WO2008119190A1 - Microfluidic platforms for genotyping - Google Patents

Abstract

The present invention provides for a method and apparatus useful for prospective clinical pharmacogenetic testing and polymorphism genotyping of patients, in particular, analysis of thiopurine s-methyltransferase gene (TPMT) in the patient. The present invention provides exemplified methods for a microchip based Restriction Fragment Length Polymorphism (RPLP) and Allele-Specific PCR (AS-PCR) tests, achpted to a microfluidic chip-based pHR and capillary electrophoresis platform to genotype the common *2, *3A, and *3C functional alleles.

Description

MICROFLUIDIC PLATFORMS FOR GENOTYPING

RELATED APPLICATION

This application claims the benefit of United States Provisional' Application Serial Number 60/907,360, filed March 29, 2007 filed under 35 U.S.C. 119(e). The entire disclosure of the prior application is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention pertains to the field of molecular diagnostics, particularly point diagnostics useful for patient genotyping.

BACKGROUND OF THE INVENTION

All of the publications, patents and patent applications cited within this application are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.

Pharmacogenetics, the characterization of gene polymorphisms that influence drug metabolism, is an emerging field with substantial clinically applicability. Single nucleotide polymorphisms (SNPs) account for 20-95% of inter-patient variability in drug response (Kalow, W., et al. Pharmacogenetics 8:283 (1998)), offering the potential to prospectively identify individuals at risk of adverse effects and those predisposed for higher drug efficacy, as well as a means to tailor drug dosing for each individual based on their SNP genotype. SNPs, stable substitutions of a single base pair, are the most common markers for both disease genes and drug response associations (Risch, NJ. Nature 405:847 (2000); McCarthy, J.J. et al. Nat Biotechnol 18:505 (2000)). Current prescription strategies are largely empirical, with the same drug dose chosen for all individuals, in part leading to adverse drug reactions and a significant negative impact on both patient health and health care costs (Pirmohamed, M. et al. Trends Pharmacol Sci 22:298 (2001)). Adverse drug reactions in the US are responsible for more than 100,000 deaths (Lazarou, J., et al. JAMA 279:1200 (1998)) and account for up to 5% of all hospital admissions (Einarson, T.R. Ann Pharmacother 27:832 (1993)). Currently, routine genotyping within a clinic is limited primarily by the need for sophisticated technology and high throughput testing strategies that are affordable only when large numbers of samples are tested together. However, clinical use requires an ability to test each patient individually at the time of presentation, before making treatment decisions and initiating therapy. One of the major factors delaying translation of pharmacogenetics to clinical practice is a lack of technology able to carry out clinical testing for individual patients in timely manner.

Microfluidic technology offers a number of unique advantages over conventional molecular biology techniques for enabling clinical genotyping. Small volumes used in microfluidic tests reduce reagent cost and time, allow for greater sensitivity and resolution in detection, and greater control of fluids and their interactions (Whitesldes, G.M. Nature 442:368 (2006)). Appropriately tailored, microfluidics is ideally suited for the genotypmg of patients one at a time, in clinically relevant timeframes.

The thiopurine s-methyltransferase gene (TPMT) gene polymorphisms are the most developed examples of clinically relevant pharmacogenetics (Wang, L. et al. Oncogene 25:1629 (2006)). TPMT is an important enzyme in the metabolism of the thiopurine drugs (6- mercaptopurine, 6-thioguanine, azathioporine) used in the treatment of haematological and autoimmune diseases. TPMT is an important inactivator of these drugs (Weinshilboum, R.M. Am J Hum Genet 32:651 (1980)). Patients with inherited TPMT deficiency accumulate toxic metabolites in hematopoietic tissues, leading to severe haematological toxicity and potentially fatal neutropenia (Evans, W.E. Ther Drug Monit 26:186 (2004)). Initially, the pharmacogenetics of TPMT were identified and characterized in acute lymphoblastic leukemia (Leonard, L.et al. Arch Dis Child 69:577 (1993)). More recently, SNPs in TPMT have been shown to be significant variables in the widespread use of thiopυrincs as immunosuppressants in the treatment of inflammatory bowel disease (Pierik, M., et al. World J Gastroenterol 12:3657 (2006)), rheumatoid arthritis (Clunie, G. P. et al. Rheumatology 43:13 (2004)), atopic eczema (Meggitt, S.J., et al. Lancet 367:839 (2006)), and organ transplantation (Thervet, E. et al. JAm Soc Nephrol 12: 170 (2001)).

TPMT activity is inherited as an autosomal co-dominant trait that exhibits genetic polymorphisms in all large populations studied (McLeod, H.L., et al. Leukemia 14:567 (2000)). Approximately 90% of individuals inherit from both parents two functional copies of the wild-type alleles, and have high activity of TPMT. 10% of individuals are heterozygotes, inheriting one non-firactional copy, a mutant allele, and have intermediate activity. 0.3% of the population have low or no detectable enzyme activity because they inherit 2 non-functional TPMT alleles (Yates, C.R. et al. Ann Intern Med 126:608 (1997)). TPMT deficient patients at risk for hematological toxicity can be treated with thiopurines, but at a 10-15 fold reduction in dose to avoid adverse events (Evans, W.E., ct al. JPediatr 119, 985 (1991)). Although approximately 23 (Schaeffeler, E., et all Hum Mutat 27:976 (2006)) variant alleles have been associated with low TPMT activity, 3 predominant alleles (TPMT*2, TPMT*3A, TPMT*3C) account for over 95% of low enzyme activity cases (McLeod, H.L., et al. Leukemia 14:567 (2000)). The 238G>C, 460G>A, and 719A>G SNPs, that alone or in combination account for the three signature alleles, are non-synonymous SNPs, which alter the amino acid sequence of the enzyme, resulting in enhanced proteolysis of the variant enzymes and a consequent reduction in functional enzyme activity (Tai, H.L., et al. Proc Natl Acad Sd USA 94:6444 (1997)).

Although phenotyping by directly measuring enzymatic activity is informative in many cases, TPMT assays are not widely available. However, it is not uncommon for patients undergoing thiopurine treatment to receive multiple blood transfusions, thus replacing host red cells with genetically unrelated donor red cells (Evans, W.E. Ther Drug Monit 26:186 (2004); Yates, CR. et al. Ann Intern Med 126:608 (1997); Cheung, S.T. et al. Eur J Gastroenterol Hepatol 15:1245 (2003); Ford, L., et al. Ann Clin Btochem 41:498 (2004)). In addition, misclassification is possible (von Ahscn, N., et al. Clin Chem 50:1528 (2004)). Given the complexity of phenoetypic assays, genotyping has advantages for clinical prediction of TPMT activity.

