GB2432365A

GB2432365A - A nucleic acid molecule for detecting polymorphisms in the UGT1A1 promoter

Info

Publication number: GB2432365A
Application number: GB0523544A
Authority: GB
Inventors: David Mark Whitcombe
Original assignee: DXS Ltd
Current assignee: DXS Ltd
Priority date: 2005-11-18
Filing date: 2005-11-18
Publication date: 2007-05-23
Also published as: GB0523544D0

Abstract

A nucleic acid molecule is disclosed which is used to detect alleles of the TA promoter region of the UGT1A1 gene. The nucleic acid molecule comprises a primer sequence comprising a sequence from the UGT1A1 gene and a probe sequence comprising a sequence the reverse of and complementary to the TA promoter of the UGT1A1 gene. The molecule also comprises first and second stem sequences which flank the probe sequence and which are complementary to each other. The nucleic acid molecule further comprises first and second fluorescent interacting moieties which are adjacent to the first and second stem sequences, respectively. Also disclosed is a method of administering irinotecan by detecting the presence of the UGT1A1*28 allele using the nucleic acid molecule of the invention. The nucleic acid molecule is based upon the Scorpions<TM> technology.

Description

A NUCLEIC ACID MOLECULE

TECHNICAL FIELD

The present invention relates to a nucleic acid molecule that can be used in the detection of the UGT1A1*28 allele and to a method of detecting the UGTIAI*28 allele using the nucleic acid molecule. The present invention also relates to a method of administering the cancer treatment Irinotecan.

BACKGROUND ART

UDP-glucuronosyl enzymes (UGTs) comprise a superfamily of important proteins that catalyse the glucuronidation of a variety of chemicals both endogenous and exogenous. The enzymes are membrane bound and catalyse the transfer of a glucuronic acid group from uridine diphosphoglucuronic acid to a functional group (eg hydroxyl, carboxyl amino etc) of the target compound. The conjugated compound thereby becomes more polar which facilitates their excretion in bile or urine.

In addition to their role in drug metabolism, these enzymes have two other biological functions: 1. protection against toxic compounds from cigarette smoke, dietary components and other environmental pollutants 2. metabolic control of a number of endogenous molecules in the body, such as bilirubin, fatty acids and both steroid and thyroid hormones.

UGT1A1 (see nucleotides 201 to 13227 of SEQ. ID NO: 9) encodes the major enzyme that catalyses the glucuronidation of bilirubin (a breakdown product of haem.) Serious mutations in this gene have been identified as causing two severe familial disorders known as Crigler-Najjar Type 1 and 2. These disorders are characterised by high levels of unconjugated bilirubins. Neither of these syndromes is particularly prevalent although the consequences may be serious (liver damage etc).

Gilberts Syndrome In addition, a milder but more common UGTIAI deficiency, Gilberts Syndrome, has been described. This has been found to be associated in large part with a variant of the promoter (see nucleotides 132 to 491 of SEQ. ID NO: 9) region upstream of the UGTIAI gene.

Specifically, a portion of the promoter region has the sequence A(TA)TAA, where n can vary between 5 and 8 (see nucleotides 132 to 174 of SEQ. ID NO: 9). This is referred to as the "TA promoter region" or "variable region". In the majority of cases n=6, but in a substantial minority of cases n=7. The larger the number of repeats, the lower the level of gene expression, see Table 1.

Table 1: Effect of Promoter Repeats on UGT1A1 Gene Expression Number of Expression Repeats -20% 6 100% 7 -70% 8 -50% These variants have a range of prevalence in different ethnic populations. See Table 2 (based on Guillemette).

Table 2: Allele frequencies in different populations (based on Guillemette) UGTI Al Allele frequency Genotype frequency (%) Allele *34 *1 *28* *33 *1/*1 *11*28 *28/*28 name Repeats 5 6 7 8 6/6 6/7 7/7 Number Population of Subjects Caucasian 77 -0.64 0.36 -40 48 12 (Scottish) Caucasian 71 0 0.613 0.387 0 34 55 11 (European) Caucasian 202 0.002 0. 698 0.295 0.005 51 37 11 (American) Caucasian 88 0 0.715 0.285 0 75 23 2 Caucasian 603 0.001 0.68 0.32 0.001 17 73 10 (American) Caucasian 71 0. 007 0.662 0.324 0.007 45 39 13 Caucasian -0.693 0.307 -46 47 7 (European) Inuit 88 -0.585 0.415 -34 49 17 (Canadian) Asian 47 0 0.84 0.16 0 70 28 2 (American) Asian 30 0 0.867 0.133 0 73 27 0 Asian 90 0 0.855 0.145 0 74 23 3 Asian 27 -0.981 0.019 -96 4 0 (Japanese) African 0.02 0.52 0.38 0.08 28 36 17 (American) African 101 0.069 0.47 0.426 0.035 26 37 19 (American) African (Sub-76 0.043 0.45 0.427 0.08 18 41 17 Saharan) African 54 0.009 0.519 0.407 0.065 24 46 17 (American) Italian 98 -0.64 0.36 -44 40 16 Amerindian 81 0 0.85 0.15 0 74 22 4 Parakana 32 0 0.672 0.328 0 38 59 3 Indian Hispanic 0 0.61 0.38 0.01 41 41 16 (Admixed) In Caucasian populations, the overall frequency of the 7TA-repeat allele (also known as the UGTIAI*28 allele) is -30%, leading to a frequency of homozygotes in the general population of -9%. The 5 and 8 repeat alleles are not frequent in any population although they are found more often in subjects of African descent.

The consequences of low UGT1A1 expression are typically mild with mild intermittent jaundice, some persistence of neonatal jaundice, but with no significant liver disease.

Irinotecan Irinotecan is a treatment for colon cancer used in conjunction with 5-fluorouracil. However, there are severe dose-limiting side effects associated with the treatment, namely diarrhoea, leucopoenia and myelosuppression. The metabolising capacity of patients is a major determinant of this toxicity. Ratain reports that patients carrying the 7-repeat allele are more prone to these side effects and those homozygous for the low expression variant are most at risk. The FDA in the USA has recently decided that the label for Irinotecan should be modified to recommend that physicians take into account the glucuronidation capacity of patients before prescribing the drug. This, in turn, requires that the number of TA repeats in the UGT1AI promoter be determined.

The Problem In order to assay for the different number of repeats, it is known in the art to use an approach which targets the length differences between the two variants. Specifically, amplification primers flanking the variant site are used to generate a product of appropriate size (100-200 base pairs), before analysis by size, usually by high resolution capillary electrophoresis. However, the problem with this approach is that it is time consuming as it requires PCR, followed by a purification step and size analysis. In addition, the method is non-homogeneous: because the tube in which the PCR takes place needs to be opened after the PCR which introduces the possibility of cross-contamination of the product.

It is therefore preferred that the different alleles be probed in a closed tube format. For single nucleotide polymorphism (SNP) analysis, a number of systems are available, such as: 5'-nuclease (TaqMan) probes (Livak;Livak et al.), Hybe-probes (Lay and Wittwer), Molecular Beacons, and Minor Groove Binding Probes (in a TaqMan configuration or as purely hybridisation probes). Each of these methods relies upon a pair of fluorescent labelled probes binding differentially to the two variant sequences; le a 6-specific probe should bind specifically to the 6-repeat target but not to the 7-repeat target and vice versa for the 7-specific probe. This relies upon the two probes having different binding efficiencies (measured by free energy (G0) or melting temperature (Tm) for their matched or mismatched targets This is difficult to achieve for this particular target for the following reasons: * The hybridisation sites for the two variants differ by a single AT/TA repeat. Because A-T base pairs tend to be weak, mismatches involving A-T pairs have a low impact on G0 and Tm.

* The high AT content of the region causes the probe site to have a low inherent Tm.

Attempts to raise the Tms of the probes by including additional sequences flanking the region of interest has the undesired consequence of reducing the impact of mismatched probing still further.

* The repetitive nature of the region makes mis-probing likely, leading to further loss of specificity In order to obtain suitable discrimination between sequences, some workers (von Ahsen, Oellerich, and Schutz) have used melting curves in which products are analysed by varying the temperature of the sample and monitoring fluorescence. Differential hybridisation of the probes is detected by the shape of the curve. The problem with this approach is that it has proven to be cumbersome and unsuitable for standardised analysis.

