WO2012104817A1 - Method for genotyping - Google Patents

Abstract

The present invention is in the field of genotyping, and in particular in the field of single base DNA genotyping. The invention provides methods for determining the identity of a nucleotide base at a specific position in a template nucleic acid using a family X DNA polymerase; family X DNA polymerases for use in determining the identity of a nucleotide base at a specific position in a template nucleic acid; thermally stable family X DNA polymerases; and kits for determining the identity of a nucleotide base at a specific position in a template nucleic acid.

Description

METHOD FOR GENOTYPING

The present invention is in the field of genotyping, and in particular in the field of single base DNA genotyping.

BACKGROUND ART

A single base substitution is a type of point mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Point mutations may be transitions, i.e. replacement of a purine base with another purine or replacement of a pyrimidine with another pyrimidine, or transversions, i.e. replacement of a purine with a pyrimidine or vice versa. Transition mutations are about an order of magnitude more common in naturally occurring mutations than transversions.

Point mutations can also be categorized functionally by the effect that they have on a polypeptide encoded by the polynucleotide. Non-sense mutations occur when a codon that previously encoded an amino acid is altered by the mutation to code for a stop, which can truncate the protein. Missense mutations occur when a codon that previously encoded one amino acid is altered by the mutation encode a different amino acid. The missense mutation results in an amino acid change, but the properties of the amino acid can remain the same (e.g. hydrophobic, hydrophilic, etc). This is termed a conservative mutation. Alternatively, the properties of the new amino acid may be different from the wild-type. This is termed a non-conservative mutation. Silent mutations occur when a codon that previously encoded one amino acid is altered by the mutation to a different codon that encodes the same amino acid.

A particularly important type of single base substitution is a single-nucleotide polymorphism (SNP). A SNP is a DNA sequence variation occurring when a single nucleotide in the genome (or other shared sequence) differs between members of a species. For a variation to be considered a SNP, it must occur in at least 1% of the population.

SNPs are often found to be the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. If the concept of personalized medicine is to be realized, it is increasingly clear that reliable identification of single nucleotide polymorphisms, the most common genetic variations between human beings, will be a key enabler.

Detection of acquired or inherited nucleic acid changes in the management of cancer is also a challenging area. Predicting response and limiting drug-induced toxicity are two important challenges faced by clinicians in the treatment of cancer. The introduction of genetic testing to individualize treatment regimens will hopefully allow better response prediction and limit drug- induced toxicity leading to improved patient outcomes.

A vast increase in knowledge of the molecular processes underlying malignant change and progression has occurred, which will be useful in the development of new therapies and in areas such as diagnosis of occult disease, monitoring of disease progress, presymptomatic disease detection (screening), and therapy selection or optimization.

Inherited (germline) changes in the genes encoding drug metabolizing enzymes, membrane transporters, and therapeutic targets are emerging as useful tools to identify patients at risk for aberrant pharmacokinetic or pharmacodynamic effects. Somatic mutations, acquired before or after the initiation of chemotherapy, can lead to clinically relevant genetic variations not found in the germline. The promise of the emerging fields of pharmacogenomics and pharmacogenetics lies in their potential to determine the right drug and dose for each patient based on inter- individual genetic variability. Therefore, it is also crucial that optimal methods are developed to detect mutations in cancer cells.

The increase in interest in SNPs has been reflected by the huge development of diverse range of SNP genotyping methods. Methods known in the art to identify and genotype SNPs have been reviewed in (Rapley and Harbron, 2004) and include:

1. - Hybridization-based methods:

Several applications have been developed that interrogate SNPs by hybridizing complementary DNA probes to the SNP site:

^■ Dynamic allele-specific hybridization (DASH)

^■ Molecular beacons

^■ DNA microarrays

2. - Enzyme-based methods:

A broad range of enzymes including DNA ligase, DNA polymerase and nucleases have been employed to generate high-fidelity SNP genotyping methods:

^■ Restriction fragment length polymorphism (RFLP)

^■ Allele-specific PCR

^■ Invader assay (Flap endonuclease)

■ Single-base extension

^■ TaqMan (5'-3' exonuclease)

^■ Ligation assay 3. - Other post-amplification methods based on physical properties of DNA:

The characteristic DNA properties of melting temperature and single stranded conformation have been used in several applications to distinguish SNP alleles. These methods very often achieve high specificity but require highly optimized conditions to obtain the best possible results.

■ Single strand conformation polymorphism (SSCP)

^■ Temperature gradient gel electrophoresis (TGGE)

^■ Denaturing high performance liquid chromatography (DHPLC)

One particular known method for identification of a single base at a specific position in a nucleic acid sequence is the single-base extension (SBE). In this method (Goelet et al, 1999; represented in Figure 1), an oligonucleotide primer hybridizes to a complementary region along the nucleic acid, to form a duplex, with the primer's terminal 3' end directly adjacent to the nucleotide base to be identified. The oligonucleotide primer is extended a single base by a family A DNA polymerase with a nucleotide terminator complementary to the nucleotide being identified (e.g. ddNTP). The terminator prevents additional nucleotides from being incorporated.

Given the importance of point mutations and SNPs in many human diseases, there is an ever present need to develop new and improved methods for the detection and identification of a single base at a specific position in a nucleic acid, i.e. a nucleotide base of interest.

DISCLOSURE OF THE INVENTION

The present invention provides an improved, new single-base extension (SBE) method for single-point genotyping using a family X DNA polymerase to perform the extension reaction.

DNA polymerases are generally classified into seven families (Rothwell and Waksman, 2005): A, B, C, D, X, Y, and reverse transcriptase (RT). Family X consists of specialized small DNA polymerases whose primary function is to fill gaps of one to a few nucleotides during DNA repair (Ramadan et al, 2004). The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. In viruses, bacteria, archaea, protozoa and lower eukaryotes as well as in plants, only one PolX is present. However, vertebrates have four members (Ροΐβ, Ροΐλ, Ροΐμ and TdT) with different specific functions in a variety of processes, such as: DNA repair, V(D)J recombination and translesion synthesis (Hiibscher et al, 2002).

Most PolX enzymes share a common modular organization (Ροΐβ core) consisting of an 8-kDa domain (which may have a size of more than or less then 8kDa, depending on the source of the PolX enzyme) and a 31-kDa polymerization domain comprising 'fingers', 'palm' and 'thumb' subdomains. Such a structural organization has been demonstrated for Ροΐβ (Pelletier et al, 1994; Sawaya et al, 1994), TdT (Delarue et al, 2002), Ροΐλ (Garcia-Diaz et al, 2004 and 2005), Ροΐμ (Moon et al, 2007) and ASFVPolX (Maciejewski et al, 2001; Showalter et al, 2001).

DNA repair processes seek out DNA lesions, removing them from the DNA strands, and repairing the genetic sequence at the site of the damaged bases. As a product of these DNA repair processes, single- and/or double- stranded gaps are created at certain points along the DNA. PolXs have evolved to accommodate these nonstandard substrates, and resolve the gaps. The unique structural feature that allows this family of enzymes to bind single- and/or double- strand gaps is the presence of an N-terminal 8 kDa domain upstream of the polymerization domain. The key role of this 8 kDa domain appears to be DNA binding, and global positioning of the enzyme on gapped or nicked substrates (Pelletier et al, 1994; Sawaya et al, 1994; Prasad et al, 1994). For this purpose, the 8kDa domain contains a Helix-hairpin-Helix motif that interacts with the DNA downstream of the gap in a non-sequence-dependent manner. Another mechanism through which the 8 kDa domain aids in DNA binding is likely by direct interaction with the 5'- phosphate moiety on the downstream end of gapped DNA.

Unlike the SBE methods known in the prior art, the present invention relies on filling a single base gap in a nucleic acid sequence. The methods of the present invention therefore require two oligonucleotides (a primer and a downstream oligonucleotide) flanking the nucleotides of interest, and a family X DNA polymerase to perform the extension reaction.

The present invention therefore provides:

" A method for determining the identity of a nucleotide base at a specific position in a template nucleic acid using a family X DNA polymerase;

^■ A family X DNA polymerase for use in determining the identity of a nucleotide base at a specific position in a template nucleic acid;

^■ A thermally stable family X DNA polymerase;

" A kit for determining the identity of a nucleotide base at a specific position in a template nucleic acid.

Methods of the invention

The present invention provides methods for determining the identity of a nucleotide base at a specific position in a template nucleic acid using a family X DNA polymerase. In a specific embodiment, the invention provides a method comprising the steps of:

(a) contacting a template nucleic acid with: (i) a primer with a 3' terminus adjacent to the base of interest and a downstream oligonucleotide with a 5' terminus downstream of the base of interest;

(ii) one or more detectable, sequence-specific terminators; and

(iii) a family X DNA polymerase;

under conditions suitable to permit annealing of the primer and the downstream oligonucleotide to the template nucleic acid flanking the base of interest, thereby creating a gap between the primer and downstream oligonucleotide;

(b) incubating the product of step (a) under conditions suitable to permit sequence specific incorporation of a detectable, sequence-specific terminator into the gap between the primer and the downstream oligonucleotide; and

(c) detecting the product of step (b).