Despite the widespread use of thiopurine drugs and numerous independent studies confirming the need for TPMT testing to avoid significant and costly toxicities, prospective TPMT genotyping has yet to be established as routine clinical practice (Becquemont, L. Drug Metab Rev 35:277 (2003); Kirchheiner, J., et al. Nat Rev Drug Discov 4:639 (2005); Woelderink, A., et al. Pharmacogenomics J 6:3 (2006)). A survey on the clinical practice of pharmacogenetic TPMT testing in four different European healthcare systems found the actual levels of committed consistent use of testing to be only 12% of all respondents (Woelderink, A., et al. Pharmacogenomics J 6:3 (2006)). A cost effective technological solution at the point of care could facilitate routine pharmacogcnctic testing. Although there are numerous methods to genotype SNPs (Chen, X. et al. Pharmacogenetic J 3:77 (2003)), most require substantive infrastructure, highly trained operators, and are based on batch processing of a large volume. of samples. The art is in need of a microfluidic chip-based method to significantly improve implementation of pharmacogenetic genotyping in a single use, point-of-care, cost effective platform that can genotype patients in a clinically relevant time-frame for informed decision making.

Despite the advantages of prospective genotype testing, pharmacogenetics remains a distant possibility for most patients. TPMT gene analysis demonstrates that although the differences in metabolism of a particular drag have been recognized and the genes and polymorphisms responsible for that variable response have been characterized, the genotyping of those polymorphisms are clinically beneficial. More particularly such testing would be cost effective for the health care system, as current patient testing is impaired by the lack of technologies feasible in a clinical setting. The development of high throughput systems for research or novel biochemistries to genotype SNP3 have failed to address the need for rapid, inexpensive, and accurate testing of a single, standalone patient in a clinical setting. Rather, their complex designs, while increasing functionality, add to the cost of fabrication, operating, and maintenance of these systems.

SUMMARY OF THE INVENTION The present art has suffered from costly and diagnostics for genotyping of patients, prior to drug administration, resulting in the possibility for inappropriate drug dosing. The art teaches correlations between genetic heterogeneity, for example SNPs, which can significantly affect metabolic activity for certain drugs, and their applicability to personalized medicine. The method and apparatus of the present invention provides for a microfluidic based, cost- effective and rapid solution for a clinician to make informed decisions for drug usage and dosage, and thus reduce the current high instance of adverse drug reactions or to inform other medical decisions. It is contemplated that the method and apparatus of the present invention prognosis based en the genetic characteristics (e.g. point mutations) of the individual organism, patient, diseased cells or pathogen.

In one aspect, the present invention provides for a method of identifying known single nucleotide polymorphisms in a sample using a microfluidic device comprising

• providing DNA derived from said sample, at least two primers and a thermostable DNA polymerase, in at least one input well on a microfluidic device thereby creating a reaction mixture; • performing at least one thermocycling event to said reaction mixture

• wherein the thermocycling event comprises bringing the temperature of said input well to a temperature sufficient to result in denaturing of substantially all of nucleic acid present in the reaction mixture, followed by bringing the temperature of said input well to a temperature sufficient to allow said at least two primers to site specifically anneal to said DNA, followed by a temperature sufficient to allow polymerase activity of said thermostable DNA polymerase on said primers annealed to said DNA; and

• introducing into said reaction mixture a restriction endonuclease at a temperature sufficient to allow site specific cleavage by said restriction endonuclease of substantially all nucleic acid present;

• identifying the presence of nucleic acids resulting from performance of said at least one thermocycling event and introduction of said restriction endonuclease from said reaction mixture; wherein said at least two primers are chosen so as to anneal upstream and downstream of a nucleic acid region suspected of containing a SNP such that in the presence of said thermostable DNA polymerase and upon performance of at least one thermocycling event, an increase in the number of copies of the nucleic acid region suspected of containing a SNP occurs thereby creating an amplified region;

wherein the thermostable DNA polymerase is not significantly irreversibly denatured or deactivated at the temperature sufficient to result in denaturing of substantially all of nucleic acid present in the reaction mixture; and

wherein the restriction endonuclease is selected such that it effects a cleavage upon binding to a sequence which may be altered due to the presence of a SNP present within said nucleic acid region suspected of containing a SNP;

such that the presence or absence of a fragment resulting from restriction endonucleaso activity indicates the presence or absence of a SNP in the sample.

In one embodiment of the present invention, the sample is a clinical sample and the DNA is chromosomal DNA. In a further embodiment, the sample is a clinical sample and the DNA is mitochondria] DNA.

In another aspect, the present invention provides for a method of identifying known single nucleotide polymorphisms in a sample using a microflυidic device comprising

• providing DNA derived from said sample, a first primer, a second primer and a thermostable DNA polymerase, in a first input well on a microflυidic device thereby creating a first reaction mixture;

• providing DNA derived from said sample, a first primer, a third primer, and a thermostable DNA polymerase, in a second input well on a microfluidic device thereby creating a second reaction mixture; • performing at least one thermocycling event to each of said first reaction mixture and said second reaction mixture, • identifying the presence of nucleic acid resulting from performance of said thermocycling event to said first and second reaction mixtures; wherein the thermocycling event comprises bringing 'he temperature of said input well to a temperature sufficient to result in denaturing of substantially all of DNA present in the reaction mixture, followed by bringing the temperature of said input well to a temperature sufficient to allow said first primer and third primer to site specifically anneal to said DNA, followed by a temperature sufficient to allow polymerase activity of said thermostable DNA polymerase on said first primer and third primers annealed to said DNA;

wherein said first primer and third primer are chosen so as anneal to a nucleic acid region suspected of containing a SNP such that in the presence of said thermostable DNA polymerase, absent the presence of a SNP, and upon performance of at least one thermocycling event, an increase in the number of copies of the nucleic acid region suspected of containing a SNP occurs thereby creating wildtype amplified region;

wherein said first primer and second primer are chosen so as anneal to a nucleic acid region suspected of containing a SNP such that in the presence of said thermostable DNA polymerase, the presence of a SNP and upon performance of at least one thermocycling event, an increase in the number of copies of the nucleic acid region suspected of containing a SNP occurs thereby creating SNP amplified region;

wherein the thermostable DNA polymerase is not significantly irreversibly denatured or deactivated at the temperature sufficient to result in denaturing of substantially all of the nucleic acid present in the reaction mixture; and

wherein said second primer is chosen such that at the temperature sufficient to allow said first primer and third primer to site specifically anneal to said DNA said second primer will anneal only to nucleic acid in which the known SNP is present;

such that the presence SNP amplified region in said first reaction mixture indicates the presence of a SNP in the clinical sample. In one embodiment of the present invention, the sample is a clinical sample and the DNA is chromosomal DNA. In a further embodiment, the sample is a clinical sample and the DNA is mitochondrial DNA.

In a preferred embodiment the amplified region is less than 400 base pairs in length.

In another aspect the present invention provides for an apparatus for identifying a known single nucleotide polymorphism comprising

• A microfluidic device having

• At least one input well capable of receiving DNA from a sample

• At least one microfluidic capillary electrophoresis channel

• A temperature cycling system in thermal communication with at least one input well

• A detecting means capable of detecting the presence of nucleic passing through said at least one microfluidic capillary electrophoresis channel

• A first computer and • An electric field generator

Wherein said electric field generator is capable of imposing an electric field parallel to at least one of said at least one microfluidic capillary electrophoresis channels;

Wherein said temperature cycling system is controlled by a second computer, and

Wherein said detecting means is in digital communication with said computer.

