonothioate modification refers to a nucleotide or nucleotide analogue with a single sulphur substitution at the scissile phosphate, whereas a phosphorodithioate modification refers to a nucleotide or nucleotide analogue with two sulphur substitutions at the scissile phosphate. The term "nucleotide" as used herein refers to deoxyribonucleotides, ribonucleotides and acyclonucleotides and includes derivatives of such nucleotides of equivalent function.

The term "nucleotide analogue" as used herein means a nucleotide which is a derivative of a naturally occurring nucleotide, which derivative comprises addition, deletion, substitution of one or more constituents of the nucleotide and which retains substantially the same function of incorporation into nucleotide chains as the naturally occurring nucleotide. In particular, a nucleotide analogue includes any modifications to or substitutes for the base, sugar or triphosphate moieties of the nucleotide from which it is derived; the analogue retaining the ability to be incorporated by a polymerase into a polynucleotide molecule.

The term "polynucleotide molecule" as used herein refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases or a mixture of the two.

The term "primer" as used herein means a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis. An "oligonucleotide" is a single- stranded nucleic acid typically ranging in length from 2 to about 500 bases. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize to the template.

The terms "3' terminus" and "3' terminal" as used herein with reference to modified single- stranded primers means one or more residues located at or near the 3' end of the primer. This term is not limited to indicate only the extreme 3' terminal residue of the primer. A "single nucleotide polymorphism" (SNP) occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. An SNP usually arises due to substitution of one nucleotide for another at the polymorphic site, and can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.

Best Mode of Performing the Invention

The present invention relates to methods of increasing the accuracy of base incorporation in base extension reactions, and in particular in single-base extension (SBE) reactions. Accordingly, the present invention provides a method of incorporating at least one nucleotide or nucleotide analogue into a polynucleotide molecule in a primer-directed extension reaction, wherein the 3' terminus of the primer is modified so as to maintain primer integrity.

In particular, the maintenance of primer integrity refers to the protection of the 3' terminus of the primer from 3' -> 5' exonuclease activity and/or protection of the primer from relative destabilization of the primer terminus. Preferably, the modification to the 3' terminus of the primer is achieved by virtue of one or more phosphorothioate modifications.

The present invention also provides for kits suitable for application of the methods of the invention, such as SNP genotyping using SBE reactions according to the methods of the present invention.

The methods and kits of the present invention can be used in a number of different genotyping and genotyping-related applications. For example, with reference to SNPs, the methods and kits can be used to determine the identity of a nucleotide at a particular site or detect a mutation at a site, thus making them particularly useful in a variety of diagnostic, evaluation and research applications. This permits the methods and kits of the present invention to find application, for example, in the diagnosis of infectious diseases or genetic disorders. The ability to accurately interrogate particular single nucleotide positions also renders the present invention useful for identification purposes, including for example, in forensic applications and determining the presence of particular pathogens (e.g. viruses, bacteria or fungi) in a sample. The methods and kits may also be employed in the quantitation of mRNA expression levels for allelic variants. In general, the present invention involves primer-directed, polymerase-catalysed extension reactions in which a target nucleic acid of interest is used as a template. The template may be a single- or double-stranded polynucleotide molecule that includes at least one variable site which is to be analysed by virtue of the extension reaction. If the nucleic acid molecule is double- stranded, after denaturation of the template to separate the strands, either of these strands may serve as the template for the methods and kits of the invention. The primer used for the primer-directed extension reaction is designed such that upon annealing with the template, the 3' end of the primer lies 5' to the position of the variant site to be analysed. Alternatively, the methods and kits of the present invention may be used in match/mismatch genotyping applications, in which case the primer is designed such that the 3' terminus thereof lies directly opposite the site of the nucleotide to be typed, rather than immediately 5' to this nucleotide.

Extension reactions of the present invention are conducted in the presence of a nucleotide mixture that includes at least one nucleotide or nucleotide analogue and a polymerase that catalyzes the incorporation of the at least one nucleotide or nucleotide analogue at the 3'-end of the primer. In a typical SBE reaction, the nucleotide mixture contains four terminator nucleotides or nucleotide analogues, most typically selected from the group consisting of dideoxyadenosine triphosphate (ddATP), dideoxythymidine triphosphate (ddTTP), dideoxyuridine triphosphate (ddUTP), dideoxycytosine triphosphate (ddCTP) and dideoxyguanosine triphosphate (ddGTP). As the nucleotide or nucleotide analogue incorporated into the primer is complementary to the nucleotide at the variant site in the template, determination of the identity of the nucleotide or nucleotide analogue incorporated allows the identification of the nucleotide present at the variant site.

A particular feature of the present invention is that the primer is modified at its 3' terminus such that the primer is protected from degradation which would otherwise result from 3' -> 5' exonuclease activity. Such 3' -> 5' exonuclease degradation may occur either by the proofreading activity inherent in the polymerase enzyme used to catalyse the addition of the least one nucleotide or nucleotide analogue at the 3' end of the primer, or alternatively by an additional enzyme supplied to provide exonuclease activity. An additional feature of the present invention is that modification of the 3' terminus of the primer enhances SBE fidelity, even in instances where non-proofreading polymerases are used.

Particular features and components of the present invention are described in more detail below.

1. Primer modification The primers of the present invention are typically oligonucleotides of, generally, 15 to 30 nucleotides in length. Such oligonucleotide primers can be prepared by any suitable method, including, for example, direct chemical synthesis or cloning and restriction of appropriate sequences. Alternatively, the primer may be prepared from a previous primer-directed extension reaction, allowing re-use of a primer previously extended. In this case, the 3' terminal modified primer may be prepared, for example, by nucleolysis of the 3'-terminal nucleotide residue or residues from a previously extended primer to obtain the original 3'-modified primer. This nucleolysis may be achieved by the 3'-5' exonuclease activity of a proofreading polymerase or by a separate 3'-5' exonuclease enzyme.

As stated above, of particular use in the methods and kits of the present invention are single-stranded primers in which the 3' terminus contains one or more phosphorothioate modifications. The modification may involve, preferably, one (phosphoromonothioate) or two (phosphorodithioate) sulphur substitutions at the scissile phosphate of the residue at the 3' terminus of the primer. In the case of a phosphoromonothioate modification, two stereoisomers are possible, commonly known as the Sp and Rp diastereomers (Eckstein, 1985). In the present invention it has been found that the Sp isomer provides greater protection from exonucleolytic attack than the Rp isomer. The Sp isomer is essentially fully protective, whereas the Rp isomer only partially protects the 3' terminus against a strong proofreading exonuclease (see Example 1 below). Accordingly, in preferred embodiments of the invention the 3' terminus of the primer is modified by the addition of the Sp isomer of a phosphoromonothioate modified nucleotide.