Any detectable, sequence-specific terminator may be used in the present invention. In a preferred embodiment of the invention, the detectable, sequence-specific terminator is a ddNTP. In a further embodiment, the detectable, sequence-specific terminator is a cleavable fluorescent nucleotide analogue.

The gap between the primer and the downstream oligonucleotide is preferably between 1 and 5 bases, such as 1, 2, 3, 4 or 5 bases. The method has been found to work most efficiently when the gap is 1 base; this is specifically recognized by family X DNA polymerases through its 8 kDa domain.

One embodiment of the methods of the present invention is illustrated schematically in Figure 2. The methods of the present invention use a primer oligonucleotide that hybridizes upstream of the nucleotide base of interest and which is extended in the SBE reaction, and a second oligonucleotide that hybridizes downstream of the nucleotide base of interest, forming a 1, 2, 3, 4 or 5-base gapped molecule that is specifically recognized by family X DNA polymerases through its 8 kDa domain.

In a preferred embodiment, the methods of the present invention use a primer oligonucleotide that hybridizes directly upstream of the nucleotide base of interest and which is extended in the SBE reaction, and a second oligonucleotide that hybridizes directly downstream to the nucleotide base of interest, forming a gapped molecule that is specifically recognized by family X DNA polymerases through its 8 kDa domain. 1-base gapped molecules are preferred because the methods using a 1 base gap are more efficient and accurate than methods using 2-5 base gapped nucleotides. The downstream oligonucleotide preferably has a phosphate group at its 5' end to strengthen the interaction between the 8 kDa domain and the downstream oligonucleotide. This is one of the mechanisms through which the 8 kDa domain aids in DNA binding on the downstream end of gapped DNA. In ternary complex structures of ηΡοΙβ (Batra et al, 2001) and ΙιΡοΙλ (Garcia-Diaz et al, 2005), the 5 '-phosphate is bound in a positively charged pocket in the 8 kDa domain. Binding is mediated by multiple hydrogen bonding interactions with basic side chains within the pocket. For ΙιΡοΙμ, the concentration of positively charged residues in this pocket is decreased relative to those in ηΡοΙβ and ΙιΡοΙλ, and there are concomitantly fewer hydrogen bonding interactions to hold this moiety in position (Moon et al, 2007). Therefore, the binding affinity of the different family X polymerases for gapped DNA substrates likely correlates to the strength of their interactions with the 5 '-phosphate.

Moreover, the downstream oligonucleotide lacks a 3' OH group to prevent incorporation of detectable, sequence-specific terminators that could interfere with the signal obtained from the DNA primer incorporating a detectable, sequence-specific terminator, and thus interfering with detection step (c) of the methods of the invention.

Typically, the primer and the downstream oligonucleotide will comprise a region that hybridises with the template nucleic acid over about 12 to 30 bases, e.g. about 15-25 bases. In certain specific embodiments, the primer hybridizes to the template over 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases. In certain specific embodiments, the downstream oligonucleotide hybridizes to the template over 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases.

The primer and the downstream oligonucleotide may also comprise further, non-hybridizing bases at the 3'and/or 5' ends. In particular, the primer may comprise further bases at the 5' end. In one embodiment, the primer comprises a homopolymeric 5' tail, for example a poly C tail, poly G tail, poly T tail or poly A tail. Heteropolymeric tails may also be used. The use of multiple primers of different lengths in the methods of the present invention permits the detection of the identity of more than one base of interest simultaneously. The different molecular weights of the primers permit specific detection of each primer and thus specific identification of each base of interest in the target sequence. The methods of the present invention therefore encompass the simultaneous identification of 1-10 or more, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more, bases of interest in the template nucleic acid using a single reaction.

The primer and the downstream oligonucleotide may be provided at a range of different concentrations in the methods of the invention. Optimal concentrations depend on the detection method used in step (c) of the method. Typically, the primer concentration will be from about 5 nM to about 450 nM concentration in the final reaction mixture in step (a). In specific embodiments using a fluorescent scanner for detection and radiolabeled primers, the primer concentration is about 5 nM to about 160 nM, e.g. about 10 nM to about 80 nM. In this specific embodiment, the primer concentration is about 5 nM, 10 nM, 20 nM, 40 nM or 80 nM. A particularly preferred concentration is 5 nM. Using cold primers and labelled ddNTPs, a typical primer concentration range is 100-300 nM.

Typically, the downstream oligonucleotide will be provided at a final concentration in the reaction mixture of step (a) at about 3 times, e.g. about 2 to 5 times, 3 times, or 4 times the concentration of the primer in order to favour gap formation. The downstream oligonucleotide may therefore be provided at about 15 nM to about 1000 nM. In specific embodiments, the downstream oligonucleotide is provided at about 15 nM to about 500 nM, e.g. about 30 nM to about 250 nM. In certain embodiments, the downstream oligonucleotide is provided at a concentration of about 15 nM, 30 nM, 40 nM, 120 nM, or 240 nM. In a particularly preferred embodiment, the downstream oligonucleotide is provided at a concentration of 15 nM.

In step (b) of the method described above, the detectable, sequence-specific terminator that is complementary to the base of interest in the template nucleic acid is incorporated onto the 3' end of the primer, resulting in an extended primer that is one base longer than the primer added in step (a)(i). In step (c), the presence or absence of the extended primer is detected.

As the person skilled in the art will be aware, the overall primer size will be related to the method of detecting and resolving different primer sequences. For example, where PAGE is used to detect the primers in step (c) of the methods of the invention, the primer and the downstream oligonucleotide will typically be from about 12 to 50 bases in length, e.g. about 15-35 bases in length. In certain specific embodiments, the primer is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases in length. In certain specific embodiments, the downstream oligonucleotide is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 bases in length.

By "conditions suitable to permit annealing of the primer and the downstream oligonucleotide to the template nucleic acid", it is meant any conditions that permit the primer and downstream oligonucleotide to hybridize with the template nucleic acid in a sequence specific manner. The person skilled in the art will be aware that optimal hybridization conditions will vary depending on the sequence of the template and the sequence of the primer and downstream oligonucleotide. Determining such conditions is within the remit of the skilled person, and may involve optimising the temperature of the annealing step and the buffer conditions. As a person skilled in the art will be aware, a double stranded template will need to be melted before the primer and downstream oligonucleotide can anneal to the nucleic acid template. By "conditions suitable to permit sequence specific incorporation of a detectable, sequence- specific terminator into the gap between the primer and the downstream oligonucleotide" it is meant any conditions that permit incorporation of a detectable, sequence-specific terminator into the gap between the primer and the downstream oligonucleotide by the family X DNA polymerase only when that detectable, sequence-specific terminator is complementary to the nucleotide of interest in the template nucleic acid sequence. The term "complement" or "complementary" when used in relation to nucleic acids refers to Watson-Crick base pairing. Thus the complement of C is G, the complement of G is C, the complement of A is T (or U), and the complement of T (or U) is A. It is also possible to use bases such as I (the purine inosine) e.g. to complement pyrimidines (C or T). Exemplary conditions include those described herein, for example, thermal cycling methods and suitable buffers are described herein. Determining further suitable conditions is within the remit of the skilled person, and may involve optimising the thermal cycling timing, temperature and cycle number and the buffer conditions.

Suitable buffers for carrying out steps (a) and (b) of the methods described above are known in the art. In one embodiment, the buffer used in steps (a) and (b) comprises about 20-100 mM Tris-HCl, pH about 7.5, about 0.5-5 mM DTT, about 2-15 mM MgCl₂, about 1-10% glycerol, and optionally about 0.025-0.5 mg/ml BSA. In a specific embodiment, the buffer comprises about 50 mM Tris-HCl, ph7.5, about lmM DTT, about 5 or 10 mM MgCl₂, about 4% glycerol, and about 0.1 mg/ml BSA.

The concentration of the template nucleic acid, the primer, the downstream oligonucleotide, the detectable, sequence-specific terminator and the family X DNA polymerase may also be optimised. One of the advantages of the methods of the present invention as compared to known SBE methods is that they provide reliable results using lower concentrations of template nucleic acid and detectable, sequence-specific terminators, in particular ddNTPs. The methods of the present invention show higher efficiency and specificity than the methods known in the art. In specific embodiments, the methods of the present invention are at least 2-10 times, e.g. at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 times or greater more efficient than the methods of the prior art.

In a specific embodiment of the methods of the invention described above, the one or more ddNTP is provided at < about 50%, 30%, 20%, 10%, 5%, 2.5% or less of the concentration used in the prior art methods. For example, the concentration of the ddNTP is about 5 nM-1 μΜ, e.g. 10 nM-500 nM, 40 nM-250 nM, 100-200 nM, in the final reaction mixture of step (a). In a specific embodiment, the one or more ddNTP is provided at a final concentration of about 40 nM, 50 nM, 75 nM, 100 nM, 125 nM or 150 nM. In certain embodiments, the ddNTPs or other sequence specific terminators are labelled with a detectable label. Any detectable label may be used, including but not limited to a radio label, a fluorescent label, streptavidin, biotin, an immunolabel, a mass label, an enzyme label. Preferred fluorescent labels include Cy3, Cy5, R6G, Rl lO, ROX, TAMRA, and Fluo. Methods to detect these labels are well known in the art (e.g. as reviewed in Lakowicz) and are also described herein.