In one embodiment, the detecting means comprises an illumination source wherein said illumination source is perpendicular to both the microfluidic device and the imaging means and said illumination source is capable of illuminating the capillary electrophoresis channel. In a more preferred embodiment, the illumination source is a diode laser and the imaging means is a CCD camera, and in an even more preferred embodiment, the same computer is in digital communication with the detecting means as is controlling the temperature cycling system. It is contemplated that the method and apparatus of the present invention may be further useful for detection of SNPs present in human and mammalian cells, as well as bacteria and virus, pathogenic or otherwise; it is also useful for detecting any type of point mutation which may be either inherited or somatically acquired in an individual organism, diseased cells, pathogens or to distinguish point mutations in the host from point mutations in the disease/pathogen.

The detection of SNPs and utility therefore, is not limited to pharmacogenomics, but is relevant in the areas of clonal expansion monitoring in oncology, mutation prevalence within cellular or organism populations, or geneology

The accompanying description illustrates preferred embodiments of the present invention and serves to explain the principles of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 shows a schematic of the microfludic chip and heating device of the present invention;

FIGURE 2 shows a schematic of the portable CE system of the present invention;

FIGURE 3 shows a 238G>C testing with a schematic diagram of RFLP genotyping with primers flanking the SNP of interest to amplify a PCR product that is then interrogated by the restriction enzyme producing specific fragments (A), with the typical band pattern on agarose gel electrophoresis of conventional RFLP (B) and representative electropherograms from microchip CE with fluorescence measured by arbitrary fluorescence units (y-axis) against time (x-axis);

FIGURE 4 shows a 460G>A testing with a schematic diagram of RFLP genotyping with primers flanking the SNP of interest to amplify a PCR product that is then interrogated by the restriction enzyme producing specific fragments (A), with the typical band pattern on agarose gel electrophoresis of conventional RFLP (B) and representative electropherograms from microchip CB with fluorescence measured by arbitrary fluorescence units (y-axis) against time (x-axis);

FIGURE 5 shows a 719A>G testing with a schematic diagram of RFLP genotyping with primers flanking the SNP of interest to amplify a PCR product that is then interrogated by the restriction enzyme producing specific fragments (A), with the typical band pattern on agarose gel electrophoresis of conventional RPLP (B) and representative electropherograms from microchip CB with fluorescence measured by arbitrary fluorescence units (y-axis) against time (x-axis);

FIGURE 6 shows a schematic diagram of AS-PCR where two separate PCR reactions allele specific primers are performed for each sample and amplification only occurs when sample template matches primer (A), the typical result from agarose gel electrophoresis of conventional AS-PCR, (B) and representative electropherograms from microchip CE with fluorescence measured by arbitrary fluorescence units (y-axis) against time (x-axis). Size standard and product peaks are as labelled (C);

FIGURE 7. shows electropherograms of RFLP genotyping of the 238G<C SNP for the TPMT *2 mutant allele performed on a portable microchip CE system.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As used herein, "microfluidic devices", sometimes termed "lab on a chip", "microfmidic chips" "microchips", "chips" or "microsystem platforms" refer to the result of applying microelectronic fabrication technologies to produce a network of wells and channels etched into glass and/or molded into polymers that are bonded to glass or silicon chips. Within these wells and microchannels, cells and reagents can be manipulated by a variety of methods including gravity feed, applying electric or magnetic fields and results detected by, for example, image analysis or optical means. Microfluidic chips provide for PCR reactions and analysis of PCR products (Footz, T.S. et al. Electrophoresis 22:3868 (2001); Obeid, P.J. et al.

Analytical Chemistry 75:288 (2003); Backhouse C.J. et al. Electrophoresis 24:1777 (2003)) . They enable high resolution separations through polymer-filled microchannels using capillary electrophoresis of e.g. multiple PCR products, and can exhibit ε high level of integration by combining multiple functions on a single chip, for example cell sorting and RT-PCR reactions for gene expression or genomic profiles of a given cell or population of cells (Backhouse, CJ. et al. Proceedings of the International Conference on MEMS, NANO and Smart Systems 377 (2003)). Within a microfluidic device, sample processing can be implemented and cells can be separated by a variety of means, including dielectrophoresis, and processed in a variety of ways, including analysis of HAS gene expression as shown here. In the future, microsystem platforms incorporating microdluidics chip-based sample processing and analysis may replace more conventional for application such as genotyping.

As used herein, the term "clinical sample" means a fluid or tissne originating from a human. The sample may either be unmodified, or alternatively the sample may be processed before introduction into the devices of the present invention. Processing is contemplate to include, but not be limited to, pH alteration, ion removal, in addition, cell separation, cell purification, cell removal, protein removal, cell lysis, enrichment of a cell of interest, nucleic acid enrichment, nucleic acid isolation, nucleic acid separation and nucleic acid purification; all of which give rise to a sample for analysis which would enrich the sample for the nucleic acid of interest, if present.

As used herein, a "single nucleotide polymorphism", or "SNP", is not limited to single nucleotide variations occurring with a given frequency within a population, rather it represents any single nucleotide variation within a nucleic acid sequence. It is explicitly contemplated that any number of disparate single nucleotide variations, including both acquired and inherited mutations, can occur within a nucleic acid sequence, and the present invention is not to be limited to detection of only one nucleic acid variation therein.

Several microchip-based techniques have been demonstrated for mutation detection, most are however appropriate for applications involving large-scale genotyping or mutational screening and also most require some amount of off-chip processing. A wide range of methods including microarray (Ramsay, G. Nat Biotechnol 16:40 (1998)), bead-based microfluidics (Verpoorte, E. Lab Chip 3:60N (2003)), and microelβctrophoretic platforms (Neuhoff, V. Electrophoresis 21 :3 (2000)) have been developed to achieve rapid detection of SNPs in microliter volumes (Ng, J.K. et al. AnalBioanal Chem 386:427 (2006)). However, to be cost-effective these platforms require extremely large sample sets (i.e. is greater than 10,000). Such large batch processing is not feasible in a clinical setting, in particular for the pharmacogenetic testing of acute lymphoblastic leukemia (ALL) patients, where even large treatment centers may see only a few hundred patients on a routine basis.

Such constraints also apply to genotyping for other types of polymorphisms. Batch processing ultimately delays the initiation of therapy and necessitates additional patient visits which threatens overall patient therapy and escalates costs. High throughput platforms require complex fabrication, extensive and expensive infrastructure, and highly trained staff for their operation and maintenance. TPMT genotyping using conventional approaches has been demonstrated by a number of techniques (Otterness, D. et al. Clin Pharmacol Ther 62:60 (1997); Spire-Vayron de Ia Moureyre, C. Hum Mutat 12:177 (1998); Schaeffeler, B., et al. Clin Chem 47:548 (2001); Haglund, S., ct al. Clin Chem 50:288 (2004)). PCR based methods initially designed to genotype a small number of samples (Spinney, L. The Scientist 19:22 (2005)) are far more suitable for clinical diagnostics than high-throughput detection methods developed for discovery, screening or validation of candidate SNPs as previously discussed.