Scorpions The present invention uses Scorpion primers (Whitcombe et al.) which are nucleic acid molecules that combine priming and probing functionalities into a single molecule. More specifically, the nucleic acid molecule comprises a primer sequence and a probe sequence.

The amplicon specific probe sequence is designed to target the extension product of its own primer sequence. The fact that the mechanism of probe binding is through a unimolecular rearrangement ensures that the kinetics and efficiency of hybridisation are good. In addition, the mode of action causes the probe to have an unexpectedly high Tm. For example a typical probe may have a Tm around 20 C higher than the bi-molecular equivalent.

As a result of this elevated Tm for the probe sequence, much shorter probes can be used, while maintaining a high and useful assay temperature. This increases the discriminatory power of the probes. In addition, the incorporation of a stem/loop structure introduces extra specificity (Bonnet et al.).

Scorpion primers of the invention have been designed to be highly specific for the two major variants of the UGT1A1 promoter and to be used in real time PCR with an annealing temperature of 60 C.

DISCLOSURE OF INVENTION

According to a first aspect of the present invention, there is provided a nucleic acid molecule comprising: (a) (i) a primer sequence comprising a sequence from the UGTIAI gene upstream of the TA promoter region of the UGT1A1 gene; and a probe sequence comprising a sequence complementary to the TA promoter region of the UGTIA1 gene; or (ii) a primer sequence comprising a sequence complementary to a sequence from the UGTIAI gene downstream of the TA promoter region of the UGT1AI gene; and a probe sequence comprising the TA promoter region of the UGTIAI gene; (b) first and second stem sequences flanking the probe sequence, the first and second stem sequences being complementary to each other; and (c) a signalling system for detecting the presence or absence of hybridisation between the stem sequences.

Conveniently, wherein the signalling system comprises first and second detectably-interacting moieties located adjacent to the first and second stem sequences, respectively, such that the first and second detectably-interacting moieties are brought sufficiently close together when the first and second stem sequences hybridise to each other that a change in a characteristic of the moieties is detectable.

Preferably, the detectably-i nteracti ng moieties are fluorescent-interacting moieties and when the first and second stem sequences hybridise to each other, the fluorescent interacting moieties are brought sufficiently close together that a characteristic of light emitted by the first and second fluorescent-interacting moieties is different from the characteristic of light when the first and second stem sequences are not hybridised to each other.

Advantageously, the first and second fluorescent-interacting moieties comprise a fluorophore group and a quencher group.

Conveniently, the nucleic acid molecule further comprises a polymerase blocking moiety.

Preferably, the polymerase blocking moiety is located just upstream of the primer sequence. 25.

Advantageously, the probe sequence is located upstream of the primer sequence.

Conveniently, the nucleic acid molecule comprises the primer sequence and probe sequence of part (a)(i) as described above.

Advantageously, the primer sequence consists of a sequence of from 6 to 50 nucleotides long, preferably 10 to 40 nucleotides long, more preferably 15 to 30 nucleotides long present within less than 100 nucleotides and preferably less than 50 nucleotides upstream of the TA promoter region.

Preferably, the probe sequence further comprises a sequence complementary to the sequence immediately adjacent the TA promoter region of the UGTIAI gene.

Conveniently, the primer sequence comprises SEQ. ID NO. 1.

Preferably, the probe sequence comprises SEQ. ID NO. 3 or SEQ. ID NO. 4.

Alternatively, the nucleic acid molecule comprises the primer sequence and probe sequence of part (a)(ii) as described above.

Advantageously, the primer sequence consists of a sequence of from 6 to 50 nucleotides long, preferably 10 to 40 nucleotides long, and more preferably 15 to 30 nucleotides long and complementary to a sequence less than 100 nucleotides and preferably less than 50 nucleotides downstream of the TA promoter region.

Conveniently, the probe sequence further comprises a sequence immediately adjacent the TA promoter region of the UGT1A1 gene.

Preferably, the primer sequence comprises SEQ. ID NO. 2.

Advantageously, the probe sequence comprises SEQ. ID NO. 5 or 6.

Conveniently, the first and second stem sequences comprise SEQ. ID NOS. 7 and 8, respectively.

According to a second aspect of the present invention, there is provided a nucleic acid molecule comprising SEQ. ID NO. 1 or SEQ. ID NO. 2 and consisting of fewer than 100 nucleotides.

Conveniently, the nucleic acid molecule consists of fewer than 50 nucleotides.

Preferably, the nucleic acid molecule consists of SEQ. ID NO. I or 2.

According to a third aspect of the present invention, there is provided a method of detecting the presence or absence of the UGTIAI*28 allele and/or the corresponding wild type allele in a sample comprising the steps of: (a) mixing a nucleic acid molecule as described above with the sample; (b) mixing a converse primer with the sample; (c) effecting a PCR reaction on the sample; and (d) detecting any change in the signalling system.

Conveniently, the nucleic acid molecule of step (a) comprises the primer sequence and probe sequence of part (a)(i) as described above and the converse primer of step (b) is a nucleic acid molecule comprising SEQ. ID NO. 2.

Alternatively, the nucleic acid molecule of step (a) comprises the primer sequence and probe sequence of part (a)(i) as described above and the converse primer of step (b) is a nucleic acid molecule comprising SEQ. ID NO. 1.

Alternatively, the nucleic acid molecule of step (a) comprises the primer sequence and probe sequence of part (a)(i) as described above and the converse primer of step (b) comprises the primer sequence and probe sequence of part (a)(ii) as described above, the signalling system of each nucleic acid molecule being capable of providing a different signal from the other and wherein step (d) comprises the step of measuring the signal from the signalling system of each nucleic acid molecule.

Preferably the signalling system is as described previously.

Advantageously, the fluorescent-interacting moieties of each nucleic acid molecule are capable of emitting light of a different wavelength and wherein step (d) comprises measuring the light emitted at each wavelength.

According to a fourth aspect of the present invention, there is provided a method of administering Irinotecan to a patient comprising the steps of: (a) obtaining a sample from the patient; (b) detecting the presence of the UGTIA1*28 allele and/or the corresponding wild type allele as described above; (c) selecting a dosage of lrinotecan based on the presence or absence of UGT1A1*28 allele; and (d) administering the selected dosage of Irinotecan.

According to a fifth aspect of the present invention, there is provided a kit comprising a nucleic acid molecule as described above, there being at least first and second sets of the nucleic acid molecules, the first set of nucleic acid molecules comprising the primer sequence and probe sequence of part (a)(i) of claim 1 and the second set of nucleic acid molecules comprising the primer sequence and probe sequence of part (a)(ii) as described above.

It is to be understood that in this specification, reference to the "UGTIA1 gene may mean a nucleic acid having at least 80%, preferably at least 90% and more preferably 100% identity with nucleotides 201 to 13227 of SEQ. ID NO: 9. Furthermore, reference to the UGT1A*28 allele" means the allele of the UGT1AI gene having a TTA repeat in the TA promoter variable region corresponding to nucleotides 163 to 174 of SEQ. ID NO: 9.

Reference to sequences upstream of the UGTIAI gene or upstream of the "TA promoter region" refer to sequences having at least 80% identity, preferably at least 90% identity and more preferably 100% identity with the nucleotides upstream of nucleotide 201 or 163 of SEQ. ID NO: 9, respectively. Similarly reference to sequences downstream of the "TA promoter region" refer to sequences having at least 80% identity, preferably at least 90% identity and more preferably 100% identity with the nucleotides downstream of nucleotide 174 of SEQ. ID NO: 9.

In this specification, the percentage "identity" between two sequences is determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) using default parameters. In particular, the BLAST algorithm can be accessed on the internet using the URL www.ncbi.nlm.nih.gov/blast.