In alternative, also preferred embodiments of the invention the 3' terminus of the primer is modified by the addition of a phosphorodithioate modified nucleotide. Because the phosphorodithioate group contains sulfur atoms at both the Rp and Sp positions, this group will confer the resistance associated with the Sp isomer. Primers with 3' terminal modifications such as phosphoromonothioate and phosphorodithioate modifications can be readily constructed by well known methods of chemical synthesis. Methods for the stereospecific chemical synthesis of Sp phosphorothioates are also known to those skilled in the art. Alternatively, the primer containing only the Sp isomer may be prepared by contacting a racemic phosphorothioate with a suitable proofreading exonuclease thereby degrading any primer containing Rp isomer.

It will be appreciated by those skilled in the art that terminal modifications to the primer other than phosphoromonothioate and phosphorodithioate modifications are also possible without departing from the scope of the present invention. For example, methylphosphonates, phosphorothiolates, phosphoramidates, boron derivatives or other nuclease-resistant modifications may be employed. in one particular embodiment of the present invention terminal modification of the primer is achieved via the incorporation of one or more residues containing a modified sugar moiety or analogue that confers proofreading exonuclease resistance. Preferably, the sugar moiety is a "locked" nucleic acid (LNA) analogue. Preferably the LNA contains a methylene or ethylene linkage between the 2'-0 position and the 4'-C position of the furanose ring of the nucleotide, such that the flexibility of the furanose ring is restricted (see Braasch and Corey, 2001 ; Morita ef al., 2002). According to this embodiment, either the terminal residue at the 3' end of the primer (N-1), the penultimate residue at the 3' end (N-2), or both the penultimate and terminal residues (N-1+N-2) of the primer are replaced with an LNA, an LNA analogue or other proofreading nuclease-resistant sugar modification or replacement, In embodiments of the methods of the invention in which the polymerase enzyme possesses proofreading activity, most preferably the LNA or LNA analogue is located at the 3' penultimate (N-2) position in the primer. N-2 LNA primers are intrinsically nuclease-resistant, but the extension products generated in base extension reactions using N-2 LNA primers are proofreadable, analogous to the behaviour of 3'-phosphorothioate modified primers. Alternatively, N-1 LNA and N-1+N-2 LNA primers may be utilised in proofreading base extension reactions in which positions after N-1 are proofread.

It should be noted that in addition to modifying the primer so as to provide protection from 3' -> 5' exonuclease activity, the template may be similarly protected. The template may be modified to provide such protection in a number of ways. For example, one or more phosphorothioate groups may be added by polymerization (Rp isomer), treatment with a terminal transferase and a phosphorothioate-dNTP (Rp isomer), or treatment with an RNA ligase and a 5'- phosphorothioate-deoxynucleotide-3'-phosphate, Alternatively, a suitable nucleotide sequence may be added to facilitate folding of the 3' end of the template, thereby making it inaccessible to the exonuclease. 2. Polymerases

Preferably the polymerase enzymes employed in the methods and kits of the present invention contain a proofreading capability by virtue of a 3' -> 5' exonuclease activity, thereby providing them with an improved accuracy of nucleotide incorporation when compared to polymerases which lack such an activity, Particularly suitable enzymes in this respect include but are not limited to T4 DNA polymerase, T7 DNA polymerase, Vent DNA polymerase, Pfu DNA polymerase, and exo⁺ Klenow fragment of £ coli DNA polymerase I. It will be appreciated that many other polymerases are known to those skilled in the art to have proofreading activity and may equally be employed in the methods and kits of the present invention.

Alternatively, the present invention also provides for methods and kits in which the polymerase used to catalyse the addition of the nucleotide or nucleotide analogue to the 3' end of the primer lacks proofreading ability, In this case, proofreading is provided by an extrinsic nuclease displaying 3' -> 5' exonuclease activity, such as exonuclease III. A number of alternative 3' -> 5' exonucleases are known to those skilled in the art and may be employed in the methods and kits of the present invention. 3. Nucleotides or nucleotide analogues for incorporation

As used within the context of the methods and kits of the present invention, the term "nucleotide or nucleotide analogue" refers in particular to any deoxynucleotides (e.g. dATP, dTTP, dUTP, dGTP and dCTP), dideoxynucleotides (e.g. ddATP, ddTTP, ddUTP, ddGTP and ddCTP), acyclonucleotides (acyATP, acyTTP, acyUTP, acyGTP and acyCTP) or derivatives thereof, so long as the nucleotide or analogue thereof can be incorporated at the 3' end of a primer during a primer- directed extension reaction, Suitable modifications which may be made to nucleotides include but are not limited to base modifications such as 7-deazaadenine, 7-deazaguanine and 5-bromouracil, sugar modifications such as 2'-fluoro-deoxyribose and 2'-amino-deoxyribose and triphosphate modifications a such as α-, β- and γ-thiotriphosphates.

Nucleotides and analogues thereof are preferably labelled in a suitable form so as to aid their detection after incorporation. The term "labelled" means that the nucleotides bear a detectable label or are modified/derivatized to permit labeling of the nucleotide following the extension reaction, the label being one that does not significantly interfere with the extension reaction. For mass spectrometric detection, "labelling" includes differentiation on the basis of intrinsic or extrinsic mass differences.

Of particular use in the present invention are labels consisting of an electrochemically active agent, a fluorescent dye, a chromophore, a hapten, chemiluminescent agent, a radioisotope or any other suitable label. Suitable electrochemically active agents include metal complexes metallocenes and organic molecules exhibiting facile electron transfer behaviour. Particularly suitable are ferrocene derivatives or complexes of a transition metal such as ruthenium or osmium. For amperometric detection, the intrinsic conduction properties of stacked nucleic acid bases is included as a suitable label. Suitable fluorescent dyes include derivatives of the fluorescein, rhodamine, bodipy, cyanine or Alexa families, or other fluorescent compounds suitable for conjugation to a nucleotide. Dyes such as FAM, ROX, TAMRA, R110, R6G, Joe, HEX, TET, Alexa, Cy3 and Cy5 are particularly preferred. Suitable haptens include digoxigenin, dinitrophenol, biotin or similar small molecules bound by antibodies or other proteins.