Restriction fragment length polymorphism (RPLP) and allele specific PCR (AS-PCR) are simple and inexpensive genotyping methods based on the well-characterized specificity of primers and enzymes to recognize specific DNA sequences. In addition, RFLP has been previously shown to be advantageous for assay miniaturization on existing microfluidic platforms (Footz, T., et al. Analyst 129:25 (2004); Pal, R. et al. Lab Chip 5:1024 (2005)). RFLP utilizes the specificity of restriction enzymes to digest DNA at specific sequences. The presence of a SNP either creates or abolishes a restriction site and the genotype is detected by determining the size of an anaplicon flanking the SNP of interest. RFLP is widely used to perform SNP genotyping and in computational models is applicable to up to 85% of SNPs in NCBI database, dbSNP (Zhang, R. et al. Nucleic Acids Res 33:W489 (2005)). AS-PCR exploits the high fidelity of DNA polymerase to elongate only in the presence of an exact match of two allele-specific primers that anneal to a SNP at its V end, in two separate PCR reactions.

The present invention provides for PCR-based SNP detection strategies adapted to a microfluidic platform; to reduce volume requirements, achieve faster analysis times and higher sensitivity. Such miniaturization demonstrates a clinically feasible genotyping platform for single patient analysis. For SO patients undergoing thiopurine treatments two non- limiting embodiments the apparatus and method of the present invention successfully genotyped SNPs using two different methods, PCR-RFLP/CE and AS-PCR/CE; using three "signature" TPMT SNPs which demonstrated ?C0% concordance with conventional methods. The present invention demonstrates an entirely microchip based apparatus and method to genotype patients at the point-of-care for the presence or absence of the SNPs, and in one non-limiting embodiment, 3 SNPs in the TPMT gene. It is specifically contemplated that the present invention may be applied to other genotyping strategies to identify polymorphism(s). One skilled in the art will recognize that the choice of primers, temperature and buffer conditions using the apparatus and general methods of the present inventions, with appropriate modification, will result in the ability to detect SNPs, or even more significant genetic changes (such as gene re-arrangements, insertions of at least one nucleotide, deletions of at least one nucleotide, etc).

Microchip based methods are faster in analysis than conventional methods of RFLP and AS- PCR and significantly more portable and cheaper than commercially available protocols, such as TaqMan®. Microfluidic "chip-based" genotyping of SNPs offers the potential for a rapid and cost effective method to prospectively genotype individual patients in the clinic for the design of personalized drug therapy. The method and apparatus of the present invention provides for increased speed of testing, reduced cost through sample volume reduction and automated processing, thus ultimately eliminating the need for the highly trained human operator necessary with the prior art. Compared to conventional RFLP of the prior art, one embodiment of the present invention, microchip RFLP as presented herein, performs a genotype of the signature TPMT SNPs, with less initial DNA and no need for a purification step between PCR and enzyme digestion at room temperature, and subsequent identification of products in seconds by microchip CE. An alternate embodiment of the present invention, AS-PCR as presented herein, demonstrates single step genotyping on a single platform, eliminating the need for downstream reactions to discriminate alleles and the need for an operator to perform manual operations. This is the first demonstration of allele-specific amplification of a PCR product on a microchip and demonstrates the specificity of DNA polymerase to the 3' end of primers on a microchip. The central biochemistry of RFLP or AS- PCR demonstrated by the apparatus and method of the present invention offers the possibility to genotype patients within minutes.

It is further contemplated that a clinical sample be used in association with the method and apparatus of the present invention. In summary, the present invention provides for conventional PCR based assays, adapted and ported to a microfluidic platform testing has been validated using patient samples and compared to conventional technologies used in a clinical setting. Pharmacogenetic SNPs have been identified by an entirely chip based genotyping system.

Patient Samples

Patient DNA samples used in this study were from the development phase of the TPMT: Azathiopurine Response to Genotyping and Enzyme Testing (TARGET) study; an In progress randomized controlled trial to assess the clinical utility and relative cost effectiveness of the TPMT pharmacogenctic test for use in patients treated with azathiopurine for inflammatory conditions, in the United Kingdom. 5 mL blood samples were collected from 80 patients with inflammatory bowel disease (IBD) after informed consent and ethical approval. All patients were recruited from the Gastroenterology Department in Manchester Royal Infirmary, Manchester, UK. DNA extraction was carried out at National Genetics Reference Laboratory (Manchester, UK), using Autopure LS Large Sample Nucleic Acid Purification Instrument (GENTRA Systems, MN) according to the manufacturer's protocol and was stored in Nunc® vials (Nalgc Nunc®, Hereford, UK) at -20°C. DNA was quantitated using RNase P-assay on a 7900 HT Fast Real-Time PCR System (Applied Biosystems, UK) according to manufacturer's protocol and was normalized to a concentration of 30-50 ng/μl. Samples were blinded and tested in an anonymous manner. Positive controls for homozygous TPMT SNPs were supplied by the Evans (St. Jude's Hospital, Memphis, USA), Ashen (University Hospital Goettingen, Germany), and Zwicker (Institut fiir Klinischc Pharmakologie, Bremen, Germany) laboratories and confirmed by direct sequencing before use.

Conventional RFLP

The RFLP protocol first established for TPMT (Yates, CR. ct al. Am Intern Med 126:608 (1997)) has been used repeatedly in a number of subsequent studies (Sirot, E. J., et al. Drug

Safety 29:735 (2006)). However, the published protocols require significant amounts of DNA and subsequent purification steps before digestion. To adapt and port these protocols on-chip, the published protocols were farther optimized and streamlined as described below. These novel conditions were validated in a conventional approach and then further adapted to the microchip.

For the 238G>C SNP, shown in Fig. 3, primers designed to flank the SNP were used to create a 293bp PCR product as previously reported (Ma, X.L. et al. Zhongguo Shi Ycm Xue Ye Xue Za ZM 11:458 (2003)), but novel PCR conditions using significantly reduced amount of DNA (8x less), without purification of PCR products prior restriction digest. Thermal cycling conditions were as follows. Initial denaturing, 95°C (5 min); 30 cycles of DNA denaturing, 95°C (30 s); primer annealing, 60°C (30 s); dNTP polymerizing, 72°C (30s); final extension, 72°C (10 min). The PCR reaction mixture contained a final concentration of IxPCR buffer, 2.0 M MgCl₂, 20OnM dATP, dGTP, dCTP, and dTTP, 0.2uM of each primer, and IOng of genomic DNA. Amplification was carried about by 0.5U (0.1 μL) of Platinum Taq DNA Polymerase (Invitrogen Life Technology, Carlsbad, USA). Following PCR, the PCR product was incubated at 550C for 1 hour using 2.5 units (0.25μL) cf BsIl (New England Biolabs, Lpswich, MA, USA) and a volume of 1OX NEBuffer 3 (New England Biolabs, Lpswich, MA, USA) to bring the final concentration of the mixture to IX. BsIl digestion of mutant DNA yields 142bp and 138bp fragments while wildtype DNA does not contain the restriction site and remains undigested (Fig.3a). Digested products were analyzed by 2% agarose gel electrophoresis stained with ethidum bromide.