BRIEF DESCRIPTION OF DRAWINGS

In order that the present invention may be further understood and so that embodiments of the invention may be more fully appreciated, embodiments of the invention will now be described with reference to the accompanying figures in which: Figure 1 is a schematic representation of a nucleic acid molecule in accordance with one embodiment of the present invention in a first configuration; Figure 2 is a schematic representation of the embodiment shown in Figure 1 in a second configuration; Figure 3 is a schematic representation of the embodiment shown in Figure 1 in a third configuration; Figure 4 is a table showing the primers used in the examples of the invention and their respective sequence ID numbers; Figure 5 is a graph showing the results of a TA6 specific PCR reaction with a forward direction Scorpions primer; matched probes and targets are shown with squares, mismatched probes and targets are shown with circles; Figure 6 is a graph showing the results of a TA6 specific PCR reaction with a reverse direction Scorpions primer; matched probes and targets are shown with squares, mismatched probes and targets are shown with circles; Figure 7 is a graph showing the results of a TA7 specific PCR reaction with a forward direction Scorpions primer; matched probes and targets are shown with squares, mismatched probes and targets are shown with circles; and Figure 8 is a series of graphs, each representing a real time PCR trace of a sample from an individual in a Duplex reaction (TA7 specific forward direction Scorpions primer associated with ROX fluorophore (circles) and TA6 specific reverse direction Scorpions primer associated with FAM fluorophore (squares)).

BRIEF DESCRIPTION OF SEQUENCE LISTINGS

SEQ. ID NOS. 1 to 8 contain the nucleotide sequences of certain components of the Scorpion primers as is shown in Figure 4.

SEQ. ID NO. 9 contains the nucleotide sequence of the UGTIAI gene (nucleotides 201 to 13227). The gene promoter is located at nucleotides 132 to 491 of which the variable TA promoter region is located at nucleotides 163 to 174 (in this example having 6TA repeats).

DETAILED DESCRIPTION

A nucleic acid molecule which forms a Scorpion primer will now be described with reference to Figure 1. It is to be noted, however, that the concept of Scorpion primers, in general, is disclosed in W099/66071, which is incorporated herein by reference.

A Scorpion primer which is a nucleic acid molecule I comprises a primer sequence 2 which is the same as a sequence from the UGTIAI gene, slightly upstream from the TA promoter sequence. Typically, the primer sequence 2 corresponds to a sequence not more than 50 or nucleotides upstream of the TA promoter sequence. The primer sequence 2 may be of any length capable of hybridising to the complementary sequence and initiating DNA polymerisation but from 6 to 50, 10 to 40 or 15 to 30 nucleotides are exemplary ranges.

Upstream of the primer sequence 2 is provided a hexethylene glycol (HEG) moiety 3, which acts as polymerase blocking moiety, to prevent movement of a polymerase moiety beyond it, during PCR.

Upstream of the hexethylene glycol moiety 3 is provided the first of a pair of fluorescent interacting moieties 4, 5 which are described in further detail below.

Upstream of the first fluorescent interacting moiety 4 is provided a first stem sequence 6.

Upstream of the first stem sequence 6 is a probe sequence 7 and upstream of the probe sequence 7 is a second stem sequence 8. Thus the first and second stem sequences 6, 8 flank the probe sequence 7. The first and second stem sequences 6, 8 have sequences complementary to each other.

The probe sequence 7 has a sequence complementary to the TA promoter region of the UGTIA1 gene and also extends slightly downstream thereof (e.g. by 3 nucleotides). This ensures that the probe sequence can be anchored to the UGTIA1 and that differences in the length of the TA promoter region result in a mismatch with the probe sequence 7.

Upstream of the second stem sequence 8 is the second fluorescent interacting moiety 5. In this embodiment, the second fluorescent interacting moiety 5 is a fluorophore group and the first fluorescent interacting moiety 4 is a quencher group, which is capable of quenching the activity of the fluorophore group 5, when the two groups are in close proximity to each other.

Examples of suitable fluorophore groups are FAM, ROX, HEX, JOE, CAL Orange and Texas Red. Examples of suitable quenching groups are dU dabcyl or BHQ1 or BHQ2.

In other embodiments, the two fluorescent interacting moieties 4, 5 are swapped round. In other words the first fluorescent interacting moiety 4 is a fluorophore group and the second fluorescent interacting moiety 5 is a corresponding quencher group.

It is to be appreciated, however, that the present invention is not limited to embodiments in which the first and second fluorescent interacting moieties comprise a fluorophore group and a quencher group. In alternative embodiments, for example the two fluorescent interacting moieties are two fluorescent resonant energy transfer (FRET) groups which interact to produce an enhanced fluorescent signal when they are sufficiently close to each other. Thus any such fluorescent interacting moieties may be used provided that a change in the proximity of the two moieties to each other results in there being a change in a characteristic of light which is emitted by one or both of the moieties 4,5. The change occurs between the situation where the first and second stem sequences 6, 8 hybridise and the two moieties 4, 5 are located relatively close to each other and the situation where the first and second stem sequences 6, 8 are not hybridised to each other and the two moieties 4, 5 are thus further apart. The change may be any of the following: the initiation of the emission of light; the termination of the emission of light; a change in the wavelength of light that is emitted; or a change in intensity of the light that is emitted.

Indeed, the present invention is not limited to fluorescent interacting moieties at all and other signalling systems may be used instead. For example, in some embodiments a single fluorescent moiety is provided in the probe sequence 7 and the coiling of the probe sequence 7 around the fluorescent moiety when the first and second stem sequences 6, 8 are hybridised to each other is detectable as a reduction in fluorescent. In other embodiments, a different type of detectably-interacting moiety is used such as radioactive isotopes whose proximity is detectable using a scintillation proximity assay.

With reference to Figure 2, the use of the nucleic acid molecule I in an assay method will now be described. In order to detect the number of TA repeats in the TA promoter region of the UGT1A1 genes in a sample, the nucleic acid 1 (or, to be precise, a plurality of copies of the nucleic acid 1) are added to the sample. The sample is exposed to light to which the fluorophore group 5 is responsive. Furthermore, any light which is emitted at the wavelength at which the fluorophore group 5 emits, is detected. The necessary further ingredients for PCR (in particular, a suitable polymerase and nucleosides) are added to the sample, as is a reverse primer, which is complementary to a sequence downstream of the TA promoter of the UGTIA1 gene and PCR is carried out.

In this example, the probe sequence 7 of the nucleic acid molecule 1 corresponds to the TA promoter region having a 6 TA repeat. In other embodiments the probe sequence 7 corresponds to another type of TA promoter region such as one having a 7 TA repeat. If there is DNA in the sample which does not have 6 TA repeats in the TA promoter region (for example, because it has 7 TA repeats) then, as PCR takes place, and the amplicons are extended from the primer sequence 2, the first and second stem sequences 6, 8 hybridise to each other. This brings the quencher group 4 and fluorophore group 5 into close proximity with one another so that the emission of light from the fluorophore group 5 is quenched and light is not emitted. The reduction of light emitted at this wavelength is therefore detected.

Referring to Figure 3, however, if the sample contains DNA with a TA promoter region having 6 TA repeats then, as the amplicon is extended from the primer sequence 2, the probe sequence 7 hybridises to the TA promoter region 9 of the amplicon. Thus the quencher group 4 and fluorophore group 5 are no longer in close proximity to each other and the fluorophore group 5 emits light in response to the light to which it is exposed. The emitted light is then detected.

It is to be appreciated that if the sample contains some DNA which has the 6 TA promoter repeat and some DNA which has the 7 TA promoter repeat (e.g. if the DNA is from an individual who is heterozygous for the allele) then the light detected from the fluorophore group 5 will be detected, albeit at a lower intensity than would be the case for an individual homozygous for the 6 TA repeat.

It is also to be understood that, in the instance where the TA promoter region does not have a 6 TA repeat (i.e. there is a mismatch with the probe sequence 7) then there may be a small amount of hybridisation between the mismatched sequences because they will nonetheless have a certain degree of complementarity with each other. For example, if the promoter region has a 7 TA repeat then the probe will be complementary over six of the TA repeats.

Nevertheless, the advantage of the Scorpion primer system is that the hybridisation between the first and second stem sequences 6, 8 is more energetically favourable than the hybridisation of the mismatched sequences and therefore occurs preferentially to mismatched hybridisation. This permits a much greater difference in Tm between the matched and mismatched probes than would occur in a non-Scorpion probe system.