4. Detection methods

Detection of the nucleotide or nucleotide analogue(s) incorporated at the 3' terminus of the modified primer may be achieved by standard methods of direct or indirect detection well known to those of ordinary skill in the art. For example, detection may be carried out by mass spectrometry, indirect colourimetric assays, fluorescence assays, chemiluminescence assays or electrochemical assays. Generally, the detection method of choice will depend largely on the type of label borne by the nucleotide or nucleotide analogue incorporated in the extension reaction. Of particular use in the present invention is matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. MALDI-TOF mass spectrometry has a major advantage as a tool to first establish the utility of proofreading in SBE genotyping - it permits the full range of primer extension products (extended, unextended and nuclease-degraded primer) to be detected directly and unambiguously. It has the disadvantage of relatively poor signal-to-noise, which limits its ability to detect low-frequency alleles in mixed populations. Detection is most effectively performed by colourimetry, fluorescence or electrochemistry, which each possess superior signal-to-noise characteristics.

5. Regeneration of the modified primer A particular advantage of the present invention is the capacity for re-use by regeneration of the primer following an extension reaction. Such a capacity for re-use has not previously been possible with methods of the prior art. Regeneration may be achieved by application of 3'-5' exonuclease activity for a sufficient period of time, The 3'-5' exonuclease activity may be derived from a proofreading polymerase or from a dedicated nuclease such as snake venom phosphodiesterase or exonuclease III. In this treatment, the primer terminus is preferentially cleaved away to produce the original 3' modified unextended primer, which is then ready for further extension reactions. This capability is particularly advantageous for applications with high-value modified surfaces and in quantitative assays where effective primer concentration must be closely controlled. 6. Kits

Kits for performing the primer-directed, polymerase-catalysed extension reactions of the methods of the present invention are also provided. Typically, such kits include one or more primers that specifically hybridize to a segment of a target nucleic acid of interest (template), The number of primers included in a kit of the present invention may vary, such that the kit can be employed for the genotyping of one or more variable nucleotide positions. If more than one primer is provided, these primers may be of the same or different primary sequence and may have the same or different modifications at their 3' termini. Whereas the template molecule is typically supplied by the user of the kit, the kit may include a suitable control template molecule for use in a control reaction and a suitable primer designed to anneal to the control template, the control reaction serving to confirm the correct functioning of the kit components and the method of the extension reaction.

Kits of the present invention also typically include labeled nucleotides or analogues thereof for incorporation at the 3' end of the primer and at least one polymerase enzyme suitable for performing the extension reaction. A suitable exonuclease enzyme may also be supplied in the kit or may be supplied by the user. The kits according to the present invention may additionally include other components for performing primer-directed extension reactions including, for example, buffers, cofactors, salts and/or diluents. Typically, the kits also include containers for housing the various components and instructions for using the kit components to conduct extension reactions according to the present invention.

The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples

Materials and Methods Oligonucleotides

The synthetic primers and templates shown in Fig. 1 were purchased in desalted form from Sigma-Genosys or Genset Pacific. All oligonucleotides were purified by RP-HPLC on a 9.4 x 250 mm Zorbax ODS column with a 0-50% gradient of acetonitrile in 50 mM UCIO4. The major eluting peak volume was reduced in a vacuum concentrator (Eppendorf) before 10 volumes of acetone were added for precipitation by centrifugation. Purified oligonucleotides were washed with acetone, dissolved in milli-Q water and desalted by spin chromatography (Micro Bio-Spin P-6, Bio- Rad). Oligonucleotides were quantitated by spectrophotometry using ε26o values provided by the supplier. Purity was checked by MALDI-TOF mass spectrometry. Polymerases and Substrates

Wild-type and exo- Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase and T7 DNA polymerase (MBI Fermentas) were purchased from Progen Industries. Sequenase 2.0 (USB) was purchased from AP Biotech. Vent {Thermococcus litoralis) and therminator DNA polymerases were purchased from New England Biolabs. A488L-Vent polymerase mutants were provided by New England Biolabs. Exonuclease III was from Promega. Deoxynucleotide triphosphates (Promega), dideoxynucleotide triphosphates (MBI) and acyclonucleotide triphosphates (NEB) were purchased commercially. Fluorescence-labelled ddNTPs were purchased from Perkin Elmer. Ferrocene-labelled nucleotides were synthesized in-house. Substrate concentrations were used as supplied by manufacturers. UV Melting

Thermal denaturation experiments were performed on a Varian Gary Bio 100 spectrophotometer equipped with a thermal accessory. Primer/template pairs at 1 μM per strand were heated to 90 °C in buffer and slow-cooled before the experiment. Melting curves were analysed with Cary software. Primer Degradation

P1 or SP1 primers or P1/LTA or SP1/LTA primer/template pairs were incubated with 1 U of polymerase in buffer. Primer remaining after incubation was quantitated relative to 100 pmol of a standard oligonucleotide (m/z 6010) added post-reaction. Primer Extension Reactions

Each primer extension reaction contained either the P1 or SP1 primer (Fig. 1), one of the LT series templates (Fig. 1) and a single triphosphate substrate. The four templates varied at position n+1 relative to the 3' end of the primer, each having a different base at this position. Primers were identical at all other positions with the exception of LTA, which also differed at position n+2 in order to avoid the intentional addition of two sequential dT residues with dTTP as substrate,

Primer extension reactions were initiated by polymerase addition. Reactions were performed in buffers supplied by the polymerase manufacturers: 50 mM Tris-HCI (pH 8.0 at 25 °C), 5 mM MgCI₂, 1 mM DTT for Klenow fragment species, 67 mM Tris-HCI (pH 8.8 at 25 °C), 6.6 mM MgCI₂, 1 mM DTT, 16.8 mM (NH )2S0 for T4 DNA polymerase, 40 mM Tris-HCI (pH 7.5 at 25 °C), 20 mM MgCI₂, 50 mM NaCI for Sequenase 2.0, 40 mM Tris-HCI (pH 7.5 at 25 °C), 10 mM MgCI₂, 1 mM DTT for T7 DNA polymerase and 20 mM Tris-HCI (pH 8.8 at 25 °C), 2 mM MgS04, 10 mM KCI, 10 mM (NH₄)2S0₄, 0.1 % Triton X-100 for Vent DNA polymerase species.