For the 460G>A SNP, shown in Fig.4, a similar RFLP assay was performed using previously reported primers (Yates, CR. et al. Ann Intern Med 126:608 (1997)) in conditions similar to those above except the final concentration Of MgCl₂ in the PCR mixture was 2.5μM and the restriction digest was performed with Mwol and NEBuffer 3 for 60 min at 60°C. Mwol digestion of wildtype DNA yields 267bp and 98bp fragments while mutant DNA does not contain the restriction site and remains undigested at 365bp.

For the 719A>G SNP, shown in Fig. 5, the same RFLP assay was performed using previously reported primers (Yates, CR. et al. Ann Intern Med 126:608 (1997)), except the final concentration of the PCR mixture contained 4.OuM of MgCl₂ and 0.02μM of the forward rrimer. The restriction digest was performed with Accl and NEBuffer 4 for 2 hours at 37°C. Accl digestion of mutant DNA yields 297bp and S6bp fragments while mutant DNA does not obtain the restriction site and

Microchip RFLP

On-chip RFLP was performed on microfluidic chips made of patterned poly(- dimethyl)siloxane (PDMS) bonded to a glass substrate as described previously (Ma, X.L. et al. Zhongguo Shi Yon Xue Ye Xue Za ZM 11:458 (2003)). A 3-port PCR chip was used in conjunction with the microvalving and thermal cycled using the custom built Peltier system, as described below. A 2 μL PCR reaction was performed in a well in the same reaction conditions as conventional RFLP, except that the amount of DNA polymerase was increased to 2.5 times to 1.25U (0.25 μL) and the forward primers used were labeled with a 5' VIC dye (Applied Biosystems, Foster City, USA). Following PCR, restriction digest was performed on the same microfluidic chip with the addition of 2.5U of the specified enzyme. The BsIl and Mwol on-chip digestions were performed at room temperature in to minutes while the Accl digest required incubation on the chip at 37°C for 2 hours. Following digestion, restriction fragments were identified by microchannel CE on a separate microchip as described below. Table 1 presents a listing of the primers used for each of the three tested SNPs.

Table 1: Summary of Primers and Restriction Enzymes for RPLP.

Conventional AS-PCR

As with RFLP, published protocols (Ya C.R. et al. Ann Intern tied 325:508 (1997)) for AS-PCR were further optimized in novel condition to port the prolocol to the microchip. Allele-specific primers for the 238G>C were used as previously reported (Yates, C.R. et al. Ann Intern Med 126:608 (1997)), with the novel conditions disclosed in "Microchip RFLP" above, however only 50ng of genomic DNA was used for each reaction, one eighth of the DNA that is required in previous conventional reports (400ng). Each sample was separately amplified in the presence of the forward wildtype, SEQ ID NO. 1, or mutant primer SEQ ID NO. 2 with a common reverse primer, SEQ ID NO. 3. The amplification occurred with the wildtype primer only in the presence of wildtype DNA (G238). Amplification occurred for the mutant primer only in the presence of mutant DNA (C238) producing a 256bp product.

TaqMan® SNP genotyping

TPMT SNP genotyping was performed by TaqMan® 5' allelic discrimination assay (Assays- by-design; Applied Biosystems, Warrington, XJK) according to manufacturer's instructions.

Briefly, each PCR reaction contained 2.5μl of TaqMan® Master Mix, 0.25 μl of 4OX assay mixture and 2.25 μl of distilled water. 5μl of this reaction mix was added to 30ng of DNA in a

96-well plate (ABgene Ltd., Epsom, UK) and PCR was performed using the following reaction conditions: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60"C for 1 minute. Upon completion of the reaction, genotype analysis was performed using the Sequence Detection Software Version 2.2.2 (Applied Biosystems), using the allele discrimination option and interpretation of genotypes were made. Integrated microchip PCR and CE for AS-PCR

AS-PCR was performed on a microchip 110 as shown in Fig. 1 made of patterned PDMS 102, fabricated using a soft lithography replica molding approach (Duffy, D.C., et al. Analytical Chemistry 70:4974 (1998)). PDMS layer 102 is irreversibly bonded to glass substrate 103. The single chip (PCR-CE chip) integrates microchip PCR with microchannel CE 101 with externally actuated pinch-off valves to manipulate fluids (not shown), as previously reported (Kaigala, G.V. et al. Electrophoresis 27:3753 (2006)). Sample is introduced into injection well 113 and transferred through microfluidic channel 114 to PCR chamber 104. On-chip AS- PCR in a volume of ~2μL was performed in PCR chamber 104, in similar conditions as described above with the same modifications for on chip RFLP, an increased amount of DNA polymerase and the use of 5' fluorescently labeled primers. Following amplification, 1 μL aliquot of the PCR product was transferred from the enclosed PCR chamber 104 into the open CE injection well 111 containing the above-mentioned mixture, 1 μL of size standard (GS 500) was included to this product and subsequently separation was performed in POP6 polymer as per the procedure described later. Various methods of fluid transfer are known in the art and contemplated by the present invention, for example a series of adjacent valves activated in sequence creating a microfluidic equivalent of peristaltic pumping are known by those skilled in the ait See, by way of non-limiting example, US 20040209354 by Mamies. Further, the details on integrated PCR-CE system are described elsewhere (Kaigala, G.V. et al. Electrophoresis 27:3753 (2006)).

AS-PCR uses unly PCR to determine genotype and requires no additional biochemical reactions to interrogate a SNP thus reducing the need for downstream reagents and incubation times. Furthermore, integration of PCR and CE eliminates the cost and delay for an operator to manually transfer PCR products to a separate platform for analysis. Ideally, the speed of AS-PCR is only limited the by the speed of mermo-cycling and CE. The apparatus and methods of the present invention demonstrate a rapid CE (requiring seconds) and with future work for rapid microchip PCR, genotype by AS-PCR is possible within minutes. In a clinical setting, a rapid pharmacogenetic test would allow for the immediate initiation of therapy without the need for the subsequent follow-up visit, which is required for batch process testing where the patient must return separately for test results. This testing strategy provides a yes/no answer. Since SNPs are present in all cells of the body, there is no need for quantitative assays.