It is to be noted that the assay method may be carried out in a single receptacle which does not need to be re-opened during the method (i.e. it is a "closed tube" method).

The above described embodiments are of "forward direction" Scorpion primers. It is to be appreciated that, in alternative embodiments of the present invention "reverse direction" Scorpion primers are provided which hybridise to the other strand of a UGTIA1 gene. In these embodiments, the primer sequence 2 of the nucleic acid 1 comprises a sequence that is complementary to a sequence from the UGT1A1 gene downstream of the TA promoter region. The probe sequence 7 is the same as the TA promoter region of the UGT1AI gene.

Such reverse direction Scorpion primers may be used in conjunction with a forward primer that is the same as a region of the UGT1A1 gene, upstreamof the TA promoter region. In this specification, the term "converse primer" is used to designate a primer of the opposite direction to its respective Scorpion primer, i.e. a reverse primer in the case of a forward direction Scorpion primer or a forward primer in the case of the reverse direction Scorpion primer.

In further embodiments of the present invention, instead of a forward or reverse direction 25, Scorpion primers being used in conjunction with aconverse primer in-order for the PCR to be effected, both a forward and reverse direction Scorpion primers are provided to carry out a duplex reaction.

In particularly preferred embodiments involving duplex reactions, the forward and reverse Scorpion primers have probe sequences specific for different alleles and each has a pair of fluorescent- reacting moieties capable of emitting light with a different characteristic. For example, the probe sequence of the forward Scorpion primer is specific for the 6 TA repeat promoter region and has fluorescent interacting moieties which emit light at a first wavelength whereas the reverse Scorpion primer has a probe sequence which is specific for the 7 TA promoter region and has fluorescent interacting moieties which emit light at a second wavelength. Such embodiments provide the highest accuracy because the difference between the signal provided on detection of a 6 TA promoter region compared with a 7 TA promoter region, or vice versa, is greatly enhanced.

Thus the present invention provides the means to test for the prevalent variants of UGT1AI*28. The assay methods of the present invention are more simple, more reliable and more robust than those described in the prior art and are "closed" tube tests which reduces the possibility of contamination of assay products. With the benefit of the results of the assay methods of the invention, it is possible to select more accurately the appropriate dosing regime for patients receiving Irinotecan.

EXAMPLES

METHODS

Primers The primers which were used in this study flank the repeat region, as shown in Table 3. The primers are also shown in Figure 4 together with their respective SEQ. ID NOS.

Table 3: Primers and Probes for UGTIA1*28 Sequence Name Sequence

F I -CCGCCTACTTATATATATATATATGGCGG-Q-

UGT_F3_6 heg-CATTAACTTGGTGTATCGATTGGTTT F2-CCGCCTACTTATATATATATATATATGGCGc3-Q-UGT_F3_7 heg-CATTAACTTGGTGTATCGATTGGTTT

F I -CCGCCATATATATATATATAAGTAGGCGG-

UGT_R3_6 Q-heg-TTGCTCCTGCCAGAGGTT F2-

CCGCCATATATATATATATATAAGTAGGCGG-Q-

UGT_R3_7 heg-TTGCTCCTGCCAGAGGTT UGT_F3 CATTAACTTGGTGTATCGATTGGTTT UGT_R3 TTGCTCCTGCCAGAGGTT Where Fl is FAM F2 is ROX, HEX, JOE, CAL Orange or Texas Red Q is dU-DABCYL or BHQ1 or 2.

heg is hexethylene glycol.

Reaction Conditions Reactions were performed in 0.2m1 vessels (single tubes, strips or plates). Reactions (25pl) typically contained: Final Item Concentration Forward primer/Scorpion 250 nM Reverse primer/Scorpion 250 nM Each dNTP 200 pM Tris-HCI (pH 8.3) 10 mM KCI 50mM MgCI2 2.5 mM Taq Polymerase 0.5 U Some reactions performed better in the presence of Qiagen Multiplex Reaction buffer that contains a proprietary mixture of Ammonium Sulphate, MgCI2, and volume exciuders such as Polyethylene glycol (MW 8000), and/or Dextran (MW 50,000); see US 2004-0115712.

PCRs were performed in Real Time PCR cyclers (Mx4000 and Mx3000P from Stratagene, or SmartCycler II from Cepheid), using standard conditions: 95 C for 10 or 15 minutes to activate the hot-start enzyme, followed by up to 50 cycles of: 95 C,60s 60 C, 60 s Analysis of control samDles Control samples were analysed by PCR followed by capillary electrophoresis using an ABI 3100 capillary sequencing instrument, according to the manufacturer's instructions.

Representatives of each apparent genotype were subjected to further analysis by sequencing.

Sequencing was performed on amplicons generated from unlabelled forward and reverse primers, by standard cycle sequencing, using big dye terminators. Reactions were separated using an ABI 3100 capillary sequencing instrument, according to the manufacturer's instructions.

RESULTS

Example 1: TA6-specifjc reactions-forward reaction

Set up for Example 1 Final

Item Identifier Concentration Forward primer/Scorpion 250 nM UGT_F3_6 Reverse primer/Scorpion 250 nM R3 dNTPs (each) 200 pM Tris-HCI (pH 8.3) 10 mM KCI 50mM MgCI2 2.5 mM Taq Polymerase 0.5 U Samples were three 6/6 homozygotes and three 7/7 homozygotes and the results are shown in figure 5.

Example 2: TA6-specifjc reactions-Reverse Reaction

Set up for Example 2 Final

Item Identifier Concentration Forward primer/Scorpion 250 nM F3 Reverse primer/Scorpion 250 nM UGT_R3_6 dNTPs (each) 200 pM Tris-HCI (pH 8.3) 10 mM KCI 50mM MgCI2 2.5 mM Taq Polymerase 0.5 U Samples were three 6/6 homozygotes and three 7/7 homozygotes and the results are shown in Figure 6.

Example 3: TA7-specific reactions-Forward Reaction

Set up for Example 3 Final

Item Identifier Concentration Forward primer/Scorpion 250 nM UGT_F3_7 Reverse primer/Scorpion 250 nM R3 dNTPs (each) 200 pM Tris-HCI (pH 8.3) 10 mM KCI 50 mM MgCI2 2.5 mM Taq Polymerase 0.5 U Samples were three 6/6 homozygotes and three 7/7 homozygotes and the results are shown in figure 7.

Example 4: Duplex Reaction

Set up for Example 4 Final

Item Identifier Concentration Forward primer/Scorpion 250 nM UGT_F3_7 Reverse primer/Scorpion 250 nM UGT_R3_6 dNTPs (each) 200 pM Tris-HCI (pH 8.3) 10 mM KCI 50mM MgCl2 2.5 mM Taq Polymerase 0.5 U The forward TA 7 specific Scorpion primer had a ROX fluorophone group. The reverse TA6 specific Scorpion primer had a FAM fluorophone group.

A plate of 96 individual DNAs from cell lines was tested on a Stratagene Mx3000P Instrument.

A column of samples that has representatives of all three genotypes were analysed individually and the results are shown in Figure 8.

Interpretation: * Samples Bi, B3, B6, B9, BlO and Bil showed higher signals for FAM than for ROX; these samples are interpreted as TA6/TA6 homozygotes.

* Samples B3, B7 and B12 showed higher signals for ROX than for FAM; these samples are interpreted as TA7/TA7 homozygotes.

* Samples B2, B5 and B8 showed similar fluorescences for both dyes; these samples are interpreted as TA6/TA7 heterozygotes.

Confirmation All 96 samples on this plate were tested by capillary electrophoresis/sizing based methods and all results were concordant.