In one embodiment of the invention, the identity of the nucleotide of interest in the template sequence is determined by conducting four separate reactions, in separate vessels, using the same template nucleic acid. Each individual reaction comprises the use of a single detectable, sequence-specific terminator. In one particular embodiment, each reaction comprises the use of a single ddNTP, i.e. one of ddATP, ddCTP, ddGTP or ddTTP. Thus, where there is a single species of template nucleic acid, only one reaction will give rise to a detectable product, i.e. the extended, 5' labelled primer, in step (c). As will be readily understood by a person skilled in the art, where the nucleotide of interest is A, only the reaction containing ddTTP will give rise to a product in step (c). Where the nucleotide of interest is C, only the reaction containing ddGTP will give rise to a product in step (c). Where the nucleotide of interest is G, only the reaction containing ddCTP will give rise to a product in step (c). Where the nucleotide of interest is T, only the reaction containing ddATP will give rise to a product in step (c).

In a further embodiment of the invention, the identity of the nucleotide of interest in the template sequence is determined by conducting a single reaction, in a single vessel, comprising four detectable, sequence-specific terminators, one complimentary to each of A, C, G and T. In this embodiment, the detectable, sequence-specific terminators are distinguishably labelled. The identity of the nucleotide of interest in the template nucleic acid can be determined by detecting the distinguishable label of the incorporated detectable, sequence-specific terminator in the product of step (c). In a preferred embodiment of this aspect of the invention, four distinguishably labelled ddNTPs are used. An important advantage of a family X DNA polymerase in this embodiment of the present invention, as compared to single base extension methods using a family A DNA polymerase in the art, is the reduction in noise in the electropherogram used to detect the labels on the four ddNTPs (see Figure 23 A, 23B and 23C). In another embodiment of the invention, the identity of the nucleotide base of interest in the template sequence is determined using pyrosequencing. Pyrosequencing (Fakhrai-Rad et al, 2002; Ahmadian et al., 2005; Langaee and Ronaghi, 2005) is a method of DNA sequencing based on the "sequencing by synthesis" principle. It relies on the detection of pyrophosphate release on nucleotide incorporation. "Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced, in the present invention this is the template sequence, and then synthesizing its complementary strand enzymatically. The pyrosequencing method is based on detecting the activity of a DNA polymerase with another chemiluminescent enzyme. Essentially, the method allows sequencing of the base of interest in the template sequence by synthesizing the complementary strand at the base of interest, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of dATPaS, dCTP, dGTP, and dTTP nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the base of interest in the template sequence. The identity of the solution which produces chemiluminescent signal allows the determination of the base of interest in the template sequence.

Single- stranded DNA template sequence is hybridized to the primer and incubated with the following enzymes: family X DNA polymerase as described herein, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5^' phosphosulfate (APS) and luciferin.

The addition of one of the four deoxynucleotide triphosphates (dNTPs) (dATPaS, which is not a substrate for a luciferase, is added instead of dATP) initiates the second step. The family X DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.

ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5^' phosphosulfate. This ATP acts as fuel to the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected and analyzed.

Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.

A further advantage of the methods of the present invention is that they are more sensitive, and can thus be used with a lower concentration of template nucleic acid than the methods of the prior art. The methods of the present invention can be used with a template nucleic acid concentration of < about 75%, 50%, 25%, 10%, 5% or less of the concentration used in the prior art methods. In a specific embodiment of the methods of the invention, the template nucleic acid is provided at about 0.01 to 1 pmol, e.g. about 0.05-0.5 pmol, or about 0.1-0.2 pmol.

The template nucleic acid can be any substantially purified nucleic acid, i.e. polymeric form of nucleotides, of any length and may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. It includes DNA, RNA, DNA/RNA hybrids. It also includes DNA or RNA analogs, such as those containing modified backbones (e.g. peptide nucleic acids (PNAs) or phosphorothioates) or modified bases. Thus the invention includes mRNA, tRNA, rRNA, ribozymes, DNA, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, probes, primers, etc. Where nucleic acid of the invention takes the form of RNA, it may or may not have a 5' cap. The template nucleic acid may be single or double stranded.

In a particular embodiment, the template nucleic acid has previously been amplified by PCR. After PCR amplification, the resulting template is in solution, along with primers, dNTPs, and PCR enzyme and PCR buffer components. To avoid participation in the subsequent primer- extension reaction, primers and unincorporated dNTPs must be removed. One of the possible methods for purifying PCR products is shrimp alkaline phosphatase (SAP) and exonuclease I treatment. Other suitable purification methods are known in the art and include silica-membrane based methods and agarose gel purification.

In a specific embodiment, the template nucleic acid is about 40 to about 150 nucleotides in length, e.g. about 50-125, about 60-80 nucleotides in length. Methods of the present invention preferably involve thermal cycling. In a specific embodiment, the thermal cycling includes the steps of incubating the mixture produced in step (a) of the method described above at a temperature sufficient to melt the nucleic acid duplex of template nucleic acid with its complementary strand or with the primer, extended primer and/or downstream oligonucleotide, followed by a step of incubating the mixture under conditions suitable to permit annealing of the primer and the downstream oligonucleotide to the template nucleic acid, followed by a step of incubating the mixture under conditions suitable to permit sequence specific incorporation of a ddNTP or other sequence-specific terminator into the gap between the primer and the downstream oligonucleotide, said steps being repeated 20-40 times, e.g. 25-35 times. In a further specific embodiment, the thermal cycling comprises the steps of incubating the mixture produced in step (a) of the method described above at about 90-98°C for about 5-30 seconds, about 45-55°C for about 1-10 seconds and about 55-70°C for about 15-60 seconds. In a preferred embodiment, the thermal cycling comprises the steps of incubating the mixture produced in step (a) of the method described above at about 96°C for about 10 seconds, about 50°C for about 5 seconds and about 60°C for about 30 seconds.

The family X DNA polymerase used in the methods of the invention can be any family X DNA polymerase that can catalyse the sequence specific insertion of a sequence specific terminator into a 1-5 base gap in a nucleic acid. In a preferred embodiment, the family X DNA polymerase used in the methods of the invention is a family X DNA polymerase that can catalyse the sequence specific insertion of a ddNTP into a 1 base gap in a nucleic acid In specific embodiments of the methods of the invention, the family X DNA polymerase is a family X DNA polymerase as defined herein in the section entitled "Polypeptides of the Invention".

The invention also provides the use of a family X DNA polymerase as defined herein for determining the identity of a nucleotide base of interest at a specific position in a template nucleic acid.

Polypeptides of the Invention

The invention also provides a thermally stable family X DNA polymerase polypeptide. In a preferred embodiment, the family X DNA polymerase is provided as a substantially isolated polypeptide. The inventors have surprisingly found that thermally stable family X DNA polymerases are particularly useful in SBE reactions, and in particular SBE reactions involving thermal cycling, and are therefore particularly useful in determining the identity of a nucleotide base of interest in a template nucleic acid sequence.

By "thermally stable" it is meant that the family X DNA polymerase has an optimum temperature for maximal enzymatic activity at about 50°C to about 80°C, e.g. about 55-75°C or 60-70°C. In a specific embodiment, the thermally stable family X DNA polymerase is also able to resist temperatures of up to about 80°C, 85°C, 90°C, 9FC, 92°C, 93°C, 94°C, 95°C or greater without substantial loss of the ability of the polypeptide to catalyse a sequence-specific SBE reaction. In a further specific embodiment, the family X DNA polymerase can resist temperatures of up to about 80°C, 85°C, 90°C, 9FC, 92°C, 93°C, 94°C, 95°C or greater for up to about 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes without being inactivated, i.e. without irreversible loss of more than 50% of maximal enzymatic activity.

In a particular embodiment of this aspect of the invention, the thermally stable family X DNA polymerase is from a thermophilic organism, for example thermophilic bacteria or archaea. In one embodiment, the bacterium is of the Thermus genus. In a preferred embodiment, the thermally stable family X DNA polymerase is from Thermus thermophilus. In alternative embodiments, the thermally stable family X DNA polymerase is from (numbers in parentheses are GenBank accession numbers): Thermus aquaticus (ZP 03496261; GI:218295448); Thermoplasma volcanium (NP_111375; GI: 13541687); Methanosarcina mazei (NP 633918; GL21227996); Ferroplasma acidarmanus (ZP_01709777; GI: 126009207); Methanothermobacter thermautotrophicus (NP 275693; GI: 15678578); Thermus scotoductus (ADW21928; GL320150550), Oceanithermus profundus (ADR37134; GI:313153283) and Meiothermus silvanus (ADH63823; GL296850808). In a particular embodiment, the family X DNA polymerase is a polypeptide comprising an amino acid sequence that is at least x% identical to SEQ ID NO: 1 or SEQ ID NO: 2, wherein x is a value selected from 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater.