The basic reaction mix for mesophilic polymerases contained 10 μM primer, 25 μM template and 1-2 U enzyme in a 10 μl volume. The basic reaction mix for thermophilic Vent polymerase contained 10 μM primer, 4 μM template and 1-2 U enzyme in a 10 μi volume. In trial experiments, reaction conditions for each combination of polymerase - substrate family (deoxy, dideoxy, acyclo) were established to accommodate the differing incorporation rates between these groups. Substrate concentrations and incubation times were varied so that incorporation of all correct substrates within each polymerase - nucleotide family utilized at least 50% of the available primer. Concentrations were then held at those values for the A, C, G and T members of that family. For the trial experiments, substrate concentrations were varied from 10 μM - 1 mM, incubation times from 3 min - 6 h and polymerase from 0.25 - 2 U per reaction. For all final reactions, nucleotide concentrations were kept at least four times above known K or Km values when available, with the enzyme concentration and incubation time varied accordingly.

For reactions utilizing extrinsic exonuclease III, 0.1-10 units of exonuclease were added to the normal polymerase reaction mixture, Mass Spectrometry

Following incubation, all primer extension reactions were stopped by addition of EDTA to 20 mM, with 5 min heating at 95 °C for mesophilic enzymes. Salts, detergents, proteins and other contaminants were removed with Ciβ Ziptips (Millipore) according to the manufacturers instructions. Samples were eluted onto a MALDI-TOF target (PerSeptive Biosystems) with a freshly-prepared solution of 45 mg/ml 3'-hydroxypicolinic acid, 5 mg/ml ammonium citrate in 45 % acetonitrile. Mass data acquisition and analyses were performed on a Voyager-DE mass spectrometer (PerSeptive Biosystems). Typically, 100 transients were collected in linear negative-ion mode with noise reduction by smoothing. Voyager analysis software was used to determine peak areas for extension product distribution and quantitation. For experiments without an added standard, incorporation was determined according to E = A_e/(A_e+A_p) and converted into a percentage, where A_p = peak area for unextended primer and A_e = peak area/s for extension product/s. Incorporation was determined as 2E for experiments in which a second aliquot of primer standard was added post-reaction. Fluorescence Polarization and Fluorescence Quenching

FP and FQ experiments were performed with a BMG PolarSTAR plate reader equipped with appropriate excitation and emission filters. Primer extension reactions were conducted in a 20 μl reaction volume in the presence of 1.5 pmol R6G- or ROX-labelled ddNTP substrate before detection in 200 μl of reading buffer, Electrochemical Detection

Detection of electrochemically-labelled SBE products was performed on a gold electrode modified with a surface assembled monolayer (SAM) consisting of a 5'-thiol-terminated antitag oligonucleotide and a mercaptohexanol (MCH) passivator. The immobilized antitag oligonucleotide was designed to be complementary to a designed oligonucleotide tag sequence carried at the 5'- end of the SBE primer (Fig. 1). The electrode was connected to a BAS electrochemical analyzer and examined using cyclic voltammetry (CV) and Osteryoung square wave voltammetry (OSWV).

Example 1 Exonuclease activity of proofreading polymerases Proofreading polymerases have not been employed in SBE assays due to primer degradation by their 3'-5' exonuclease activity (Syvanen et al., 1990; Haff & Smirnov, 1997). To examine this issue qualitatively, P1 and SP1 primers or P1/LTA and SP1/LTA primer/template pairs were incubated with proofreading polymerases and primer degradation monitored by MALDI-TOF mass spectrometry, Results for primer incubation with T4 DNA polymerase, which has a strong exonuclease activity, are shown in Figure 2. As expected, T4 DNA polymerase extensively degrades the P1 primer - the intact P1 peak at m/z 5478 (Fig. 2a) is almost completely degraded to a mixture of shorter products in less than 10 min (Fig. 2b). The masses of these products correspond to the 5' end of the progressively shortened P1 sequence, as expected. The majority of primer is degraded to trinucleotide or smaller fragments in less than one hour under the present conditions (Fig. 2c). In contrast, the 3'-phosphorothioate primer SP1 at m/z 5494 (Fig. 2d) remains largely intact after incubation with T4 DNA polymerase for 1 h (Fig. 2e) or 16 h overnight (Fig. 2f). T7 and Vent DNA polymerases show similar behaviour to T4 DNA polymerase (data not shown). Vent and other thermophilic DNA polymerases are typically employed when primer is present in substantial excess. Although a large amount of intact SP1 primer clearly remains after long-term incubation with T4 DNA polymerase, there is some degradation apparent in the appearance of new peaks, mostly at m/z < 1200 corresponding to products smaller than tetramers (Fig. 2e,f). To investigate further, quantitative degradation experiments were performed for two proofreading polymerases. As shown in Figure 3, exo⁺ Klenow fragment (KF) causes essentially no degradation of SP1 over 16 h, consistent with efficient inhibition of its relatively weak 3'-5' exonuclease by both the Rp and Sp phosphorothioate diastereomers (Kunkel et al., 1981 ; Brautigam & Steitz, 1998). In contrast, the strong exonuclease activity of T4 DNA polymerase causes primer levels to decrease to a value near 50 % within 1 h, followed by minimal degradation out to 16 h (Fig. 3). This biphasic behaviour by T4 DNA polymerase is consistent with a mechanism of 3'-5' exonuclease inhibition where the Sp isomer is much more stable than the Rp isomer to degradation (Brautigam & Steitz, 1998), The Rp isomer therefore only partially protects the 3' primer terminus against a strong proofreading exonuclease, while the Sp isomer is essentially fully protective.

The net effect of PS exonuclease-resistance is to produce an "all or nothing" population of essentially stable PS primers and very short non-priming degradation products - any slow degradation of the PS primer terminus (Fig. 2e,f; Fig. 3) is followed by rapid degradation of the remaining PO groups (Fig. 2b). Primer stability is somewhat improved by the presence of template (data not shown), but the all-or-nothing population distribution of PS primerremains (see below).