REFERENCES

Bonnet G, Tyagi 5, Libchaber A, Kramer FR. 1999. Thermodynamic basis of the enhanced specificity of structured DNA probes Proc. Nat!. A cad. Sd. U. S. A 96:6171-6 Guillemette C. 2003. Pharmacogenomics of human UDP-glucuronosyltransferase enzymes Pharmacogenomics J 3:136-58 Lay MJ, Wittwer CT. 1997. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR C!in Chem 43:2262-7 Livak KJ. 2003. SNP genotyping by the 5'-nuclease reaction Methods Mo! BioL 21 2:129-47 Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization PCR Methods App!. 4:357-62 Ratain MJ. 2002. Irinotecan Dosing: Does the CPT in CPT-1 1 Stand for "Can't Predict Toxicity"? J Clln Oncol 20:7-8 von Ahsen N, Oellerich M, Schutz E. 2000. DNA Base Bulge vs Unmatched End Formation in Probe-based Diagnostic Insertion/Deletion Genotyping: Genotypi ng the UGT1A1 (TA)n Polymorphism by Real-Time Fluorescence PCR C!in Chem 46:1939-45 Whitcombe D, Theaker J, Guy SP, Brown T, Little S. 1999. Detection of PCR products using self-probing amplicons and fluorescence Nat. BiotechnoL 17:804-7