In specific embodiments, the family X DNA polymerase is a polypeptide that comprises an amino acid sequence that is at least y% identical to SEQ ID NO: 1 or SEQ ID NO: 2, wherein ^ is a value selected from 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, and further comprises asparagine (N) at a position equivalent to 266 in SEQ ID NO: 1. The identity of the position equivalent to 266 in SEQ ID NO: 1 can be determined by aligning the sequence of interest to SEQ ID NO: 1 as described herein (see Figure 9). For example, in the equivalent sequence from Thermus aquaticus, the position equivalent to 266 in SEQ ID NO: 1 is position 266. In the Thermoplasma volcanium sequence, however, it is position 269.

In particular, the thermally stable family X DNA polymerase comprising asparagine at position 266 (numbering according to SEQ ID NO: 1) provides improved insertion efficiency of pyrimidines and improved pyrimidine usage when compared to the wild-type family X DNA polymerase. In certain embodiments, the thermally stable family X DNA polymerase comprising asparagine at position 266 is at least about 2-10 times more efficient, e.g. > about 2, 3, 4, 5, 6, 7, 8 or 9 times more efficient, at inserting a pyrimidine in an appropriate single nucleotide gap when compared to the wild-type family X DNA polymerase.

The present invention also encompasses functionally active fragments of the polypeptides of the invention. The fragments may be at least about 100 amino acids in length, e.g. about 100, 150, 200, 250, 300, 350, 400, 450, 500 amino acids in length. By "functionally active", it is meant that the fragment retains the biological functions of the family X DNA polymerase, i.e. the functionally active fragment is able to catalyse a sequence-specific SBE reaction.

A polypeptide of the invention or functionally active fragment thereof may, compared to SEQ ID NO: 1 or 2, include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid substitutions. A conservative amino acid substitution is defined as the replacement of one amino acid with another which has a related side chain, provided that the amino acid substitution does not affect the biological functions of the family X DNA polymerase, i.e. does not affect the ability of the polypeptide to catalyse a sequence-specific SBE reaction. Genetically-encoded amino acids are generally divided into four families: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine, arginine, histidine; (3) non-polar i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In general, substitution of single amino acids within these families does not have a major effect on the biological activity. Moreover, the polypeptides or functionally active fragments thereof may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to SEQ ID NO: 1 or 2. Furthermore, the polypeptides or functionally active fragments thereof may include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to SEQ ID NO: 1 or 2. The deletions, insertions and substitutions may be at any position that does not interfere with the biological function of the family X DNA polymerase.

The term "polypeptide" refers to amino acid polymers of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Polypeptides can occur as single chains or associated chains. Polypeptides of the invention can be naturally or non-naturally glycosylated (i.e. the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring polypeptide). The polypeptides of the invention may be isolated or purified.

Polypeptides of the invention are preferably provided in purified or substantially purified form i.e. substantially free from other polypeptides (e.g. free from naturally-occurring polypeptides), particularly from other DNA polymerase polypeptides or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure i.e. less than about 50%), and more preferably less than about 10%> (e.g. 5% or less) of a composition is made up of other expressed polypeptides.

Polypeptides of the invention may be attached to a solid support. Polypeptides of the invention may comprise a detectable label (e.g. a radioactive or fluorescent label, or a biotin label).

The invention provides polypeptides comprising a sequence -X-Y- or -Y-X-, wherein: -X- is an amino acid sequence as defined above, i.e. a polypeptide of the invention, and -Y- is not a sequence as defined above i.e. the invention provides fusion proteins.

The invention provides a process for producing polypeptides of the invention, comprising the step of culturing a host cell of to the invention under conditions which induce polypeptide expression. The invention provides a process for producing a polypeptide of the invention, wherein the polypeptide is synthesised in part or in whole using chemical means.

Nucleic acids

The invention also provides nucleic acids encoding a family X DNA polymerase polypeptide of the invention. In a specific embodiment, the family X DNA polymerase encoded by the nucleic acid comprises asparagine (N) at a position equivalent to 266 in SEQ ID NO: 1. In a preferred embodiment, the nucleic acid is an isolated nucleic acid. The invention also provides a nucleic acid comprising nucleotide sequences having sequence identity to such nucleotide sequences. Such nucleic acids include those using alternative codons to encode the same amino acid. As defined above for the template nucleic acids, the nucleic acids of the invention encompass any polymeric form of nucleotides of any length.

In a specific embodiment, the nucleic acid of the invention comprises a nucleotide sequence that is at least z% identical to the nucleic acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, wherein z is a value selected from 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater.

The invention also provides nucleic acid which can hybridize to these nucleic acids. Hybridization reactions can be performed under conditions of different "stringency". Conditions that increase stringency of a hybridization reaction of widely known and published in the art. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25°C, 37°C, 50°C, 55°C and 68°C; buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6 x SSC, 1 x SSC, 0.1 x SSC, or de-ionized water. Hybridization techniques and their optimization are well known in the art (US patent 5,707,829).

The invention includes nucleic acid comprising sequences complementary to these sequences (e.g. for antisense or probing, or for use as primers).

Such antisense, primer and probe sequences may be fragments of the nucleic acids of the invention or the reverse complement thereof. In certain embodiments, the nucleic acid fragments are at least about 15-100 nucleotides in length, e.g. about 20-80, about 25-70, about 20, 25, 30, 35, or 40 nucleotides in length.

Nucleic acid according to the invention can take various forms (e.g. single-stranded, double-stranded, vectors, primers, probes, labelled etc.). Nucleic acids of the invention may be circular or branched, but will generally be linear. Unless otherwise specified or required, any embodiment of the invention that utilizes a nucleic acid may utilize both the double-stranded form and each of two complementary single-stranded forms which make up the double-stranded form. Primers and probes are generally single-stranded, as are antisense nucleic acids.

Nucleic acids of the invention are preferably provided in purified or substantially purified form i.e. substantially free from other nucleic acids {e.g. free from naturally-occurring nucleic acids), particularly from other nucleic acids encoding DNA polymerases or host cell nucleic acids, generally being at least about 50% pure (by weight), and usually at least about 90% pure.

Nucleic acids of the invention may be prepared in many ways e.g. by chemical synthesis {e.g. phosphoramidite synthesis of DNA) in whole or in part, by digesting longer nucleic acids using nucleases {e.g. restriction enzymes), by joining shorter nucleic acids or nucleotides {e.g. using ligases or polymerases), from genomic or cDNA libraries, etc.

Nucleic acid of the invention may be attached to a solid support {e.g. a bead, plate, filter, film, slide, microarray support, resin, etc.). Nucleic acid of the invention may be labelled e.g. with a radioactive or fluorescent label, or a biotin label. This is particularly useful where the nucleic acid is to be used in detection techniques e.g. where the nucleic acid is a primer or as a probe.

Nucleic acids of the invention may be part of a vector i.e. part of a nucleic acid construct designed for transduction/transfection of one or more cell types. Vectors may be, for example, "cloning vectors" which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors" which are designed for expression of a nucleotide sequence in a host cell, "viral vectors" which is designed to result in the production of a recombinant virus or virus-like particle, or "shuttle vectors", which comprise the attributes of more than one type of vector. Preferred vectors are plasmids. A "host cell" includes an individual cell or cell culture which can be or has been a recipient of exogenous nucleic acid. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. Host cells include cells transfected or infected in vivo or in vitro with nucleic acid of the invention.

Where a nucleic acid is DNA, it will be appreciated that "U" in a RNA sequence will be replaced by "T" in the DNA. Similarly, where a nucleic acid is RNA, it will be appreciated that "T" in a DNA sequence will be replaced by "U" in the RNA.

Nucleic acids of the invention can be used, for example: to produce polypeptides in vitro or in vivo; as hybridization probes for the detection of nucleic acid in biological samples; to generate additional copies of the nucleic acids; to generate ribozymes or antisense oligonucleotides; as single-stranded DNA primers or probes; or as triple-strand forming oligonucleotides.

The invention provides a process for producing nucleic acid of the invention, wherein the nucleic acid is synthesised in part or in whole using chemical means.

The invention provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors) and host cells transformed with such vectors.

Applications of the methods, uses, and polypeptides of the invention

The methods, polypeptides and/or uses thereof are for determining the identity of a nucleotide base of interest at a specific position in a template nucleic acid. In particular, the methods and/or polypeptides can be used to genotype a S P. The methods and/or polypeptides can be used to genotype a SNP in a particular species of nucleic acid or population of template nucleic acids.