Example 2 Deoxynucleotide incorporation in SBE reactions

SBE reactions were first examined for each of the four natural deoxynucleotide triphosphates opposite four different templates to cover all 16 possible substrate-template interactions. Results for extension of the P1 and SP1 primers by KF, T4, T7, and Vent DNAP are summarized in Tables I and II (below). For the mesophilic polymerases, template was present in substantial excess over primer (25 μM vs 10 μM), with near-saturating substrate (200 μM). The behaviour of KF, which displays the greatest misincorporation, will now be described in detail for illustration. As shown for the P1/LTT primer/template pair, two major classes of error are observed upon extension by exo- KF (Fig. 4a,e). Misincorporation of dG opposite the T of the LTT template occurs at the remarkably high level of 74% (Fig. 4a), the worst misincorporation result for any primer/template/polymerase combination (Table I). The average incorrect/correct extension ratio (l/Ca g) for the P1/exo- KF set is 30 % including those combinations without measurable misincorporation (Table I). Replacement of the P1 primer by the SP1 primer produces a small but significant reduction in misincorporation for exo- KF. Misincorporation of dG declines to 67% for LTT/dGTP (Fig. 4b) and the l/C_avg drops to 24% (Table I). This reduction is likely associated with weakening of the 3' primer terminus/template duplex by the SP1 phosphorothioate group, which also causes the extent of correct incorporation to drop slightly but much less significantly (Table I). On top of this mild PS primer effect, use of proofreading exo⁺ KF with the SP1 primer causes a strong reduction in misincorporation of dG to 21% (Fig. 4c), with an l/Cavg decrease to 7% and two previously significant misincorporations which drop to below the limit of detection (Table I). Correct SP1 extension by exo⁺ KF yields a clean result (Fig. 4d). While the relative benefit of proofreading is apparent in the examples of Figure 4, it is clear that KF exonuclease activity is not adequate to entirely eliminate misincorporation under the present conditions. To determine the effect of stronger proofreading, we examined the exo⁺ T4 DNAP/SP1 pairing for all substrates. As indicated in Table I, T4 DNAP reduces all misincorporations to near or below the limit of detection, with an l/Cavg Of 1 %.

Even better improvement in assay performance is observed upon comparison of the Sequenase 2.0/P1 system with the T7 DNAP/SP1 proofreading system. Sequenase 2.0 (T7-) intrinsically makes fewer errors than KF (Table I), but all errors for T7 DNAP/SP1 are reduced to below the limit of detection. Figure 5 shows that this improvement is obtained without any detectable degradation-extension events, For conditions favouring both correct substrate incorporation (Fig, 5a-d) and misincorporation (Fig. 5e-h), only clean products are observable in the mass window where any aberrant products would be expected to appear. To demonstrate the utility of proofreading in cycling applications, thermophilic T. litoralis (Vent) DNAP variants run with excess primer yield results consistent with those obtained for the mesophilic polymerases (Table I) - a significant improvement in misincorporation upon replacing P1 primer (l/C_aVg = 20%) by SP1 primer (l/C_avg = 12 %), and a major improvement upon introducing proofreading activity (l/Cavg = 2%).

Deoxynucleotide substrates display a novel second type of error, where extension with the correct nucleotide is followed by further extension with mispairing (Fig. 4e-g, Table II). As expected, highly efficient double extension (Δm/z 626) of the P1 primer by exo- KF (Fig. 4e) is reduced significantly by substitution of SP1 primer (Fig. 4f) and more substantially in the proofreading exo⁺ KF/SP1 system, so that a single correct dA extension (Δm/z 313) predominates (Fig. 4g). The strong proofreading activity of T4 DNAP reduces double incorporation to below detectable levels (Fig. 4h, Table II). In contrast to KF, T7 DNAP displays no significant double incorporation even in non-proofreading form (Table II).

Table I. Primer extension with dNTP substrates. Exonuclease-deficient (-) and proofreading (+) variants of Klenow fragment (KF), T4 DNAP (T4), T7 DNAP (T7) and Vent DNAP (V) are indicated. Correct nucleotide incorporation for each template is shown in bold. "- " entries indicate values below the limit of detection (< 5%). a. 10 μM primer, 25 μM template, 200 μM substrate, 1 U polymerase, 60 min at 37 °C. b. 10 μM primer, 4 μM template, 200 μM substrate, 1 U polymerase, 25 cycles of 30 s at 85 °C, 1 min at 53 °C, 1 min at 63 °C.

Table II. Incorrect double addition of dNTP substrates by exonuclease-deficient (-) and proofreading (+) variants of Klenow fragment (KF), T4 DNA polymerase (T4) and T7 DNA polymerase (T7), The identity of the template nucleotide for misincorporation is shown in brackets. "-" entries indicate values below the limit of detection (< 5%).

Example 3 Dideoxynucleotide and acyclonucleotide terminators in SBE reactions

Many SBE methods use dideoxynucleotide (ddNTP) terminators to ensure that only the base immediately adjacent to the primer terminus is extended. Values and trends for extension of the P1 and SP1 primers by ddNTPs (Table III) tend to mirror the behaviour of dNTPs (Table I), although not perfectly. The relatively low fidelity of the exo- KF/P1 pairing (l/Cavg = 46%) is improved first by substitution of SP1 primer (l/C_aVg = 31%) and further by substitution of proofreading activity (l/C_avg = 9%). Better performance is observed for the other DNAPs, with the exo⁺ T4 DNAP/SP1, exo⁺ T7 DNAP/SP1 and exo⁺ Vent/SP1 pairs displaying no or almost no observable misincorporation. This is a substantial improvement over their exo- DNAP/P1 counterparts (Table III). Very high ddNTP concentrations were employed in these experiments to ensure substrate saturation.