SEQUENCE LISTING

<110> DxS Limited <120> A Nucleic Acid Molecule <130> WA/MP1O1513GE <160> 9 <170> Patentln version 3.1 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Part of Scorpion Primer <400> 1 cattaacttg gtgtatcgat tggttt 26 <210> 2 <ii> 18 <212> DNA <213> Artificial Sequence <220> <223> Part of Scorpion Primer <400> 2 ttgctcctgc cagaggtt 18 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Part of Scorpion Primer <400> 3 tacttatata tatatatat 19 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Part of Scorpion Primer <400> 4 tacttatata tatatatata t 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Part of Scorpion Primer <400> 5 atatatatat atataagta 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Part of Scorpion Primer <400> 6 atatatatat atatataagt a 21 <210> 7 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Part of Scorpion Primer <400> 7 ccgcc 5 <210> 8 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> Part of Scorpion Primer <400> 8 ggcgg 5 <210> 9 <211> 13227 <212> DNA <213> Homo sapiens <220> <221> gene <222> (201) (13227) <223> UGT1A1 <220> <221> promoter <222> (132) (491) <223> <220> <221> misc_feature <222> (163) (174) <223> Variable region <400> 9 attccagcca gttcaactgt tgttgcctat taagaaacct aataaagctc caccttcttt 60 atctctgaaa gtgaactccc tgctaccttt. gtggactgac agctttttat agtcacgtga 120 cacagtcaaa cattaactt gtgtatcgat tggtttttgc catatatata tatataagta 180 ggagagggcg aacctctggc aggagcaaag gcgccatggc tgtggagtcc cagggcggac 240 gcccacttgt cctgggcctg ctgctgtgtg tgctgggccc agtggtgtcc catgctggga 300 agatactgtt gatcccagtg gatggcagcc actggctgag catgcttggg 9ccatccagc 360 agctgcagca gaggggacat gaaatagttg tcctagcacc tgacgcctcg ttgtacatca 420 gagacggagc attttacacc ttgaagacgt accctgtgcc attccaaagq gaggatgtga 480 aagagtcttt t9ttagtctc gggcataatg tttttgagaa tgattctttc ctgcagcgtg 540 tgatcaaaac atacaagaaa ataaaaaagg actctgctat gcttttgtct gctttccc 600 acttactgca caacaaggag ctcatggcct ccctggcaga aagcagcttt gatgtcatgc 660 tgac9gaccc tttccttcct tgcaqcccca tcgtggccca gtacctgtct ctgcccactg 720 tattcttctt gcatgcactg ccatycagcc tggaatttga ggctacccag tgccccaacc 780 cattctccta cgtgcccagg cctctctcct ctcattcaga tcacatgacc ttcctgcagc 840 gggtqaagaa catgctcatt gccttttcac ayaactttct gtgcgacgtg gtttattccc 900 cgtatgcaac ccttgcctca gaattccttc ayagagaggt gactgtccag gacctattga 960 gctctgcatc tgtctggctg tttagaagtg actttgtgaa ggattaccct aggcccatca 1020 tgcccaatat ggtttttgtt ygtggaatca actgccttca ccaaaatcca ctatcccagg 1080 tgtgtattgg agtgggactt ttacatgcgt atattctttc agatgtatta ctttggatcg 1140 attaactagc cccagatata tgctgagcaa gcattctgag ataatttaaa atgccctctt 1200 ttgttaattt ttgactccta ggtttgagtc tgtctttggc atcatcttct ggatgatttc 1260 ttg9tatctg agatttcggg aaagcattcc ttggacattt tactctgtgt gctcca9tgg 1320 atagtaatca attagaaaca acaagctgtt aaatgccata ggcaca9aat gctgggtttg 1380 gggcaccctg cagaaaactc agttgaagcc tgcaccttgc cctggattca gtcaggcagg 1440 caatgttcag gactgatgaa atcattcttt gatgatgata yatcctggaa atgaaagttg 1500 cctttgtgac cctggttaaa gctccagttt ctaaatattc tgataagaag ctaaatcctg 1560 cagtccgttc tcttctaatg agtgaatcac cagacagtca ggttctgaca tgatacagaa 1620 aggttgtagg tttcattctc aagctattag gtttattttt cccctacaga gtttgaagta 1680 tgcaaaaagt agcattcaca tcctcatcga aatctcagca gaggatagaa aagaacagga 1740 gaggctcctt cagatggagc gttagggaat tactctttga ggagtgaca tttcagagag 1800 cgttcattca cttatcctgc aaagattggc tgaggatcta ctggcagccc aggcacttcc 1860 caggtgctgc gtctg9ctcc cattaagggg actgatatca ccttc9gagg tgaccttatt 1920 tccactatac ctccaatgtg atttgtattt tatttttttt aattttctgt gcattttcct 1980 tcatagcaca tcaaatatgg cagccatttc acttagatag ttgttgattg tccgcttcac 2040 atcatgagcc atgtggggac ctgtgtgact ttgcattaat cacatccact gtatgcggcg 2100 tcctcaacac ctgccaatg9 gtctgcatgt atttggcgcc ccataaatct cagcacctaa 2160 ggcacagaat aggcacccac cgaatatgtg ttacattaat gaatgagaag aaaggtgcca 2220 accgaggtct agttaatggg tcgagagtaa tccacaatag ctctttttag ttctttgtac 2280 tccagctatt acataccaat atgtatatag aaacatatgt aaaatttttt ggttgctttt 2340 tctacaaaat agagtaacag tgtattccca ctgcccactt accgataatg tcatggatat 2400 cactccagte ttaaatgcta ttacttttta aactatgaaa tagtatttca tggtacttgt 2460 gtaccaca9t gtattctyct ggagatctag tctagttccc cacagaggaa cattacaatt 2520 tgtattccag gagttttgtt gtt9tgacct caaacacttc ctttaaaaag ataagctatt 2580 ttgtagttta aaaaacattt gttctgtttc tttctcattc atcttttctt aagtatttta 2640 cacggttttt tttttttggt cactactgtg aatgt9ttat ttttttgcat ttctatctct 2700 agctgatt.at ctactcatta ctcagctatc tcatcaaaat attgattttc ataataaaaa 2760 ataataggca gtcatttgct gataaagaaa ttttggtttc ttctcttata aattccatgc 2820 caaatatcag 9gctattgaa tttattagaa tctctaaaaa cayttgaata attctggcaa 2880 taggaaagat gcccgtctg ctqctatttt agtggaaatt gattatcatt tcattatttt 2940 gcattatgtt agccattgtt ttctgaacag gctttattga tttagataat ttccttcttt 3000 gcgtgaggat gtttgtagga gaggcaccga actttatcag ctgcctttct ggcatttatt 3060 qatataacca taaaagtcta agtggtgaac tgtgttgact acatatttgt tgttgccttg 3120 tttggtgcag tcaggcttag gtgtqaaaat atgtttttaa attgtacctt ttagtaacct 3180 gttttgtctt gttgcatgtt ttaatctgaa attccacttt ttggatatta atattaccac 3240 ttctgtatta tttttgttta catttcccta gcacatcttt agtactcctt tgtcttcaag 3300 ctttcttcct ttttaaacaa catggcactg gtatttttaa tccagtcagg cagttgcttt 3360 aataagtgca ttttgcctat ttgaatctaa caattaatag atttgattgt aactctctca 3420 gtttacttta tgtttagttq actttgccat tctccttttt ccggatttct actggttggt 3480 caagttactg ttcttatttt ctctttcttc ctttgttaac taaaaatgcc actctgcact 3540 accattcctc ttgtgttgat ggtcctattc tcaatactct tgataaaact cctgaacttt 3600 aagaataaag ataaaacttt tattgcacaa agaagtccae agagaaagca caacctggca 3660 ttggcgtgtc tttggtgtgt ctgaaggaaa agagatagtg gaacaacatt gggagaaaag 3720 gaatgaaact caagaattcc aagatgttcc tcccctgcca gggtaagata gcagtggttc 3780 acagacaatc gcaatgctgg gtctgagaaa aataactaaa cagaagatta gtgaggacca 3840 aggcttcgag atggccagga gaggaaagct tgggagcagg gaaggttgag atatatgtgg 3900 gttactgqga atgcgtgatg gtgaagtcac agatgaccca catggtgtct aagtgctaaa 3960 gaagaattce gggaaaatga aatgcatttg ggaagggaaa atctaattaa aagcctaaac 4020 taaaaataca aaattcttgg taaagtttag gagttatgtt aaatgtctca ttttggctgg 4080 tgaagtctca tcagaacagg gaaattctct cattcagggg catctcatct tttctttgaa 4140 gggaatcaat ggtgggggat tggagtgtta ttttcagtta atatgttgct tcactctttg 4200 gtcattccgg taactgtgaa gtcagggtga agtttaaggg aagctttgcc aagtagggga 4260 tggacttcac ctttattgag cctcatagta gctggctcag gtaggagttg gccgtgatga 4320 caacttctct gcagtttgcc ctgcgtgaat ctccagatga acttttgtgc catttaaact 4380 ttcgtgatct cctgctattt aacttcgaat gtttatggac ctgtgggttc aattttgtgt 4440 gaatcacatc ctgctgattg ctgagtgggc gtgtgggagg gtgtgcctgg aggagaactt 4500 agactcggcc ttttccagat gagcttcagt gtaagagtgg gtttcatgaa gagcaaaggt 4560 ccta9gaaat ttaagtaagc catttaccaa cgctcagaag aaagaacttg aagagcactt 4620 ggaaatgagc tgtgtctccc caagaaa9ag ggagagaaag aggggagaga tgtggtgcag 4680 accctaggga ggaaggagtt cagaaaaacc atcctcaggg tgttcttgct acaaaccaaa 4740 aaatgcagca tggtggtggg gaggatgact ctgtcctccc tgacttttag atgagcccaa 4800 9ggaaaaggc aaagacaaag cccttaagag ccagaggact cacgaggcc tggggctggt 4860 gagagtggcg gggagagagg gctcaccttg gagaaggat ggtcagtgtc tggggctttc 4920 ctggtcatgt tccaaatcag gcttggcagg agtcctgctg tgcaaattgc gtttgctgag 4980 ccctgtcaga ggtctcctgt gtctcacatc tagggtgacc agcatcctgg cttcctcagg 5040 actgttcagg ttttagcact gaacatcaca tgtcctaggg aacccctcag tttgggcaag 5100 ccctyccaca tcacacaatc atattagtgc cctcagtatt ctttgcaaac ataaaaccat 5160 agactcagta atcccattac tgggtatata cccaaagaaa tataaattat tctactataa 5220 agacacatgc acatatttgt ttattgcagc actattcaca ataacaaagt catggaacca 5280 acccagatgc ccatcaatgg tagattggat aaagaaaatg tggtacatat acaccatgga 5340 atactatgca gccataacaa ggaatgagat catattcttt gcaaggacat ggatgaagct 5400 ggaagccatc atcctccaca aactaacaca ggaacagaaa atcaaacacc gcatgttctc 5460 actcataagt gggayttgaa cagtgagaat gcgtagacgc agggagggga acaacacaca 5520 ccagggcttg tggcggggtg aggggtgagg ggaggaactt agaggatagg tcaataggtg 5580 cagcaaacca ccatggcata tgtatcccag aacttcaagt aaataataat aataataatt 5640 aataataata ataataataa ataaacccat aaagccattt gagagattct tgggggattc 5700 attggaccac tgaaaatcta cagtgagaaa agaattgcca tgttgatgaa acaggaaaac 5760 tttccttgtc cccctcacag agcatgtgac agcgggaggg gctcactttc tcagtgcgcc 5820 actgctcaaa cctctagggg agcatacaga cgggcaggtt gtggggctct gacctcaccg 5880 gcagt9tcta gaggtggatg tttacagctc ctgaagctcc agtgggcgtg ggttatggcc 5940 ttcttttagt tttgccctct atagtcagct tgtgttaacc agctcaatta caccctctac 6000 cttgtcgcaa ggacagaggq ctttctgtat cctgggggct tgccttggtg taccagaa9a 6060 atcgaatccc acctgggctt ggagaatgag tgcaaggatt tattgagtgg atgtagctct 6120 cagcagatgg