When the methods and/or polypeptides of the invention are used to identify the frequency of a particular base at a specific position in a template nucleic acid, for example when identifying the frequency of a SNP in a population of template nucleic acids, it is important that the method and/or polypeptide incorporates all ddNTPs with a known and comparable efficiency. The present invention provides an advantage over the prior art methods in this regard. In particular, the thermally stable family X DNA polymerase comprising asparagine (N) at position 266 (numbering according to SEQ ID NO: 1) provides improved insertion efficiency of pyrimidines and improved pyrimidine usage when compared to the wild-type family X DNA polymerase. The methods and/or polypeptides of the invention are particularly useful for determining the identity of a specific base in a template nucleic acid sequence, wherein said specific base is an SNP that plays a role in disease. The present invention is therefore useful in diagnosis of disease, monitoring the progression of disease, presymptomatic disease detection (screening), and therapy selection and optimization. For example, certain alleles or SNPs in specific genes determine an individual's response to therapy. In particular, the SNP may be present in a gene encoding a drug metabolizing enzyme, a membrane transporter or a therapeutic target. Thus, the present invention can be used to identify individuals at risk of aberrant pharmacokinetic or pharmacodymanic effects. The methods of the invention may also be used to identify individuals who would benefit from therapy with a particular drug.

SNPs may also occur in neoplastic disease. The present invention can also be used for detection of acquired or inherited nucleic acid changes in the management of cancer. Predicting response and limiting drug-induced toxicity are two important challenges faced by clinicians in the treatment of cancer. The introduction of genetic testing to individualize treatment regimens based on the methods of the present invention will allow better response prediction and limit drug-induced toxicity leading to improved patient outcomes.

General

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

The term "about" in relation to a numerical value x is optional and means, for example, x+10%.

Sequence alignments can be carried out using the Multalin tool at (http://bioinfo.genopole- toulouse.prd.fr/multalin/multalin.html).

References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids is the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987) Supplement 30. A preferred alignment is determined by the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith- Waterman homology search algorithm is taught in Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.

References to a percentage sequence identity between two nucleic acid sequences mean that, when aligned, that percentage of bases is the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987) Supplement 30. A preferred alignment program is GCG Gap (Genetics Computer Group, Wisconsin, Suite Version 10.1), preferably using default parameters, which are as follows: open gap = 3; extend gap = 1.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Original single-base extension (SBE) method

Figure 2. SBE method of the invention (X-Pol SBE method)

Figure 3. SBE without thermal cycling Figure 4. Optimum temperature for TthPolX DNA polymerase activity and TthPolX resistance to high temperature

Figure 5. 5 '-phosphate increases TthPolX activity on gapped molecules

Figure 6. TthPolX prefers to bind 5'-phosphate gapped molecules

Figure 7. TthPolX DNA polymerase activity on 1 -nucleotide gapped molecules

Figure 8. TthPolX preference for purines in 1 -nucleotide gap-filling reactions

Figure 9. Multiple amino acid sequence alignment of the palm/thumb region of family X

DNA polymerases

Figure 10. View of ΙιΡοΙβ active site complexed with gapped DNA and ddCTP

Figure 11. Alternating position of Asn279 at hPoip active site

Figure 12. TthPolX mutant S266N DNA polymerase activity on 1-nucleotide gapped molecules

Figure 13. Improved nucleotide insertion efficiency and pyrimidine usage by TthPolX mutant S266N Figure 14. Comparison of the nucleotide insertion efficiency between TthPolX wild-type versus mutant S266N

Figure 15. Nucleotide insertion fidelity by TthPolX mutant S266N

Figure 16. X-Pol SBE is more accurate using thermal cycling

Figure 17. X-Pol SBE accuracy on different sequence contexts

Figure 18. X-Pol SBE, TthPolXversus S266N protein mutant

Figure 19. X-Pol SBE is more efficient with 5'P downstream DNA

Figure 20. X-Pol SBE is compatible with fluorescent ddNTPs

Figure 21. X-Pol SBE efficiency increases with oligonucleotide doses

Figure 22. X-Pol SBE method shows higher efficiency than the original one

Figure 23. X-Pol SBE method requires a lower concentration of primers and shows less background noise than ABI Prism® SNaPshot™ Multiplex kit MODES FOR CARRYING OUT THE INVENTION

Cloning of TthPolX

Sequence analysis of the Thermus thermophilus HB8 genome (DDBJ/EMBL/GeneBank AB107660.1; GL29603630) and Thermus thermophilus HB27 genome (DDBJ/EMBL/GeneBank AEO 17221.1; GL46197919) revealed one ORF from each genome, TTHA1150 and TTC0785 respectively, encoding a protein that belongs to the PolX family. Using this sequence information, we synthesized two primers for amplification of the TthPolX gene by PCR from Thermus thermophilus genomic DNA. The gene fragment amplified by PCR using Expand High Fidelity polymerase (Roche) was ligated into the pGEM T-easy vector (Promega) by TA cloning and confirmed by sequencing. Using the Ndel and EcoRI sites, the fragment bearing the target gene was ligated into pET28 vector (Novagen), which allows the expression of recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni²⁺-affinity resins. Site-directed mutations were introduced into TthPolX expression plasmid by a PCR-based method (QuikChange Site-Directed Mutagenesis kit, Stratagene).

Overproduction and purification of TthPolX

Expression of TthPolX was carried out in the Escherichia coli strain BL21-CodonPlus (DE3)- RIL (Stratagene), with extra copies of the argU, ileY, and leuW tRNA genes. Expression of TthPolX was induced by the addition of 1 mM IPTG to 1.5 liters of log phase E. coli cells grown at 30°C in LB to an Abs6oo_nm of 0.5. After induction, cells were incubated at 30°C for 5 h. Subsequently, the cultured cells were harvested, and the pelleted cells were weighted and frozen (-20°C). Just before purification, which was carried out at 4°C, frozen cells (5 gr) were thawed and resuspended in 20 ml buffer A (50 mM Tris-HCl, pH 7.5, 5% glycerol, 0.5 mM EDTA, 1 mM DTT) supplemented with 0.5 M NaCl and protease inhibitors, and then disrupted by sonication on ice. Cell debris was discarded after a 5-min centrifugation at 3000 rpm. Insoluble material was pelleted by a 20-min centrifugation at 11000 rpm. DNA was precipitated with 0.4% polyethyleneimine (10% stock solution in water, pH 7.5) and sedimented by centrifugation for 20 min at 11000 rpm. The supernatant was diluted to a final concentration of 0.25 M NaCl with buffer A and precipitated with ammonium sulphate to 50% saturation to obtain a polyethyleneimine-free protein pellet. This pellet was resuspended in buffer A without EDTA and 30 mM imidazole, and loaded into a HisTrap FTP column (5 ml, GE Healthcare) equilibrated previously in this buffer and 1 M NaCl. After exhaustive washing with buffer A and 1 M NaCl, proteins were eluted with a linear gradient of 30-250 mM imidazole. The eluate containing TthPolX was diluted with buffer A to a final 0.1 M NaCl concentration, and loaded into a monoS 4.6/100 PE column (1.7 ml, GE Healthcare), equilibrated previously in buffer A and 0.1 M NaCl. The column was washed, and the protein eluted with a linear gradient of 0.1-0.5 M NaCl. Fractions containing TthPolX were pooled, diluted to 0.2 M NaCl and loaded into a HiTrap Heparin HP column (5 ml, GE Healthcare), equilibrated previously in the same buffer. The column was washed, and the protein eluted with a linear gradient of 0.2-0.5 M NaCl. Fractions containing TthPolX were pooled, diluted again to 0.2 M NaCl and loaded into the same column, equilibrated previously in buffer A and 0.2 M NaCl. TthPolX was eluted with buffer A with 1 M NaCl. This fraction contains highly purified (>99%) TthPolX. Protein concentration was estimated by densitometry of Coomassie Blue-stained 10% SDS-polyacrylamide gels, using standards of known concentration. The final fraction, adjusted to 50% (v/v) glycerol, was stored at -70°C. The same protocol was used to purify the protein mutant S266N.

Overproduction and purification ofhPoi and hPolX

ηΡοΙβ and ηΡοΙλ proteins were overexpressed and purified to homogeneity essentially as previously described (Abbotts et al, 1988; Garcia-Diaz et al, 2002). DNA substrates

Synthetic oligonucleotides purified by PAGE were obtained from Sigma. 1 -nucleotide gapped molecules were generated by annealing PI primer (5' GATCACAGTGAGTAC - SEQ ID NO: 5) to four T13 templates (5' AGAAGTGTATCTCNTACTCACTGTGATC where N is A, C, G or T - SEQ ID NO: 6) and to downstream oligonucleotide DG1 (5' AGATACACTTCT - SEQ ID NO: 7) or DG1P (DG1 with a 5'-phosphate group). PI primer was fluorescently (Cy5) or radioactively (³²P) labelled at its 5 '-end. The primer was hybridized to template and downstream oligonucleotides to generate different gapped molecules in the presence of 50 mM Tris-HCl, pH 7.5, and 0.3 M NaCl and heating to 80°C for 10 min before slowly cooling to room temperature over night. DNA polymerization assays on activated DNA

The standard assay contained, in 25 μΐ, 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 13.3 nM [a-³²P] dATP, and 625 ng of activated calf thymus DNA. After incubation, reactions were stopped by adding 10 mM EDTA, and the samples were filtered through Sephadex G-50 spin columns. Polymerization activity was proportional to the amount of radioactivity present in the excluded volume, determined by counting Cerenkov radiation. For determining the optimum temperature for TthPolX activity, reactions were initiated by adding 50 nM TthPolX and incubated for 20 min at the indicated temperatures. For determining TthPolX resistance to high temperatures, reactions were initiated by adding 100 nM TthPolX and preincubated the indicated times at 90°C. After that, reactions were incubated for 10 min at 65°C.