Sequencing of microchip PCR product

Expected restriction digestion fragment patterns confirmed the identity of conventional and microchip PCR products for RFLP and further confirmed by nucleotide sequencing. Sequencing was performed conventionally using the BigDye Terminator v3.1 Cycle

Sequencing Kit on a capillrjy-bnsed ABI Prism 3100 DNA Sequencer (Applied Biosystems, Foster City, USA). The product is listed as SEQ ID NO. 1

Microchip heating instrumentation

A custom-built dual thermoelectric module (TEM) 106, 107 (9500/127/085B, FerroTec, Nashua, USA)) temperature cycling system 109 provides for rapid temperature transitions both during heating (5-6°C/s) and cooling (3-4°C/s) along with stable hold temperatures (±0.1 °C of the set point) within the thermocylced area 105. Thermal management, i.e. both heating and cooling is achieved using the TEM 106, 107 by appropriately controller the flow (and amount) of current by the drive electronics that is coordinated by a microcontroller (18F458, Microchip technologies Inc., USA). The TEMs are physically arranged in a cascade mode for improved performance and stacked between highly pure copper plates 112 to ensure uniform spreading of the heat across the TEM 106, 107; all supported by heat sink 108. Temperature within this TEM stake is measured using temperature sensors (LMSO, National Semiconductors, USA). The current flow through the TEMs regulates the heat-flow and thus the temperature within the chip. To ensure this current flow is sufficient to allow thermal cycling within the PCR reaction chamber with stable hold times and rapid transitions, the current flow is regulated by custom-built, software controlled, Proportional Integral Derivative (PID) controller and resides within the drive electronics, in the microcontroller Fluid handling is performed using robotic arms that make use of the elastic nature of PDMS, externally applying pressure at the valving points the fluid is confined during thermal cycling within the PCR chamber. It is also contemplated as part of the present invention, that pneumatic or hydraulic force may be used to impose a deformation, with the prior art describing a number of possible methods to enact the preferred embodiment of the present invention, with valving based upon the deformation of an elastic layer in a microfhiidic chip. Other valving methodologies are also contemplated by the present invention, including by way of non-limiting example, electromechanical or thermally actuated valves known in the art. Fluid handling and microchip fabrication is known in the art, for example PCT/CA2007/000959 which is incorporated by reference.

Microchip capillary electrophoresis (CE)

The microfluidic chips of the preferred embodiment of the present invention consist of 4 reservoirs or wells 111, 115, 116, 117, each ~3 μL in volume that are connected by two microchannels 101, 114 50 μm wide and 20μm deep in a simple cross configuration as described previously (Footz, T. et al. Electrophoresis 22:3868 (2001)). Fragment analysis was performed on these microchips using the μTK (Fig. 1) which provides a high voltage power supply and laser induced fluorescence (LIF) system that uses an excitation of 532nm and detection at 578nm. The untreated channel 101 of the microchips were filled with fresh aliquots of POP-6 polymer (Applied Biosystems, Foster City, USA) that were heated at 65°C for 10 minutes to ensure complete dissolution of all precipitates and to lower viscosity to facilitate loading of the polymer into the microchannels. The sample loading well 111 contains a mixture of 1 μL of the digested VIC-labeled PCR product, 0.5μL of a fluorescently labeled DNA ladder, GeneScan 500 TAMRA (Applied Biosystems Foster City, USA), 1.2 μL HiDi formamide (Applied Biosystems, Foster City, USA) and 0.3 μL of a IX genetic analysis buffer with EDTA (GABE, Applied Biosystems, Foster City, USA) that was denatured at 96°C for 5 minutes and rapidly cooled to approximately 40°C. Sample injection with 0.4 kV (60 s) followed by separation at 6kV (240 s) was performed with detection being at performed at 76mm from the intersection (Footz, T. et aL Electrophoresis 22:3868 (2001)). It is contemplated that detection may be performed at any number of distances from the intersection, the selection of the distance based upon the speed, resolution and presence of other nucleic acids of similar size as the nucleic acid of interest in the particular application. Portable CE system

A portable CE system (Fig.2) has. been designed which is based on a high voltage DC-to-DC converter 206, receiving DC power through power input 207 and capable of applying the electric fields contemplated herein, a minimum, parallel to at least one of the microchannels of microfluidic device 201. Electrophoresis is performed in this device according to the same experimental parameters as described for the μTK. As in the μTK, the maximum potential for electrophoresis is 6kV for the separation of the DNA fragments and the size standards. A means for illumination 205 is used as the excitation source for detecting the fluorescently tagged DNA. Different from the μTKs however, is that illumination occurs perpendicular to detection. This has been done to simplify the optics within the system. Beyond this, the cost of the system has also been significantly reduced by using imaging mean.: 204, capable of producing a digital signal upon receiving light from fluorescently tagged DNA. Optionally lensing means and filtering means are interposed between imaging means 204 and microfluidic device 201. In a preferred embodiment, imaging means 204 is a CCD based digital camera and illumination means 205 is a laser diode, and in an even more preferred embodiment the laser diode emits electromagnetic radiation at a wavelength of 635nm. Image processing is done externally on a PC in digital communication with the portable CE system through cable 208, to produce an electropherogram from the acquired images. Each image that is acquired is reduced to a single data-point in the electropherogram. The electropherogram is generated in real-time by applying the sampling period of the camera. The sampling period must be sufficient to fully capture the resulting signal. Satisfying the Nyquist criterion for this type of signal required a sampling period less than 1 second when electrophoresis is performed for 6kV experiments.

One skilled in the art will recognize that lens means can be fulfilled by those materials or apparatus capable of receiving and transmitting electromagnetic radiation of given wavelengths (for example, light) wherein the path of the transmitted light converges to a distal point. It is contemplated that lens means may be of various materials, by way of non-limiting example, glass or plastic; though fluids maintained in particular shapes and sizes are known in the art to perform as lens means. Though most lens means effect the convergence of the transmitted electromagnetic radiation through a difference in the refractive index of the lens means compared to the material surrounding the lens means, this is not contemplated as being a limitation necessary for the practising of present invention.

Illumination means are those devices or apparatus known in the art capable of emitting electromagnetic radiation containing, at least, a desired wavelength. This includes various incandescent or fluorescent sources, though in a preferred embodiment the illumination source provides a coherent emission of electromagnetic radiation, for example a laser. As disclosed herein, the laser may be a diode laser emitter, though the laser may also be sleected from gas, chemical, dye, metal-vapour, solid-state or semi-conductor lasers. One skilled in the art will recognize that the generation of the electromagnetic radiation is not required to be within the portable CE system, rather the electromagnetic radiation may be generated outside of the portable CE system and directed into and through the illumination means, by way of example, through fibre-optics. The desired wavelength is determined according to the absorption properties of the microfluidic device, as well as the absorption and emission spectra of any fluorescent marker associated with the nucleic acid sample present within the microfluidic channels.

Imaging means are those devices known in the art capable of receiving electromagnetic radiation of a desired wavelength, and converting the electromagnetic radiation into an electronic or digital signal capable of transmission to a computer, display, recording or storage device. It is contemplated that lens means and imaging means may be incorporated as a single device.

Through the present invention utilizes a fluorescent marker for detection of nucleic acid within a microfluidic channel, other means for nucleic acid detection are known in the art, and contemplated as part of the present invention. Such nucleic acid detection means include, but are not limited to, surface plasmon resonance and electrochemical reduction. It is contemplated by the present invention that detection of nucleic acids includes such other means of detection.

Filtering means are those materials which are semi-transmissive of electromagnetic radiation, wherein only electromagnetic radiation of a desired wavelength is transmitted through, to, for example, the imaging means. It is contemplated that filtering means is used to substantially restrict the electromagnetic radiation received by the imaging means to that electromagnetic radiation resulting from the fluorescence of the fluorescent markers associated with nucleic acids in the micfofluidic channels. Therefore it is contemplated that the filter means will be selected so as to be transmissive to at least the wavelength of light resulting from fluorescence of the fluorescent markers associated with nucleic acids in the microfluidic channels.