In acyclonucleotide terminators (acyNTPs), the furanose sugar moiety is replaced by a 2- hydroxyethoxymethyl group. These species are coming into more widespread use (Gardner & Jack, 2002). Table IV summarizes the results of acyNTP incorporation experiments. For these substrates, misincorporation by KF occurs at much lower levels than for corresponding dNTP or ddNTP substrates, while Vent DNAP misincorporates at equivalent or slightly higher levels. All four proofreading systems display misincorporation levels below the limit of detection, the best performance for any substrate family. Template Substrate % ddNTP incorporation

Polymerase KF- KF- KF+ T4+ T7- T7+ V- V- V+

Primer P1^a SP1^a SP1^a SP1b P1b SP1 P1^c SP1^C SP1^C

LTA ddATP 36 27 - - - - - - - ddCTP 53 32 8 - 11 - - - - ddGTP 26 17 - - - - 17 10 - ddTTP 76 73 73 57 62 67 91 82 72

LTC ddATP 25 19 - - 9 - - - - ddCTP 23 13 - - 10 - - - - ddGTP 100 100 100 76 100 83 100 100 92 ddTTP 21 13 - - - - 35 28 -

LTG ddATP 19 16 - - - - 36 26 9 ddCTP 100 100 88 72 83 80 100 100 100 ddGTP 50 26 - - 11 - 27 19 - ddTTP 19 16 - - - - 23 12 -

LTT ddATP 87 79 73 77 89 86 79 70 63 ddCTP 79 51 32 - 22 - 32 22 - ddGTP 100 75 41 - 18 - 9 - - ddTTP ^s 49 26 9 - 12 - 25 14 -

Incorrect avg 42 28 8 0 8 0 17 11 1

Correct avg 91 88 84 71 84 79 93 88 ■ 82 l/Cavg / 46 31 9 0 9 0 18 12 1

Table III. Primer extension with ddNTP substrates by exonuclease-deficient (-) and proofreading (+) variants of Klenow fragment (KF), T4 DNA polymerase (T4), T7 DNA polymerase (T7) and Vent DNA polymerase (V). Correct nucleotide incorporation appears in bold. "-" entries indicate values below the limit of detection (< 5%). a. 10 μM primer, 25 μM template, 1 mM substrate, 2 U polymerase, 4 h at 37 °C. b. 10 μM primer, 25 μM template, 1 mM substrate, 2 U polymerase, 6 h at 37 °C. c. 10 μM primer, 4 μM template, 1 mM substrate, 2 U polymerase, 35 cycles of 30 s at 85 °C, 1 min at 53 °C, 1 min at 63 °C.

Table IV. Primer extension with acyNTP substrates. Polymerase identities are the same as those listed for Tables I and III. "-" entries indicate data below the limit of detection (< 5%). a. 10 μM primer, 25 μM template, 500 μM substrate, 2 U polymerase, 4 h at 37 °C. b. 10 μM primer, 25 μM template, 500 μM substrate, 2 U polymerase, 6 h at 37 °C. c. 10 μM primer, 4 μM template, 200 μM substrate, 2 U polymerase, 35 cycles of 30 s at 85 °C, 1 min at 53 °C, 1 min at 63 °C.

Example 4 Use of an extrinsic exonuclease

In addition to the application of intrinsic 3'-5' exonuclease activity, we also examined whether proofreading could be separately performed by an added nuclease. These experiments were carried out with exo- KF, which displays relatively poor misincorporation behaviour, SP1 primer and the widely available £ coli exonuclease III. Results are summarized in Table V and Figure 6. In general, increasing amounts of added exo III activity diminish errors. For the strong misincorporation of dGTP opposite the LTT primer (Fig. 6a), 5 U of exo III are sufficient to reduce the error to a low level (Fig. 6c). The same observation holds for double addition of dGTP opposite LTC (Fig. 6b,d). These improvements are associated with minimal effects on correct extension reactions with both dNTP and ddNTP substrates (Table V).

Table V. Extrinsic proofreading by exonuclease III added to SP1 primer extension by exo- Klenow fragment. Data for correct nucleotide incorporation are shown in bold. Conditions as per Table I (deoxynucleotides) and Table III (dideoxynucleotides). "-" entries indicate values below the limit of detection (< 5%).

Example 5 Concentration Dependence of Idling Turnover Proofreading polymerases are capable of repetitive extension/cleavage reactions known as "idling turnover". Depending upon the ratio of exonuclease activity to polymerase activity, there is the potential for correctly-added nucleotides to be excised from the primer terminus, reducing correct product yield. This process results in the net conversion of triphosphate substrate into monophosphate. With the exonuclease activity of T7 DNAP for example being close to that of the polymerase activity, it is important to examine whether high yields of correct extension products can be maintained over a range of experimental conditions including low substrate concentrations.

To establish baseline behaviour in the absence of proofreading, we first monitored primer extension by Sequenase 2.0 (1 U) in the presence of excess SP1/LTG primer/template (2.5 μM/10 μM) and the correct dCTP substrate at concentrations between 2.5 and 160 μM (Fig. 7a). Equivalent incorporation is observed for substrate concentrations ≥40 μM, with somewhat lower incorporation at 10 μM. By comparison, lowering the substrate concentration to 2.5 μM results in a substantial drop in the rate and level of extension at all timepoints (Fig. 7a).

For the corresponding experiment with proofreading T7 DNAP (Fig. 7b), the net level of incorporation is only marginally lower than that for the exo- variant at substrate concentrations ≥40 μM, without any significant difference in extension after 90 min. However, while behaviour out to 30 min incubation is consistent under all conditions tested, a substrate concentration of 10 μM results in a slight decline of net extension at protracted timepoints, an effect which is more pronounced at 2.5 μM substrate concentration (Fig. 7b). This behaviour can be attributed to idling turnover under strong substrate limitation. A similar pattern is observed for reactions with further limitation of enzyme (Fig. 7c,d), where 0.25 U T7 DNAP causes net primer extension to decline somewhat following long incubation at low substrate concentrations (Fig. 7d).

While the majority of the data in the previous examples relate to experiments in which the substrate concentration is >100 μM, the results in this Example demonstrate that proofreading polymerases can also be employed at much lower concentrations without significant signal degradation. Idling turnover by T7 DNAP causes minimal loss of correct signal intensity for a dCTP concentration of 2.5 μM (one-seventh Km) out to incubation times of 30-60 min. This maintenance of signal is important for SBE implementations using expensive labelled substrates or where substrate incorporation is essentially driven to completion.