DNA polymerization assays on l-nucleotide gapped molecules

The incubation mixture contained, in 20 μΐ, 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 5 nM of the DNA hybrid indicated in each case, 40 nM TthPolX wild-type or mutant S266N, and the indicated concentration of each dNTP. Reaction mixtures were incubated for 10 min at 37°C and stopped by adding 10 μΐ of stop solution (10 mM EDTA and 97.5% deionized formamide). Extension of the labelled primer strand was analyzed by 8 M urea and 20% PAGE, and visualized using a Typhoon 9410 scanner (GE Healthcare).

Electrophoretic mobility shift assay (EMSA) analysis

To analyze Jt/zPolX/DNA interactions, different 5'P-labelled DNA polymerization substrates (5 nM) were incubated with various concentrations of TthPolX for 10 min at 37°C, in 12.5 μΐ of 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 4% glycerol and 0.1 mg/ml BSA, and then, the incubation mixture was adjusted to 10% glycerol and loaded in 4% native polyacrylamide gels in 5x TAE and 50 mM EDTA (pH 7.5). After electrophoresis at 180 mA/4°C, the protein-DNA complexes, producing a shift in the position of the labelled free DNA, were detected by autoradiography.

Graphic representation of the insertion efficiency for each of the four dNTPs by TthPolX wild-type and mutant S266N

Gel band intensities were quantified using a Typhoon 9410 scanner and ImageQuant TL software (GE Healthcare). The observed rate of nucleotide incorporation (inferred from the amount of extended primer) was then plotted as a function of nucleotide concentration. The values plotted are the mean of at least three independent experiments.

On Figure 13, the value that represents the difference in catalytic efficiency between S266N mutant and wild-type enzyme (highlighted in green) was calculated as an average of the ratio of S266N extension product to wild-type extension product for each dNTP concentration.

Multiple amino acid sequence alignment of the palm/thumb subdomain region of human/bacterial/archaeal family X DNA polymerases

Numbers indicate the amino acid position relative to the Ή-terminus of each DNA polymerase. Because of the large number of sequences, only selected representatives from the Eubacteria and Archea genera are aligned with human family X DNA polymerases. Names of organisms are abbreviated as follows (numbers in parentheses are GenBank accession numbers): Tth, Thermus thermophilus (YP 144416); Taq, Thermus aquaticus (ZP 03496261); Bsu, Bacillus subtilis ( P_390737); Lmo, Lysteria monocytogenes (YP_013839); Ssa, Staphylococcus saprolyticus (YP 301742); Sau, Staphylococcus aureus (YP 001246578); Dre, Desulfotomaculum reducens (YP_001112987); Aae, Aquifex aeolicus ( P_213981); Tde, Thiobacillus denitrificans (AAZ97399); Dra, Deinococcus radiodurans ( P 294190); vo, Thermoplasma volcanium ( P_111375); Mma, Methanosarcina mazei ( P_633918); Tac, Ferroplasma acidarmanus (ZP_01709777); Mth, Methanothermobacter thermautotrophicus ( P_275693). Conserved residues are indicated with red (>85% of the aligned polymerases), blue (85-50% of the sequences), and green (50-25% of the sequences) letters. Alignment was made by using the Multalin tool (http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html) and further adjusted by hand. An asterisk indicates the residue that has been mutated in Tt PolX (Figure 9).

3D-structural analysis and predictions of specific polymerase residues

To study the potential interactions of the conserved asparagine at position 279 of ηΡοΙβ (see Figure 9), we use crystal structures of ΙιΡοΙβ complexed with a 1 -nucleotide gapped-DNA and the complementary incoming nucleotide (PDB: 1BPY and 2FMP).

To prepare Figure 10 only residues aligned on Figure 9 (255-296) were selected. Electrostatic potential of the molecular surface is shown in blue (positive) and red (negative). The molecular surface of ΙιΡοΙβ Asn279 is shown in yellow. The template, downstream and primer strands are shown in gray and the incoming ddNTP is shown in light blue.

On Figure 11 only primer-terminus, templating base and the incoming nucleotide are shown to study alternate positions of Asn279 at ηΡοΙβ active site. Atoms are coloured following CPK colour scheme. Hydrogen bonds are shown as dashed lines.

Figures 10 and 11 were created using the Swiss PDB Viewer program

PCR template for single-base extension reaction

S P-containing fragments were obtained by specific PCR amplification. The fragments have lengths ranging from 66 to 79 base pairs. PCR amplification fragments were treated with shrimp alkaline phosphatase (SAP) and exonuclease I to eliminate dNTPs and PCR primers, prior to use at single-base extension.

Single-base extension (SBE) reaction

The standard assay contained, in 20 μΐ, 50 mM Tris-HCl pH 7.5, 5 or 10 mM MgCl₂, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 5 nM of the fluorescently labelled primer oligonucleotide, 15 nM of the downstream oligonucleotide, the amplification fragment indicated in each case, 50 nM Tt PoXX or 100 nM ΙιΡοΙλ/ηΡοΙβ, and 50 nM of each of the four ddNTPs. Reactions conducted without thermal cycling were incubated for 30 min at 37°C, and oligonucleotides were previously hybridized to template DNA by heating to 90°C for 5 min before slowly cooling to room temperature over night in the presence of 50 mM Tris-HCl, pH 7.5, and 0.3 M NaCl. Reactions conducted with thermal cycling followed these steps: 96°C for 10 seconds, 50°C for 5 seconds and 60°C for 30 seconds. This cycle was repeated 25 times for each reaction. Reaction mixtures were stopped by adding 10 μΐ of stop solution (10 mM EDTA and 97.5% deionized formamide). Extension of the labelled primer strand was analyzed by 8 M urea and 20% PAGE, and visualized using a Typhoon 9410 scanner (GE Healthcare).

Comparison between X-Pol SBE method and ABI PRISM® SNaPshot^lM Multiplex Kit from Applied Biosystems.

SBE reactions were performed in parallel, using either SNaPshot™ reagents or a mixture containing: 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 0.1 μΜ R1 10-ddGTP/R6G-ddATP, 1 μΜ TAMRA-ddCTP/ROX-ddUTP, 50 nM TthFolX S266N and the indicated amounts of upstream and downstream primers. All reactions included 1 μΐ of cleaned PCR product as template DNA. Reactions were conducted with thermal cycling following these steps: 96°C for 10 seconds, 50°C for 5 seconds and 60°C for 30 seconds. This cycle was repeated 25 times for each reaction. After SBE reaction, samples were run on a capillary electrophoresis instrument and the fluorescent nucleotides inserted in each case were detected.

X-Pol single-base extension method for genotyping

Single-base extension (SBE) is a method for determining the identity of a nucleotide base at a specific position along a nucleic acid. For instance, the method is widely used to identify single- nucleotide polymorphisms (SNPs). In the original method (Goelet et al, 1999; represented on Figure 1), an oligonucleotide primer hybridizes to a complementary region along the nucleic acid, to form a duplex, with the primer' s terminal 3 ' end directly adjacent to the nucleotide base to be identified. The oligonucleotide primer is extended a single base by a DNA polymerase with a nucleotide terminator complementary to the nucleotide being identified (e.g. ddNTP). The terminator prevents additional nucleotides from being incorporated.

Many different approaches can be taken for determining the identity of a terminator, including fluorescence labelling, mass labelling for mass spectrometry, measuring enzyme activity using a protein moiety, and isotope labelling. Commonly, the target region is previously amplified by PCR followed by the SBE reaction. After PCR amplification, the resulting template is in solution, along with primers, dNTPs, and enzyme and buffer components. To avoid participation in the subsequent primer-extension reaction, primers and unincorporated dNTPs must be removed. One of the possible methods for purifying PCR products is shrimp alkaline phosphatase (SAP) and exonuclease I treatment.

The novelty of X-Pol SBE method described here (Figure 2), resides in the use of a second oligonucleotide that hybridizes directly downstream to the nucleotide base to be identified, forming a 1 -nucleotide gapped molecule that is specifically recognized by family X DNA polymerases through its 8 kDa domain. This downstream oligonucleotide has a phosphate group at its 5' end to strengthen the interaction between the 8 kDa domain and the downstream oligonucleotide. This is one of the mechanisms through which the 8 kDa domain aids in DNA binding on the downstream end of gapped DNA. In ternary complex structures of ΙιΡοΙβ (Batra et al, 2001) and hPo (Garcia-Diaz et al, 2005), the 5'-phosphate is bound in a positively charged pocked in the 8 kDa domain. Binding is mediated by multiple hydrogen bonding interactions with basic side chains within the pocket. For ΙιΡοΙμ, the concentration of positively charged residues in this pocket is decreased relative to those in ΙιΡοΙβ and ηΡοΙλ, and there are concomitantly fewer hydrogen bonding interactions to hold this moiety in position (Moon et al, 2007). Therefore, the binding affinity of the different family X polymerases for gapped DNA substrates likely correlates to the strength of their interactions with the 5'-phosphate.