gggaagccag aaggggatgg aatgggaagg gtttcccctg gagtcaqacc 6180 gctcagtggc ccgggctcgg tggcccg9gc tcggtggcct g9gctctcct ccgactgcct 6240 cagccaaact ccgcgttgtt ctgctggtca gtggcctgcc ggtgcctgtt ggtgagttct 6300 tctcaatgtc cagctgtcct tgcgtccctc cgctgatgtg ctcctcccga tgtccagcta 6360 cctgtgtgtc tgcctgctag ggtcttgggg tttttatagg cacatgatgg gggcgtggca 6420 ggccagggtg gttttgggaa atgaaacatt taggcaggaa aacaaaaatg cctgtcctca 6480 cctaggtcca tgggcacagg tctgggggtg gagccctcgc cagggaccac accctcttct 6540 acccagcact tcccttccct acttccatat catttaaagg gaccacgccc ttcccagctc 6600 ttcccttctg tatcactgat gccttgctct gtgttctcta agtggaatta tcactgtgtg 6660 tatgtacagg tgtgtgcatg tgtgtgcatg tacctgtgct tttcttttgg aaaactagca 6720 cattacctgg attttgcatc tcaaggataa ttctgtaagc aggaaccctt cctcctttag 6780 aaggaa9taa aggagaggaa aatgctgtaa aacttacata ttaataattt tttactctat 6840 ctcaaacacg catgccttta atcatagtct taagaggaag atatctaatt cataacttac 6900 tgtatgtagt catcaaagaa tatgagaaaa aattaactga aaatttttct tctggctcta 6960 ggaatttgaa gcctacatta atgcttctgg agaacatgga attgtggttt tctctttggg 7020 atcaatggtc tcagaaattc cagagaagaa agctatggca attgctgatg ctttgggcaa 7080 aatccctcag acagtaagaa gattctatac catggcctca tatctatttt cacaggagcg 7140 ctaatcccag acttccagct tccagattaa ttctcttaat tggaacctta gatttggctt 7200 ttccctgcca cttcccaact attaatccaa aggttttttt tgttgttgtg gttgttgtca 7260 ttgttttcaa tttgactctc aaatactcta ttaaactatg atccaccaca ctcagaagta 7320 tcattttctc taagagactc aaaagtgtat tagggagaat ttatttaaaa ataaaataaa 7380 tgggatattg tttcttcata ttaaatagaa gtatttctcc aaaaagctgt tggttagaac 7440 actgaattta tgtcttacat ttctgctctt atagttctgc atccacttgt ttcattaagc 7500 aaactttccc ttaaagtqca ggaaagtgaa aaaatcctaa gtgcacagct tgataaatta 7560 tcacaaattc acgta9tgca tacacccttg taactaaacc tccaaaacaa gatgccggaa 7620 gttgccagtc ctcagaagcc ttcacagtta ctgatcctcc cactctgtta aagactgttc 7680 cttcagagga cccctgtttt ctagttagta tagcagattt gttttctaat catattatgt 7740 tctttcttta cgttctgctc tttttgcccc tcccaggtcc tgtggcggta cactggaacc 7800 cgaccacga atcttgcgaa caacacgata cttgttaagt 9gctacccca aaacgatctg 7860 cttg9tatgt tgggcggatt ggatgtatag gtcaaacca9 ggtcaaatta agaaaatggc 7920 ttaagcacag ctattctaaa ggattgttga gcttgaaaat attat9gcca acatatccta 7980 cattgctttt tatctagtgg ggtatctcaa cccacatttt cttctgcaaa tttctgcaag 8040 ggcatgtgag taacactgag tctttgga9t gttttcagaa cctagatgtg tccagctgg 8100 aaactcagag atgtaactgc tgacatcctc cctattttgc atctcaggtc accc9atgac 8160 ccgtgccttt atcacccatg ctggttccca tggtgtttat gaaagcatat gcaatggcgt 8220 tcccatggtg atgatgccct tgtttggtga tcagatggac aatgcaaagc gcatggagac 8280 taagggagct ggagtgaccc tgaat9ttct ggaaatgact tctgaagatt tagaaaatgc 8340 tctaaaaca gtcatcaatg acaaaaggta agaaagaaga tacagaagaa tactttggtc 8400 atggcattca tgataaaatt gtttcaaata tgaaaacatt tacgtagcat ttaatagcgt 8460 tgtttcaaat ataaaaacaa atacataaaa atctggattt ttatttcttc tttttttttt 8520 tttttttttt ttgagatgga gtcttgctct gtcacctagg ctggagtgca gtggtgcaat 8580 cttggcttac tgcaacctcc acctcccacg ttcaagcagt tctgcctcag cctccgtgta 8640 gctyygatta caggtgtcca ccaccacgcc cggttaattt ttgtattttt tagtagagaa 8700 a9ggtttcac catgtttgtc a9gctggtct tgaactcctg acttcaggtg atccacctgc 8760 ctcggcctgc caaagtgctg agattacagg catgagccag cgcgtctgac ctggatttat 8820 aaataagata atttayaggt tattattcac tttataaaag gattctttag tttctatata 8880 atttatcata taatttattt agaattttat ttcccccatt agatttaaaa ctccaattta 8940 cataaaaagt tgccataata gacatctgat ccataagttt cctgcacaga aagaaatact 9000 ccattataag aagcatagta tctttaagag aaaaacaact caaatgctta gaagtacagc 9060 tttttgca9c actggaacct gtgagaaatt ttgtccatgg agtttatgaa tgaaggagct 9120 ataagatatc acagacaaag tcttagaata agagcaaayg aaaatttgct caaatgtggc 9180 cctgaaaacg attcaaaggg caaatgattt ctggattaaa gtagtatat tactgtcaag 9240 ctcactggta ata99cttat tagaacctta tgggaagaag tggtggccag tggtagattt 9300 catccgacaa tagatactgt gtgcatatgt gcgtgtgcgt ttgtgcatgt ggctgtgctc 9360 atgtgtgggt gcacacgtgt gcattcatat gcgtgtgtgt. gtgtgtgcgt gtgtttatga 9420 gagtgtccat tgctttctcc catggttacc tcctttagaa agaagcagca gtcaggaaga 9480 cagatgtgaa gagctggagc atgttcagat gagaggagac ggaacacggg gacacaccag 9540 cttgagcaag ggacaacagg ggaggactga tgactgactt cccacctttg aggtgctaat 9600 gtgtgtgtgg tggcactgga taaaagatca atgttggcta ggcaccatgg cacacgcctg 9660 tagtcccagc cactctggag gctaaggcgg gaggattgct tgagcccaga agttggaggc 9720 tgctatgagc cgtgatcatg ccactgcact ccagcaacct gggcaacaga gtgagaccct 9780 gtctcaaaaa aaaaaaaaaa aatgaaaagt ccacataacc tgagcatcat gtgcccagag 9840 cgttgggtgg tgtggtccca ttccttcctt ccagcggctt cttctggcca cctcaatgtc 9900 aggatgtcct qctcacatat caataccatt aaaacctgac ttctttccct gcactgttga 9960 agctccttct tgaggctcac attatggata taattttgat tctttcttca gtggtataga 10020 taactacttg taacctaaga acaacttggt gaaagtcctc taatacatta ttttttaaaa 10080 aaacacaaat caatgagctc aacttattaa ctaactttca tctattcatt tttgagccat 10140 ccctgtctga ttgtgaatct ccatgattcc aacactctga gctggggata gtgcctacac 10200 aaaataaaaa gaagtggaaa attttcaaac atcagtttat gctgacaacc aggccataat 10260 aggtgctcaa ttactattga atgaatgaat gaaagttctg gccaggtacg gtggctcatg 10320 cctgtagtcc caacactttg ggaggccgag gcaggtggat cacttgaggt taggagttcg 10380 aaaccaacct gaccaacatg aagaaacctt atctctacca aaaaaatata aaaaaattac 10440 ccaggcatgg tggtgtatgc ctgtaatccc agctatttgg gaggctgagg caggaaaatc 10500 acttgaacct gagagycgga ggttgcagtg agctgagatt gtgccactcc actccagcct 10560 qggcgacaga gtgagactcc gtcttactta aaaaaaaaaa aaagaaggtt ccaagaaaat 10620 tcatcttaag gtttatgtaa aaggaagatg atatttaaca tgattcatgy ccaagtacta 10680 atattacatt ataataatgt ttccaaataa cattatagat atgtttaaag acagtgtatt 10740 aggctgttct tgcattgctg taaagaaata cccaagactg ggtaatttat aaagaaaaga 10800 ggtttcattg gctcgtgttt ctgcaggctg tacaggaagc ttagtgctga catcacttgg 10860 ctgccggggg aacctcaggg agcttttact catggcagaa ggcaatgcgg gagcttgcat 10920 gtcacatggc aaaagcagga gcgagagaga gttggggggg aaggtgccac acacttttta 10980 atgaccggct ctcacaataa ctcatgaaaa ctcactatca ggaagacagc actaaagcac 11040 aagggatccg accccatgat ccaaacacct cccaccaggc cccatctcca gcactgggga 11100 ttacaattca acatgagatc tgagtgtgga caaatatcca aactgtatca gtcaacagcg 11160 atcataatta gtcctgaata ggagtgcctt tttttttctt tcttctccct tttcttttct 11220 acttcctcct ccttttccct ctcctcttca atctcctctt cattcctgta gcaccaaggg 11280 ttgaagcacc taacccgttt tggattgaga tgttctgatt gggcaatgaa cactgtccag 11340 aataaacaga aatccatttt gcactaagtg gctgcacaga ccctgcctca tgctaaatct 11400 agcacccaga tagtttaatg tttcaatgac tgaattacaa atatatcatc accttggatt 11460 tggcacttac aaatggctgt taatttggcc agaggtggtt gtttacaact tcaaatagga 11520 gactattcat aatttctgac gtgacatttt cctttcttta ttttactgta tgaaaatata 11580 atgaaattec tcacaaaata tcactaaaaa gaaaagaaga agagtaggaa gcaaggttaa 11640 aatatttcta aaatataatt ttggtctttc tttttctccc ttccttcctc cgtccctctc 11700 tcctttcctc tctccctccc t.ccctccctc ccttcctcct ttccttgctt ccttccctcc 11760 ttctcttcct tctttttcaa gagatcaata acatttatta agaataagtt tcttaattat 11820 aacctttcag gtgataatag taacacagcc tgggcaacac aataagacct tgtttctaca 11880 aaaaatttaa aaattggcca gacatagtyy tgcatgacta attccagcta ctctggaggc 11940 tgaggcagga ggatggcttg agcccaggag ttggaggctg cagttagcca tgcttgtgcc 12000 actacactcc agcccgggca acagggcaag actctgtatc taaaaacaac aacaacaaca 12060 ataatagaaa caggtttcct ttcccaagtt tggaaaatct ggtagtcttc ttaagcagcc 12120 atgagcataa agagaggatt gttcatacca caggtgttcc aggcataacg aaactgtctt 12180 tgtgtttagt tacaaggaga acatcatgcg cctctccagc cttcacaagg accgcccggt 12240 ggagccgctq gacctggccg tgttctgggt ggagtttgtg atgaggcaca agggcgcgcc 12300 acacctgcgc cccgcagccc acgacctcac ctggtaccag taccattcct tggacgtgat 12360 tggtttcctc ttggccgtcg tgctgacagt ggccttcatc acctttaaat gttgtgctta 12420 tggctacc9g aaatgcttgg gaaaaaagg gcgagttaag aaagcccaca aatccaagac 12480 ccattgagaa gtgggtggga aataaggtaa aattttgaac cattccctag tcatttccaa 12540 acttgaaaac agaatcagtg ttaaattcat tttattctta ttaaggaaat actttgcata 12600 aattaatca9 ccccagagtg ctttaaaaaa ttctcttaaa taaaaataat agactcgcta 12660 gtcagtaaag atatttgaat atgtatcgtg ccccctctg9 tgtctttgat caggatgaca 12720 tgtgccattt ttcagaggac gtgcagacag gctggcattc tagattactt ttcttactct 12780 gaaacatgc ctgtttggga gtgcgggatt caaaggtggt cccacggctg cccctactgc 12840 aaatggcagt tttaatctta tcttttggct tctgcagatg gttgcaattg atccttaacc 12900 aataatggtc agtcctcatc tctgtcgtgc ttcataggtg ccaccttgtg tgtttaaaga 12960 agggaagctt tgtaccttta gagtgtaggt gaaatgaatg aatggcttgg agtgcactga 13020 gaacagcata tgatttcttg ctttggggaa aaagaatgat gctatgaaat tggtgggtgg 13080 tgtatttgag aagataatca ttgcttatgt caaatggagc tgaatttgat aaaaacccaa 13140 aatacagcta tgaagtgctg ggcaagttta ctttttttct gatgtttcct acaactaaaa 13200 ataaattaat aaatttatat aaattct 13227