EXAMPLE 1: Microchip RFLP

The TPMT genotype was determined in a population of 80 patients undergoing thiopurine immunosuppression. This population was enriched with known variants but the genotype was kept blinded until results were compared between microchip RFLP (Fig. 3 a, 4a, 5a) and conventional RFLP which acted as an internal reference. For this study, as Ae external gold standard a commercially available Taqman® assay was performed according to the manufacturer's protocol at the National Genetics Reference Laboratory, Manchester, UK. Conventional RFLP, as yet another control for the intermediate step, produced restriction fragment patterns on gel electrophoresis as expected from previous reports (Fig. 3b, 4b, 5b).

Three SNPs were used 238G>C (Fig. 3), 460G>A (Fig. 4) and 719A>G (Fig. 5).

Microchip RFLP produced electropherograms with patterns of fluorescent intensity peaks (Fig. 3c, 4c, 5c) similar to band patterns on the conventional gel electrophoresis, enabling clear genotyping based on fragment size. Restriction digest fragments in all cases were of greater or equal fluorescence intensity to DNA size standards facilitating rapid and easy determination of restriction fragment patterns even with a monochromatic detection system as in the μTK. Microchip RFLP uses 8 fold reduced levels of patient DNA and required no purification steps between PCR and restriction digest, in contrast to conventional techniques in previous reports (Yates, CR. et a). Ann Intern Med 126:608 (1997)). Compared to conventional RFLP, which requires an additional purification step after PCR and a one-hour incubation at elevated temperature, BsIl and Mwol digests on the chip were performed directly on the same chip without purification and at room temperature in 10 minutes. This significant shortening of the digestion time likely reflects the reduced volume of the reaction, and the consequent reduced requirement for DNA, thereby allowing the reaction to reach completion a shorter time and at room temperature. Further acceleration of the overall assay protocol on microchip occurs during on-chip CE which takes seconds rather then the minutes to hours required for conventional CB on a capillary DNA analysis system or slab gel electrophoresis. Overall, PCR-RFLP/CE takes 3 hours on-chip, using the apparatus and method of the present invention, as compared to at least 9 hours using conventional methods and conventional instruments. It is contemplated that further reduction in time is possible, with satisfactory results possible in under one hour.

The size of restriction digest fragments resolved in the present work, 87bp to 365bp, demonstrates that a wide range of fragment sizes can be resolved on-chip. Further, the close proximity of the fluorescently labeled primer for the 719A>G SNP demonstrates the versatility m being able to resolve restriction digest fragments smaller than 100bp by microchip CE. This is a first step to establish the use of mismatch primers (Shinder, G.A., et al. MoI Carcinog 7:263 (1993)), primers with an internal mismatch to the template to create an artificial restriction site, for SNPs having no natural restriction site.

Among the 80 patients tested by microchip RFLP, 1 was heterozygous for the 238G>C SNP (TPMT*2), and 5 were both heterozygous for the 460G>A SNP, and 719A>G SNPs (TPMT*3A). No bomozygotes were detected for any of the three signature SNPs among the population set studied. Microchip RFLP detected homozygotes for all three SNPs in DNA from known positive controls, derived from patients known to be deficient in TPMT activity. Table 2 shows a comparison between microchip RFLP, conventional RFLP, and Taqman® demonstrating 100% concordance between all of the methods. In comparison to gold standards, microchip RFLP matches the sensitivity and specificity for genotyping TPMT SNPs in clinical samples.

Table 2: Comparison of genotyping results between conventional RFLP, microchip RFLP

EXAMPLE 2: Microchip-based Integrated AS-PCR-CE

To demonstrate SNP genotyping with a single biochemical reaction on a single integrated microchip, AS-PCR was shown to successfully genotype (in duplicates) the 238G>C SNP. DNA polymerase will only elongate in the presence of an exact match between the primer and the template, particularly at the 3* end. AS-PCR utilizes this high fidelity of DNA polymerase with 2 allele specific primers whose 3' nucleotide anneals to either the wildtype or mutant allele. These allele specific primers are used in 2 separate PCR reactions with a common reverse primer. Amplification in the presence of the specific primer indicates the genotype. The specificity of allele-specific primers were tested on-chip on known positive controls and confirmed the amplification of PCR product by a primer only in the presence of the corresponding template (Fig. 6a). This test was performed on an integrated microchip wherein both PCR and CE can be performed and referred to as PCR-CE chip. Results from the integrated PCR-CB chips produced electropherograms (Fig. 6c) clearly showing the expected product peak at 256bp. The results correlate with that observed using conventional techniques (Fig. 6b). This sizing is performed relative to the DNA size ladder used during the CE. In me case of the homozygotes (wt/wt and mt/mt), there is a significantly more intense product peak compared to the heterozygote (wt/mt). These weaker intensity peaks in the heterozygote are expected since there is approximately half as much template compared to homozygotes, however, the product peak remains clearly distinguishable from size standards to enable making a reliable allele determination.

For the future, multiplexing is an attractive method to simplify the genotyping reactions described in this study mat would definitely result in further reduced reagent usage. Multiplex AS-PCR, has the potential for simultaneous testing of multiple SNPs through the use of creative primer design and tagging. Multiple SNPs have been distinguished using, for example, oligonucleotide tagged PCR primers with fluorescent tags to create fragments of specific size that are discriminated by gel electrophoresis. It is contemplated that the use of multiple fluorescent labels with the apparatus and methods of the present invention is possible, to discriminate between witdtype and mutant alleles in addition to distinguishing different SNPs by fragment length to in a single multiplex reaction designed to genotype multiple SNPs. Such implementation would be possible using the methods and compounds for multiple fluorescent labelling, presently known in the art.

EXAMPLE 3 : Portable microchip CE

Purified RFLP products were analyzed on the portable CE system of the present invention to demonstrate its efficacy for clinically based genotyping. For all three variants sufficient resolution in separation, and appropriate sizing with monochromatic detection is observed. This has been demonstrated in the discrimination capability between alleles (mt/mt, wt/mt, wt/wt) of the 238G>C SNP (Fig. T). As shown in Fig. 7, size standard and product peaks are as labelled, with fluorescence extracted from the integrated CCD camera intensity values (y- axis) against time (x-axis). A single, fully digested, peak at 142bp indicates a homozygote mutant and is labelled in Fig. 7, as in other figures, as Mt/Mt. A full length product peak at 281bp and a digested peak at 142bp indicates the presence of both alleles and is labelled in Fig. 7, as in other figures, as MtAVt. A single full length product peak at 281 bp indicates a homozygote wildtype and is labelled in Fig. 7, as in other figures, as Mt/Mt Using AS-PCR, homozygotes are clearly distinguishable from size standards. As discussed above, for heterozygotes, the PCR product peak intensities are reduced but still remain clearly distinguishable from size standards. The resolution in separation of the portable system compared favorably with the μTK, and is more than sufficient for a confident allele call using RFLP. Multicolor detection systems are being developed to detect PCR products from multiplexed reactions, for point-of-care diagnostics.