Moreover, downstream oligonucleotide lacks a 3 ' OH group to prevent incorporation of labelled nucleotides that could interfere with the signal obtained by the DNA primer, the latter being critical to identify the base of interest.

SBE requires thermal cycling for an efficient genotyping

In order to check the viability and efficiency of this new SBE method, we performed several SBE reactions to genotype the SNPs listed on Table 1 using different genomic DNA samples.

For this purpose, we chose ΙιΡοΙλ and ηΡοΙβ as the DNA polymerases for the reaction, due to their strong 5 '-phosphate binding and high-fidelity polymerization activity (low error rate). However, hPo and ηΡοΙβ were not able to "read" the SNPs tested in each case, as none of the primers showed relevant extension with complementary ddNTPs under different assay conditions (Figure 3).

Template DNA for SBE reactions is duplex DNA (two complementary strands), as it is generated through PCR amplification. Consequently, primer and downstream oligonucleotides for SBE reaction compete for the template strand with the complementary one. Moreover, the oligonucleotides length (;¾15-20 nts) is much shorter than the complemenatery strand (;¾60-79 nts). Thus, during previous hybridization step (90°C for 5 min and slow cooling to RT), the complementary strand probably expels primer and downstream oligonucleotides due to its higher affinity to template strand. This fact probably prevents ηΡοΙλ and ηΡοΙβ polymerization during SBE reaction.

The fact that the original SBE method is currently performed with thermal cycling, together with the results obtained using ΙιΡοΙλ and ηΡοΙβ without thermal cycling, indicates that the use of thermal cycling during SBE reactions is essential. As a result, we looked for a new DNA polymerase that would have family X features and thermostability, in order to be employed in the SBE method described in this report.

Table 1

Thermus thermophilus DNA polymerase X for SBE

The recent completion of many bacterial genome sequences revealed the presence of genes encoding polymerases that belong to family X. Sequence analysis of bacterial PolXs revealed that they are structurally organized in two differentiated domains, the universal Ροΐβ-like core and a C-terminal PHP (Ramadan et al, 2004).

Contrary to the case of mammalian PolXs, the study of the cellular and molecular functions of bacterial PolXs has made little progress. A recent study revealed that PolX from the heat-stable organism Thermus thermophilus (JtAPolX) has DNA/RNA polymerase activity and Mn²⁺- dependent exonuclease activity (Nakane et al, 2009). On the basis of these results, we chose Tt PolX as the candidate DNA polymerase for the new SBE method described in this report.

Once we cloned, overexpressed in E. coli and purified to homogeneity Tt PolX (see Materials and Methods), we checked if the enzyme had the X family polymerization properties necessary for this new SBE method: specific recognition and high-fidelity polymerization (low error insertion rate) on 5 '-phosphate gapped DNA molecules. Moreover, the enzyme should be thermostable to perform DNA synthesis under thermal cycling conditions.

Firstly, we used calf thymus activated DNA as substrate to test the optimum temperature for TthPolX polymerization activity. As shown in Figure 4, TthPolX showed its maximum activity when the assay temperatures were from 60 to 70°C. Moreover, TthPolX was able to resist at 90°C for several minutes before being inactivated. These results allow TthPolX to be employed in SBE reactions with thermal cycling, as it can resist the denaturing steps and polymerize at high temperatures after the annealing step.

As shown in Figure 5, TthPolX shows a significant increase in the polymerization capacity when a phosphate group is present at the 5 '-side of the gap, compared with the same molecule having a hydroxyl group at the 5 '-end of the gap.

In ηΡοΙλ and ΙιΡοΙβ a 5 '-phosphate-dependent increase in processivity is structurally and functionally related to the presence of the N-terminal 8 kDa domain (Sawaya et al, 1994; Prasad et al, 1994; Hubscher et al, 2002; Garcia-Diaz et al, 2004). Since TthPolX also contains a 8 kDa domain, and is stimulated by the presence of a 5 '-phosphate group in the 1 -nucleotide gapped substrate, we tested if this stimulation was primarily due to differences in the DNA-binding capacity, a step preceding dNTP binding and catalysis. The formation of stable Jt/zPolX/DNA complexes, assessed by EMS A, was higher at all enzyme concentrations tested when the 1- nucleotide gapped DNA had a 5'-phosphate group (Figure 6).

These results demonstrate that TthPolX comprises all the properties necessary to accomplish the new SBE extension method with thermal cycling.

TthPolX shows higher dNTP insertion efficiencies when the incoming dNTP is a purine

Once we showed that TthPolX could be a good candidate for SBE, we studied dNTP insertion efficiency for each of the four possible dNTPs on 1-nucleotide gapped DNA molecules with 5 '- phosphate.

Figure 7 shows the dNTP insertion efficiency of TthPolX for each of the four dNTPs. When the incoming dNTP was a purine (dATP or dGTP), the reaction efficiency was higher than when the dNTP was a pyrimidine (dCTP or dTTP). This difference was investigated using defined DNA molecules and single turnover conditions, where the enzyme concentration is higher than the concentration of DNA. Single turnover analysis of dNTP incorporation clearly revealed that TthPolX shows strong preference for purines. Quantification of the incorporation efficiency of each complementary dNTP demonstrates a strong imbalance in correct dNTP incorporation with a strong preference for purines: dG»dA»dT>dC (Figure 8). Thus, when the incoming nucleotide is a pyrimidine (dCTP or dTTP), the catalytic efficiency decreases dramatically. Serine 266 of TthPolX could be responsible for the low dNTP insertion efficiency when the incoming dNTP is a pyrimidine

Multiple amino acid sequence alignment of the palm/thumb subdomain region of human/bacterial/archaeal family X DNA polymerases (see Materials and Methods) was performed to identify residues that could be responsible for the different catalytic efficiency showed by TthPoXX when using purines versus pyrimidines as the incoming dNTP (Figure 9). This alignment revealed that TthPolX has a serine at position 266 (indicated with an asterisk) when most of the polymerases have an asparagine at the equivalent position. This difference could provide a structural basis (further discussed later) for the decrease in the catalytic efficiency when the incoming dNTP is a pyrimidine. Asparagine 279 ofhPolp interacts with templating base and incoming nucleotide

The spatial location of ηΡοΙβ Asn279 would permit its interaction with both templating base and incoming nucleotide (Figure 10). This fact would imply a role of Asn279 in the formation of the correct base pair between the templating base and the incoming nucleotide. Moreover, previous studies showed its critical role in discriminating between correct and incorrect dNTPs (Kraynov et al, 1997).

The side chain of ΙιΡοΙβ Asn279 can interact with both templating base and incoming nucleotide. In fact, at the PDB entry 2FMP, the gamma carbon of Asn279 side chain is rotated 180° and the NH₂ group forms a hydrogen bond with the O group from the incoming nucleotide (Figure 11).

Most family X polymerases (see Figure 9) have also an asparagine at the equivalent position of ηΡοΙβ Asn279. This conservation from bacteria to humans supports the important role of this residue during catalysis. Conversely, the serine present at this position in TthPoXX would not be able to produce the same interactions with the nascent base pair, due to different distances and reactive groups present in the side chain (Ser: OH; Asn: O and NH₂). Thus, Ser266 from TthPolX could never form a hydrogen bond with the incoming nucleotide like the one formed by ηΡοΙβ Asn279 (Figure 11). TthPolX mutant S266N shows higher dNTP insertion efficiency than wild-type enzyme

In order to check if TthPoYX Ser266 is the residue responsible for the different insertion efficiency between purines and pyrimidines, we mutated Ser266 to Asn by site-directed mutagenesis, restoring the consensus of family X polymerases.

TthPolX mutant S266N shows higher dNTP insertion efficiency than wild-type enzyme under linear and saturated conditions with the four possible dNTPs (Figure 12). This increase is bigger when the incoming dNTP is a pyrimidine (dCTP or dTTP).

Quantification of the incorporation efficiency of each complementary dNTP demonstrates that the TthPolX mutant S266N inserts the four dNTPs onto DNA with higher efficiency than the wild-type enzyme (Figure 13). The improvement was bigger when the incoming dNTP was a pyrimidine (3.4-fold for dCTP, and 5.1-fold for dTTP), while the values for purines just increased 1.5-fold for dATP and 2-fold for dGTP.