Example 6 Extension and Proofreading of LNA-Modified Primers

Two homologous LNA-modified primers were incubated with an appropriate template, polymerase and substrate before PAGE detection of primer extension events. As shown in Fig. 8 lanes 7 and 10, primers with a penultimate (N-2) LNA residue are intrinsically resistant to 3'-5' exonuclease proofreading activity. Due to the frame-shift effect of nucleotide incorporation, this resistance is also observed following extension of a primer with an LNA residue at the 3'-terminal (N-1) position. In Fig. 8 lane 13, primer PAI-C3 carrying a 3'-terminal LNA has been extended to yield a proofreading-resistant product that is one nucleotide longer than the starting primer (Fig. 8 lane 1) and is also one nucleotide longer than an extended and subsequently proofread PAI-C5 primer carrying an LNA residue at position N-2 (Fig. 8 lane 14). N-1 LNA primers are therefore not suitable for proofreading SBE applications because they generate an exonuclease-resistant, non- proofreadable N-2 product (Fig. 8 lane 13). On the other hand, N-2 LNA primers are intrinsically nuclease-resistant, but their extension products are proofreadable (Fig. 8 lane 14), analogous to the behaviour of 3-PS primers. Example 7 Fluorescence Detection of Proofreading SBE Reactions

A fluorescence polarization (FP) assay was used to examine the effect of proofreading activity with fluorescence-tagged nucleotides. Fig. 9 shows a timecourse for degradation of R6G- ddCTP-terminated primers by exo+-Vent DNAP. Extended natural DNA primer is rapidly degraded (diamonds) and extended 3 -PS primer is degraded at a similar rate (triangles), indicating successful proofreading. In contrast, extension of a 3 -LNA primer yields a non-proofreadable fluorescent product (squares).

Example 8

Electrochemical Detection of Proofreading SBE Products in a Tag Array Format

Electrochemical detection of proofreading SBE can be performed in a number of different modes. In an electrochemical tag array format, a tagged SBE primer is extended with an electrochemically-labelled nucleotide/s in solution, followed by capture of the now electrochemically-active extended primer by an electrode surface carrying an antitag sequence. Fig, 10 shows Osteryoung square wave voltammograms (OSWV) of antitagged-gold electrodes following capture of a 3'-PS primer extended with a residue of Fc1-dUTP or VF1-dUTP, two alternative ferrocene-labelled nucleotides. The peak near 400 mV (Fig. 10A) is due to the derivatized ferrocenecarboxylate moiety of FC1-dUTP, while the peak near 200 mV (Fig. 10B) is due to the vinylferrocene moiety of VF1 -dUTP.

References

Braasch, D. A. and D. R. Corey (2001). "Locked nucleic acid (LNA): fine tuning the recognition of DNA and RNA." Chemistry & Biology 8: 1 -7.

Brautigam, C. A. and T. A. Steitz (1998). "Structural principles for the inhibition of the 3 -5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates." Journal of Molecular Biology 277: 363-377. Bray, M. S., E. Boerwinkle, et al. (2001). "High-throughput multiplex SNP genotyping with MALDI- TOF mass spectrometry: practice, problems and promise." Human Mutation 17: 296-304.

Eckstein, F. (1985). "Nucleoside phosphorothioates." Annual Review of Biochemistry 54: 367-402. Haff, L. A. and I. P. Smirnov (1997). "Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry." Genome Research 7: 378-388. Hsu, T. M., X. Chen, et al. (2001). "Universal SNP genotyping assay with fluorescence polarization detection." Biotechniques ZV. 560-570. Hu, Y.-W., E. Balaskas, et al. (1998). "Primer specific and mispair extension analysis (PSMEA) as a simple approach to fast genotyping." Nucleic Acids Research 26: 5013-5015. Hu, Y.-W., E. Balaskas, et al. (2000). "Comparison and application of a novel genotyping method, semiautomated primer-specific and mispair extension analysis, and four other genotyping assays for detection of hepatitis C virus mixed-genotype infections." Journal of Clinical Microbiology 38: 2807-2813.

Kunkel, T. A., F, Eckstein, et al. (1981). "Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis." Proceedings of the National Academy of Sciences, U.S.A. 78: 6734-6738, Lindroos, K., U. Liljedahl, et al. (2001). "Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries." Nucleic Acids Research 29: e69.

Morita, K., C. Hasegawa et al. (2002) "2'-0,4'-C-ethylene-bridged nucleic acids (ENA): highly stable nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug." Bioorganic and Medicinal Chemistry Letters 12: 73-76. Pastinen, T., A. Kurg, et al. (1997). "Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays." Genome Research 7: 606-614.

Shapero, M. H,, K, K. Leuther, et al. (2001). "SNP genotyping by multiplexed solid-phase amplification and fluorescent minisequencing." Genome Research 11: 1926-1934. Sun, X., H. Ding, et al. (2000). "A new MALDI-TOF based mini-sequencing assay for genotyping of SNPs." Nucleic Acids Research 28: e68. Syvanen, A.-C, K. Aalto-Setala, et al. (1990), "A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E," Genomics 8: 684-692.

Claims

1. A method of incorporating at least one nucleotide or nucleotide analogue into a polynucleotide molecule in a primer-directed extension reaction, the method comprising the steps of:

(b) incorporating at least one nucleotide or nucleotide analogue into said duplex molecule at the 3' end of said primer in the presence of a polymerase enzyme; wherein the 3' terminus of said single-stranded primer is modified so as to maintain primer integrity.

2. The method according to claim 1 wherein the modification to the 3' terminus of the primer is one or more phosphorothioate linkages in which at least one residue at the 3' terminus of the primer contains at least one sulphur substitution at the scissile phosphate.

3. The method according to claim 2 wherein the phosphorothioate linkage is a phosphorodithioate linkage.

4. The method according to claim 2 wherein the phosphorothioate linkage is a phosphoromonothioate linkage.

5. The method according to claim 4 wherein the phosphoromonothioate is the Sp isomer.

6. The method according to any one of claims 2 to 5 wherein the phosphorothioate linkage resides in the 3' terminal residue of the primer,

7. The method according to claim 1 wherein the modification to the 3' terminus of the primer is provided by a non-native group selected from the group consisting of: methylphosphonates, phosphorothiolates, phosphoramidates and boron derivatives.

8. The method according to claim 1 wherein the modification to the 3' terminus of the primer provided by one or more replacement sugar moieties located at the 3' terminal position, the 3' penultimate position, or any mixture of positions that includes one or both of these locations.

9. The method according to claim 8 wherein the sugar moiety is a bicyclic "locked" nucleic acid analogue.

10. The method according to claim 9 wherein the locked nucleic acid analogue contains a methylene or ethylene linkage between the 2'-0 position and 4'-C position of the furanose ring of the nucleic acid.

11. The method according to claim 9 or 10 wherein the polymerase enzyme contains proofreading activity and the locked nucleic acid analogue is located at the 3' penultimate position of the primer.

12. The method according to any one of claims 1 to 11 wherein the template molecule is modified so as to provide protection from exonuclease activity.

13, The method according to claim 12 wherein the template molecule includes one or more phosphorothioate linkages at the 3' terminus.