Claims

CLAIMS

1. A nucleic acid molecule comprising: (a) (i) a primer sequence comprising a sequence from the UGTIAI gene upstream of the TA promoter region of the UGTIAI gene; and a probe sequence comprising a sequence complementary to the TA promoter region of the UGTIAI gene; or (ii) a primer sequence comprising a sequence complementary to a sequence from the UGT1AI gene downstream of the TA promoter region of the UGT1A1 gene; and a probe sequence comprising the TA promoter region of the UGT1A1 gene; (b) first and second stem sequences flanking the probe sequence, the first and second stem sequences being complementary to each other; and (c) a signalling system for detecting the presence or absence of hybridisation between the stem sequences.

2. A nucleic acid molecule according to claim 1 wherein the signalling system comprises first and second detectably-interacting moieties located adjacent to the first and second stem sequences, respectively, such that the first and second detectably-interacting moieties are brought sufficiently close together when the first and second stem sequences hybridise to each other that a change in a characteristic of the moieties

is detectable.

3. A nucleic acid molecule according to claim 2 wherein the detectably-interacting moieties are fluorescent-interacting moieties and when the first and second stem sequences hybridise to each other, the fluorescent interacting moieties are brought sufficiently close together that a characteristic of light emitted by the first and second fluorescent-interacting moieties is different from the characteristic of light when the first and second stem sequences are not hybridised to each other.

4. A nucleic acid molecule according to claim 3 wherein the first and second fluorescent-interacting moieties comprise a fluorophore group and a quencher group.

5. A nucleic acid molecule according to any one of the preceding claims further comprising a polymerase blocking moiety.

6. A nucleic acid molecule according to claim 5 wherein the polymerase blocking moiety is located just upstream of the primer sequence.

7. A nucleic acid molecule according to any one of the preceding claims wherein the probe sequence is located upstream of the primer sequence.

8. A nucleic acid molecule according to any one of the preceding claims wherein the nucleic acid molecule comprises the primer sequence and probe sequence of part (a)(i)of claim 1.

9. A nucleic acid molecule according to claim 8 wherein the primer sequence consists of a sequence of from 6 to 50 nucleotides long, preferably 10 to 40 nucleotides long, more preferably 15 to 30 nucleotides long present within less than 100 nucleotides and preferably less than 50 nucleotides upstream of the TA promoter region.

10. A nucleic acid molecule according to claim 8 or 9 wherein the probe sequence further comprises a sequence complementary to the sequence immediately adjacent the TA promoter region of the UGTIAI gene.

11. A nucleic acid molecule according to any one of claims 8 to 10 wherein the primer sequence comprises SEQ. ID NO. 1.

12. A nucleic acid molecule according to any one of claims 8 or 11 wherein the probe sequence comprises SEQ. ID NO. 3 or SEQ. ID NO. 4.

13. A nucleic acid molecule according to any one of claims I to 7 wherein the nucleic acid molecule comprises the primer sequence and probe sequence of part (a)(ii) of claim 1.

14. A nucleic acid molecule according to claim 13 wherein the primer sequence consists of a sequence of from 6 to 50 nucleotides long, preferably 10 to 40 nucleotides long, and more preferably 15 to 30 nucleotides long and complementary to a sequence less than 100 nucleotides and preferably less than 50 nucleotides downstream of the TA promoter region.

15. A nucleic acid molecule according to claim 13 or 14 wherein the probe sequence further comprises a sequence immediately adjacent the TA promoter region of the UGTIAI gene.

16. A nucleic acid molecule according to any one of claims 13 to 15 wherein the primer sequence comprises SEQ. ID NO.
2.

17. A nucleic acid molecule according to any one of claims 13 to 16 wherein the probe sequence comprises SEQ. ID NO. 5 or 6.

18. A nucleic acid molecule according to any one of the preceding claims wherein the first and second stem sequences comprise SEQ. ID NOS. 7 and 8, respectively.

19. A nucleic acid molecule comprising SEQ. ID NO. I or SEQ. ID NO. 2 and consisting of fewer than 100 nucleotides.

20. A nucleic acid molecule according to claim 19 and consisting of fewer than 50 nucleotides.

21. A nucleic acid molecule according to claim 19 and consisting of SEQ. ID NO. 1 or 2.

22. A method of detecting the presence or absence of the UGTIAI*28 allele and/or the corresponding wild type allele in a sample comprising the steps of: (a) mixing a nucleic acid molecule of any one of claims I to 20 with the sample; (b) mixing a converse primer with the sample; (c) effecting a PCR reaction on the sample; and (d) detecting any change in the signalling system.

23. A method according to claim 22 wherein the nucleic acid molecule of step (a) is a nucleic acid molecule according to claim 8 and the converse primer of step (b) is a nucleic acid molecule comprising SEQ. ID NO. 2.

24. A method according to claim 22 wherein the nucleic acid molecule of step (a) is a nucleic acid molecule according to claim 13 and wherein the converse primer of step (b) is a nucleic acid molecule comprising SEQ. ID NO. 1.

25. A method according to claim 22 wherein the nucleic acid molecule of step (a) comprises the primer sequence and probe sequence of claim I part (a)(i) and the converse primer of step (b) is nucleic acid molecule in accordance with claim I part (a)(ii), the signalling system of each nucleic acid molecule being capable of providing a different signal from the other and wherein step (d) comprises the step of measuring the signal from the signalling system of each nucleic acid molecule.

26. A method according to claim 25 wherein the signalling system is as defined in claim 2 or
3.

27. A method according to claim 26 wherein the fluorescent-interacting moieties of each nucleic acid molecule are capable of emitting light of a different wavelength and wherein step (d) comprises measuring the light emitted at each wavelength.

28. A method of administering Irinotecan to a patient comprising the steps of: (a) obtaining a sample from the patient; (b) detecting the presence of the UGTIAI*28 allele and/or the corresponding wild type allele in accordance with any one of claims 22 to 27; (c) selecting a dosage of Irinotecan based on the presence or absence of UGTIA1*28 allele; and (d) administering the selected dosage of Irinotecan.

29. A kit comprising nucleic acid molecule according to any one of claims I to 21, there being at least first and second sets of the nucleic acid molecules, the first set of nucleic acid molecules comprising the primer sequence and probe sequence of part (a)(i) of claim I and the second set of nucleic acid molecules comprising the primer sequence and probe sequence of part (a)(ii) of claim 1.