As a summary, Figure 14 shows a comparison of the nucleotide insertion efficiency of the TthPolX S266N mutant versus the wild-type enzyme. We can conclude that the substitution of the original Ser266 of TthPolX to the conserved residue present in most family X polymerases improves overall catalytic efficiency. However, the improvement is bigger when the incoming nucleotide is a pyrimidine. When using dTTP, the efficiency is even superior in comparison to dATP insertion. As a result of this, the nucleotide insertion efficiency of the TthPolX mutant S266N is not only higher, but also more equilibrated between purines and pyrimidines. TthPolX S266N preferentially incorporates complementary nucleotides

As an analysis of the capacity of TthPoYX mutant S266N to catalyze faithful DNA synthesis, each of the four dNTPs was assayed individually as a substrate to be incorporated opposite the four possible templating bases. Figure 15 shows that in all cases, mutant S266N preferentially inserted the correct dNTP, and not the incorrect ones (even at a 1000-fold higher concentration). Thus, TthPoYX S266N performs DNA synthesis following the Watson-Crick base pairing rules.

TthPolX S266N is capable of performing SBE reactions efficiently and accurately

Figure 16 shows the great capacity of TthPoYX S266N for genotyping different SNPs following X-Pol new SBE method with thermal cycling, in comparison with the results obtained by hPo and ηΡοΙβ without thermal cycling. Samples were previously genotyped by ABI PRISM® SNaPshot™ Multiplex Kit (Applied Biosy stems), and the results obtained with both methods matched completely, supporting the high-fidelity of TthPoYX S266N. Moreover, as shown in Figure 17, only the correct ddNTP is inserted on primer's terminal 3 '-end in each case. Figure 18 compares the SBE efficiency of TthPolX wild-type versus mutant S266N. As in the results previously obtained on defined molecules (Figures 12 to 14), mutant S266N also showed higher SBE efficiency than TthPolX wild-type in all the sequence contexts analyzed (S Ps A to E from Table I). TthPolX S266N completely extended the primers in four of the five reactions (SNPs A, B, C and E), while TthPolX wild-type just performed efficient SBE reactions on 3 cases (SNPs A, C and E). Remarkably, TthPolX wild-type was not able to genotype SNP D under the same conditions, probably because it required the insertion of a pyrimidine (ddCTP).

Figure 19 shows the crucial role of the phosphate group (P) present at the 5 '-side of the gap during SBE reaction. When the 5 '-phosphate group is replaced with an OH residue, the efficiency of the reaction is significantly decreased in all cases analyzed. Moreover, if downstream oligonucleotide is absent (T/P), TthPolX S266N is not able to insert the correct ddNTP under the same assay conditions. This assay remarks the essential role of downstream oligonucleotide during SBE reaction using TthPolX.

Figure 20 shows the ability of TthPolX S266N to perform SBE with fluorescent ddNTPs on different sequence contexts. TthPolX completely extended all primers with the complementary fluorescent ddNTP, showing the same efficiency as when using cold ddNTPs.

The ability to perform SBE with fluorescent ddNTPs opens the possibility to change the oligonucleotide concentration in the reaction, as primers can be unlabelled. Figure 21 shows how the signal emitted by the extended primers, labelled by the addition of one fluorescent ddNTP, increases with primer doses. This fact would allow increasing SBE sensitivity when the assay conditions were suboptimal.

Finally, we compared the efficiency and accuracy of the original SBE method with the new X- Pol SBE method presented in this report. The original method with just one oligonucleotide (primer) and the enzyme Ampli Taq Gold (Applied Biosciences), required higher ddNTPs (10- fold) and template DNA (2-fold) concentrations to obtain lower extension levels than TthPolX S266N with two oligonucleotides (Figure 22).

X-Pol SBE method for genotyping shows better performance than prior systems available in the market

In order to test the improved features of X-Pol SBE method for genotyping, we performed several SBE reactions in parallel, either using X-Pol SBE method or ABI PRISM® SNaPshot™ Multiplex Kit from Applied Biosystems.

In singleplex reactions, using the same PCR products, we could observe an increase in the electropherogram signal and genotyping efficiency relative to genotyping primer concentration when using the X-Pol system, compared to the SNaPshot™ system (Figure 23). For instance, peaks similar to those obtained with 50 nM upstream primer in the SNaPshot™ kit were obtained with only 5/15nM upstream/downstream primers with X-Pol method (Figures 23 A and 23B). Thus, the use of the expensive, fluorochrome-labelled upstream primers is reduced tenfold.

In multiplex reactions, the concentrations of primers for the different SNPs have to be carefully adjusted, regardless of the genotyping kit used.

With the control DNA samples used, purified from peripheral blood, both singleplex and multiplex genotyping reactions showed 100% correlation when performed with either SNaPshot™ or X-Pol system.

An important advantage of the X-Pol system is the reduction in noise in the electropherogram. The much cleaner baseline obtained with X-Pol method is beneficial in general, allowing a better signal to noise ratio, thus enhancing the sensitivity of the assay (Figures 23B and C). In addition, the quality of the genotyping improves, particularly when multiple SNPs are heterozygous in a sample, or when the ratio between the two alleles of an SNP is significantly different from 1. This is the case, for instance, in the study of tumour DNA, where somatic mutations result in various percentages of mutated alleles over the wild-type sequence. Moreover, the cleaner profile becomes very relevant when using suboptimal DNA samples, such as those obtained from paraffin-embedded tumour tissues.

The results presented herein identify the essential features of X-Pol SBE method: the presence of a downstream oligonucleotide, and a family X DNA polymerase (e.g. Tt PolX S266N) that specifically recognizes the 1-base gapped molecules generated and efficiently fills the gap with the complementary ddNTP, regardless of the ddNTP type (purine or pyrimidine).

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Claims

CLAIMS:

1. A method for determining the identity of a nucleotide base of interest at a specific position in a template nucleic acid comprising the steps of:

(a) contacting a template nucleic acid with:

(i) a primer with a 3' terminus adjacent to the base of interest and a downstream oligonucleotide with a 5' terminus downstream of the base of interest;

(ii) one or more detectable, sequence-specific terminators; and

(iii) a family X DNA polymerase;

under conditions suitable to permit annealing of the primer and the downstream oligonucleotide to the template nucleic acid flanking the base of interest, thereby creating a gap between the primer and downstream oligonucleotide;

(b) incubating the product of step (a) under conditions suitable to permit sequence specific incorporation of a detectable sequence specific terminator into the gap between the primer and the downstream oligonucleotide; and

(c) detecting the product of step (b).

2. A method according to claim 1, wherein the gap is a single base gap.

3. A method according to claim 1 or claim 2, wherein the detectable sequence-specific terminator is a ddNTP.

4. A method according to any one of claims 1 to 3, wherein step (b) comprises thermal cycling.

5. A method according to clam 4, wherein the thermal cycling comprises the steps of incubating the product of step (a) at:

(A) about 96°C for about 10 seconds;

(B) about 50°C for about 5 seconds; then

(C) about 60°C for about 30 seconds;

and repeating steps (A) to (C) 20-34 times.

6. A method according to any one of claims 1 to 5, wherein the downstream oligonucleotide comprises a 5' phosphate moiety.

7. A method according to any one of claims 3 to 5, wherein the one or more ddNTPs are detectably labelled.

8. A method according to any one of claims 3 to 7, wherein the one or more ddNTP is one of ddATP, ddCTP, ddGTP or ddTTP.

9. A method according to any one of claims 3 to 7, wherein the one or more ddNTP is all of ddATP, ddCTP, ddGTP or ddTTP, and wherein each of ddATP, ddCTP, ddGTP or ddTTP are distinguishably labelled.

10. A method according to any one of claims 7 to 9, wherein the detectable label is a radio label, a fluorescent label, streptavidin, biotin, an immunolabel, a mass label, or an enzyme label.

11. The use of a family X DNA polymerase for determining the identity of a nucleotide base of interest at a specific position in a template nucleic acid.

12. A method or use according to any one of claims 1 to 11, wherein the family X DNA polymerase is thermally stable.

13. A method or use according to claim 11, wherein the family X DNA polymerase is from Thermus thermophilus.

14. A method or use according to claim 11 or 13, wherein the family X DNA polymerase is a polypeptide comprising an amino acid sequence that is at least 60% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

15. A method or use according to claim 14, wherein the family X DNA polymerase comprises asparagine at position 266 (numbering according to SEQ ID NO: 1).

16. A method or use according to claim 14, wherein the family X DNA polymerase comprises a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

17. A thermally stable family X DNA polymerase.

18. A thermally stable family X DNA polymerase according to claim 17, wherein the thermally stable family X DNA polymerase is from Thermus thermophilus

19. A family X DNA polymerase according to claim 17 or claim 18, wherein said family X DNA polymerase is a polypeptide comprising an amino acid sequence that is at least 60% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

20. A family X DNA polymerase according to claim 19 comprising asparagine at position 266 (numbering according to SEQ ID NO: 1).

21. A family X DNA polymerase according to claim 19 comprising a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

22. A nucleic acid encoding a family X DNA polymerase of any one of claims 17 to 21.

23. A kit for determining the identity of a nucleotide base at a specific position in a template nucleic acid comprising a family X DNA polymerase according to any one of claims 17 to 21.

24. The kit of claim 23, further comprising sequence specific terminators, and reagents for carrying out the methods of claims 1 to 16.

25. A method or use according to any one of claims 1 to 16, or kit of claim 23 or claim 24, wherein nucleotide base of interest in the template nucleic acid is a single nucleotide polymorphism (SNP).