While particular embodiments of the present invention have been described in the foregoing, it is to be understood that other embodiments are possible within the scope of the invention and are intended to be included herein. It will be clear to any person skilled in the art that modifications of and adjustments to this invention, not shown, are possible without departing from the spirit of the invention as demonstrated through the exemplary embodiments. The invention is therefore to be considered limited solely by the scope of the appended claims.

Claims

What is claimed is:

1. A method for identifying a known single nucleotide polymorphisms (SNPs) present in a sample using a microfluidic device comprising

- providing DNA derived from said sample, at least two primers and a thermostable DNA polymerase, in at least one input well on a microfluidic device thereby creating a reaction mixture;

- performing at least one thermocycling event to said reaction mixture

wherein the thermocycling event comprises bringing the temperature of said input well to a temperature sufficient to result in denaturing of substantially all of the nucleic acid present in the reaction mixture, followed by bringing the temperature of said input well to a temperature sufficient to allow said at least two primers to site specifically anneal to said DNA, followed by a temperature sufficient to allow polymerase activity of said thermostable DNA polymerase on said primers annealed to said DNA; and

- introducing into said reaction mixture a restriction endonuclease at a temperature sufficient to allow site specific cleavage by said restriction endonuclease of substantially all nucleic acid present;

- identifying the presence of nucleic acids resulting from performance of said at least one thermocycling event and introduction of said restriction endonuclease from said reaction mixture;

wherein said at least two primers are chosen so as to anneal upstream and downstream of a nucleic acid region suspected of containing a SNP such that in the presence of said thermostable DNA polymerase and upon performance of at least one thermocycling event, an increase in the number of copies of the nucleic acid region suspected of containing a SNP occurs thereby creating an amplified region; wherein the thermostable DNA polymerase is not significantly irreversibly denatured or deactivated at the temperature sufficient to result in denaturing of substantially all of the nucleic acid present in the reaction mixture; and

wherein the restriction endomuclease is selected such that it effects a cleavage event upon binding to a sequence which may be altered due to the presence of a SNP present within said nucleic acid region suspected of containing a SNP;

such that the presence or absence of a fragment resulting from restriction endonuclease activity indicates the presence or absence of a SNP in the sample.

2. The method of claim 1 wherein the sample is a clinical sample and the DNA is chromosomal DNA.

3. The method of claim 2 wherein the amplified region is less than 400 base pairs in length.

4. The method of claim 3 wherein said primers are SEQ ID NO. 5 and SEQ ID NO, 6, the restriction endonuclease is Bs11 and the SNP is 238G>C associated with reduced TPMT enzyme activity.

5. The method of claim 3 wherein said primers are SEQ ID NO. 7 and SEQ ID NO. 8, the restriction endonuclease is Mwol and the SNP is 460G>A associated with reduced TPMT enzyme activity.

6. The method of claim 3 wherein said priiners are SEQ ID NO. 9 and SEQ ID NG. iθ_: the restriction endonuclease is Acc1 and the SNP is 719A>G associated with reduced TPMT enzyme activity.

7. A method for identifying a known single nucleotide polymorphisms (SNPs) present in a sample using a microfluidic device comprising - providing DNA derived from said sample, a first primer, a second primer and a thermostable DNA polymerase, in a first input well on a microfluidic device thereby creating a first reaction mixture;

- providing DNA derived from said sample, a first primer, a third primer, and a thermostable DNA polymerase, in a second input well on a microfluidic device thereby creating a second reaction mixture;

- performing at least one thermocycling event to each of said first reaction mixture and said second reaction mixture,

- identifying the presence of nucleic acid resulting from performance of said thermocycling event to said first and second reaction mixtures;

wherein the thermocycling event comprises bringing the temperature of said input well to a temperature sufficient to result in denaturing of substantially all of the nucleic acid present in the reaction mixture, followed by bringing the temperature of said input well to a temperature sufficient to allow said first primer and third primer to site specifically anneal to said DNA, followed by a temperature sufficient to allow polymerase activity of said thermostable DNA polymerase on said first primer and third primers annealed to said DNA;

wherein said first primer and third primer are chosen so as anneal to a nucleic acid region suspected of containing a SNP such that in the presence of said thermostable DNA polymerase, absent the presence of a SNP, and upon performance of at least one thermocycling event, an increase in the number of copies of the nucleic acid region suspected of containing a SNP occurs thereby creating wildtype amplified region;

wherein said first primer and second primer are chosen so as anneal to a nucleic acid region suspected of containing a SNP such that in the presence of said thermostable DNA polymerase, the presence of a SNP and upon performance of at least one thermocycling event, an increase in the number of copies of the nucleic acid region suspected of containing a SNP occurs thereby creating SNP amplified region; wherein the thermostable DNA polymerase is not significantly irreversibly denatured or deactivated at the temperature sufficient to result in denaturing of substantially all of the nucleic acid present in the reaction mixture; and

wherein said second primer is chosen such that at the temperature sufficient to allow said first primer and third primer to site specifically anneal to said DNA said second primer will anneal only to DNA in which the known SNP is present;

such that the presence SNP amplified region in said first reaction mixture indicates the presence of a SNP in the sample.

8. The method of claim 7 wherein the sample is a clinical sample and the DNA is chromosomal DNA.

9. The method of claim 8 wherein the SNP amplified region or wildtype amplified region is less than 400 base pairs in length.

10. The method of claim 9 wherein the presence of SNP amplified region in said first reaction chamber and absence of wildtype amplified region in said second reaction chamber indicates a clinical sample homozygous for the SNP.

11. The method of claim 9 wherein the presence of SNP amplified region in said first reaction chamber and presence of wildtype amplified region in said second reaction chamber indicates a clinical sample heterozygous for the SNP.

12. The method of claim 9 wherein said first primer is SEQ ID No. 3, said second primer is SEQ ID NO. 2, said third primer is SEQ ID NO. 1 and the SNP is 238G>C A associated with reduced TPMT enzyme activity.

13. An apparatus for identifying a known single nucleotide polymorphism in a sample comprising

A microfluidic device having

At least one input well capable of receiving DNA from a sample At least one microiluidic capillary electrophoresis channel

A temperature cycling system in thermal communication with at least one input well

A detecting means capable of detecting the presence of nucleic passing through said at least one microfluidic capillary electrophoresis channel

A first computer and

An electric field generator

Wherein said electric field generator is capable of imposing an electric field parallel to at least one of said at least one microfluidic capillary electrophoresis channels;

Wherein said temperature cycling system is controlled by a second computer; and

Wherein said detecting means is in digital communication with said computer.

14. The apparatus of claim 13 wherein said detecting means comprises an illumination source, and an imaging means wherein said illumination source is perpendicular to both the microfluidic device and the imaging means and said illumination source is capable of illuminating said capillary electrophoresis channel.

15. The apparatus of claim 14 wherein said illumination source is a diode laser and said imaging means is a CCD camera.

16. The apparatus of claims 13, 14, and 15 wherein said first and second computers are the same computer.