14, The method according to any one of claims 1 to 11 wherein the template molecule contains a 3' terminal nucleotide sequence adapted to fold into a nuclease-resistant conformation.

15, The method according to any one of claims 1 to 14 wherein a single nucleotide or nucleotide analogue is incorporated into the duplex molecule in step (b).

16, A method of determining the identity of at least one nucleotide or nucleotide analogue incorporated into a polynucleotide molecule in a primer-directed extension reaction, the method comprising the steps of:

(a) hybridising a single-stranded polynucleotide primer with a single-stranded polynucleotide template molecule to generate a primer-template duplex molecule;

(b) incorporating at least one nucleotide or nucleotide analogue into said duplex molecule at the 3' end of the primer in the presence of a polymerase enzyme; and

(c) detecting of the identity of the at least one incorporated nucleotide or nucleotide analogue, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity.

17. The method according to claim 16 wherein a single nucleotide or nucleotide analogue is incorporated into the duplex molecule in step (b).

18. The method according to claim 16 or 17 wherein the modification to the 3' terminus of the primer is one or more phosphorothioate linkages in which at least one residue at the 3' terminus of the primer contains at least one sulphur substitution at the scissile phosphate.

19. The method according to claim 16 or 17 wherein the modification to the 3' terminus of the primer is provided by one or more bicyclic "locked" nucleic acid analogues located at the 3' terminal position, the 3' penultimate position, or any mixture of positions that includes one or both of these locations.

20. A method of genotyping by primer-directed base extension, the method comprising the steps of: (a) hybridising a single-stranded polynucleotide primer with a single-stranded polynucleotide template molecule to generate a duplex primer-template molecule;

(c) detecting the identity of the at least one incorporated nucleotide or nucleotide analogue, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity.

21. The method according to claim 20 wherein the modification to the 3' terminus of the primer is one or more phosphorothioate linkages in which at least one residue at the 3' terminus of the primer contains at least one sulphur substitution at the scissile phosphate,

22. The method according to claim 20 wherein the modification to the 3' terminus of the primer is provided by one or more bicyclic "locked" nucleic acid analogues located at the 3' terminal position, the 3' penultimate position, or any mixture of positions that includes one or both of these locations.

23. A method of single nucleotide polymorphism typing in a target polynucleotide molecule, the method comprising the steps of:

(a) hybridising a single-stranded polynucleotide primer with said target polynucleotide molecule in single-stranded form such that a duplex primer-target molecule is generated with the 3' end of the primer immediately adjacent the single nucleotide polymorphism in the target polynucleotide molecule;

(b) incorporating one nucleotide or nucleotide analogue into said duplex molecule at the 3' end of the primer in the presence of a polymerase enzyme; and (c) detecting the identity of the incorporated nucleotide or nucleotide analogue to thereby determine the identity of the single nucleotide polymorphism in the target polynucleotide molecule, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity.

24. The method according to claim 23 wherein the modification to the 3' terminus of the primer is one or more phosphorothioate linkages in which at least one residue at the 3' terminus of the primer contains at least one sulphur substitution at the scissile phosphate.

25. The method according to claim 23 wherein the modification to the 3' terminus of the primer is provided by one or more bicyclic "locked" nucleic acid analogues located at the 3' terminal position, the 3' penultimate position, or any mixture of positions that includes one or both of these locations.

26. A' method for detecting a mutation at a specific nucleotide position in a target polynucleotide molecule, the method comprising the steps of:

(a) hybridising a single-stranded polynucleotide primer with said target polynucleotide molecule in single-stranded form such that a duplex primer-target molecule is generated with the 3' end of the primer immediately adjacent the specific nucleotide position in the target polynucleotide molecule; (b) incorporating one nucleotide or nucleotide analogue into said duplex molecule at the

3' end of the primer in the presence of a polymerase enzyme; and

(c) detecting the identity of the incorporated nucleotide or nucleotide analogue to thereby detect a mutation at the specific nucleotide position in the target polynucleotide molecule, wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity.

27. The method according to claim 26 wherein the modification to the 3' terminus of the primer is one or more phosphorothioate linkages in which at least one residue at the 3' terminus of the primer contains at least one sulphur substitution at the scissile phosphate.

28. The method according to claim 26 wherein the modification to the 3' terminus of the primer is provided by one or more bicyclic "locked" nucleic acid analogues located at the 3' terminal position, the 3' penultimate position, or any mixture of positions that includes one or both of these locations,

29. A method for diagnosing a genetic disorder or predisposition to said genetic disorder in an individual wherein said genetic disorder is caused by mutation of a single nucleotide, the method comprising the steps of:

(a) isolating a polynucleotide molecule containing said single nucleotide from said individual;

(b) hybridising a single-stranded polynucleotide primer with said polynucleotide molecule in single-stranded form such that a duplex molecule is generated with the 3' end of the primer immediately adjacent the single nucleotide in the polynucleotide molecule;

(c) incorporating one nucleotide or nucleotide analogue into said duplex molecule at the s 3' end of the primer in the presence of a polymerase enzyme; and

0 30. The method according to claim 29 wherein the modification to the 3' terminus of the primer is one or more phosphorothioate linkages in which at least one residue at the 3' terminus of the primer contains at least one sulphur substitution at the scissile phosphate.

31. The method according to claim 29 wherein the modification to the 3' terminus of the 5 primer is provided by one or more bicyclic "locked" nucleic acid analogues located at the 3' terminal position, the 3' penultimate position, or any mixture of positions that includes one or both of these locations.

32. A kit for incorporating at least one nucleotide or nucleotide analogue into a polynucleotide o molecule in a primer-directed extension reaction comprising:

(a) a single-stranded oligonucleotide primer;

(b) a mixture of nucleotides or nucleotide analogues for incorporation; and

(c) a polymerase enzyme; wherein the 3' terminus of the single-stranded primer is modified so as to maintain primer integrity. 5

33. The kit according to claim 32 wherein the modification to the 3' terminus of the primer is one or more phosphorothioate linkages in which at least one residue at the 3' terminus of the primer contains at least one sulphur substitution at the scissile phosphate.

34. The kit according to claim 32 wherein the modification to the 3' terminus of the primer is provided by one or more bicyclic "locked" nucleic acid analogues located at the 3' terminal position, the 3' penultimate position, or any mixture of positions that includes one or both of these locations.