Sample processing for nucleic acid amplification

[0001] The invention relates to the field of diagnostic assays, in particular, nucleic acid amplification-based assays conducted using biological samples.

[0002] Nucleic acids are used frequently in the clinical setting in, for example, the identification of genetic mutations and in the diagnosis of bacterial and/or viral infections. In general, such methods have used nucleic acids isolated or extracted from biological samples.

[0003] The most commonly used method for isolating DNA from a biological sample (e.g., blood) involves lysing the cells contained in the sample with a combination of a proteolytic enzyme and a detergent followed by extracting the mixture with an organic solvent, e.g., phenol and chloroform, so that the DNA enters the aqueous phase and the protein enters the organic phase. The DNA in the aqueous phase is then concentrated by alcohol precipitation and resuspended in a suitable volume for analysis. Such methods are, however, time-consuming and require the use toxic reagents.

[0004] Methods of stabilizing DNA in body fluids are known (e.g., U.S. Patent Application Publication No. 2003-104251).

[0005] Provided herein are methods for using biological samples as a source of nucleic acid template in nucleic acid amplification assays without having to extract the nucleic acids from the sample. The biological sample is contacted with a solution that functions to make the nucleic acid in the sample available for use in amplification without the need for extraction of the nucleic acids. The solution will be referred to herein as “nucleic acid processing solution” (NAPS). Such methods require less sample handling and are quicker and more cost-efficient than methods that require nucleic acid extraction.

[0006] In a first aspect, there is provided a method for amplifying nucleic acids from a cell sample, wherein the method comprises, contacting the cell sample with a nucleic acid processing solution (NAPS) having a chelating agent, a denaturing agent, and a buffering agent to form a modified cell sample, wherein the NAPS has a pH between about 5 and about 11, and using the modified cell sample as a template in a nucleic acid amplification reaction.

[0007] In a second aspect, there is provided a method for amplifying nucleic acids from a cell sample, wherein the method comprises,

[0008] contacting the cell sample with a nucleic acid processing solution (NAPS) having a chelating agent, a denaturing agent, and a buffering agent to form a modified cell sample, wherein the NAPS has a pH between about 5 and about 11, and wherein the modified cell sample provides the template nucleic acids, and

[0009] amplifying the template nucleic acids, wherein the template nucleic acids have not been extracted from the modified cell sample.

[0010] In a third aspect, there is provided a method for amplifying nucleic acids from an acellular body fluid sample, wherein the method comprises, contacting the acellular body fluid sample with a nucleic acid processing solution (NAPS) having a chelating agent, a denaturing agent, and a buffering agent to form a modified acellular body fluid sample, wherein the NAPS has a pH between about 5 and about 11, and using the modified acellular body fluid sample as a template in a nucleic acid amplification reaction.

[0011] In a fourth aspect, there is provided a method for amplifying nucleic acids from an acellular body fluid sample, wherein the method comprises,

[0012] contacting the acellular body fluid sample with a nucleic acid processing solution (NAPS) having a chelating agent, a denaturing agent, and a buffering agent to form a modified acellular body fluid sample, wherein the NAPS has a pH between about 5 and about 11, and wherein the modified acellular body fluid sample provides the template nucleic acids, and

[0013] amplifying the template nucleic acids, wherein the template nucleic acids have not been extracted from the modified acellular body fluid sample.

[0014] In particular embodiments, the NAPS has a pH from about 5.0 and about 11.0, preferably about 6.0 to about 11.0, preferably 7.5 to about 10.0, and more preferably about 7.0. In embodiments wherein DNA is the template for direct PCR, a pH from about 7.0 to about 10.0, or about 7.5, about 8.0, or about 8.0 to about 9.0 can be used. In embodiments wherein RNA is the template for direct or indirect PCR (e.g., RT-PCR), a pH from about 5.0 to about 7.0, desirably from about 6.5 to about 6.8 can be used. A buffer can be added to the NAPS to maintain the pH in a constant range. Such buffers are well-known in the art and include HEPES, TRIS, carbonate buffer, or BES.

[0015] The NAPS includes one or more chelating agents to form complexes with metal ions. Without wishing to be bound thus, it is believed that the chelating agent prevents metal ions from binding to DNA, removes metal ions that have already bound to DNA, or bind to metal ions (e.g., Fe(II)/Fe(III) or Cu(I)/Cu(II)) strongly enough to inhibit their redox cycling, and hence, the formation of reactive oxygen species. The chelating agent can be selected from the group consisting of: ethylenediamine tetraacetic acid (EDTA), cyclohexane diaminetetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetet-raacetic acid (TETA), and desferrioximine, or chelator analogs thereof. Desirably, the chelating agent is cyclohexane diaminetetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA), tetraazacyclododecanetetraace-tic acid (DOTA), and desferrioximine, and most desirably, the chelating agent is cyclohexane diaminetetraacetate (CDTA). Stronger chelators (i.e., chelators with a higher dissociation constant than EDTA when bound to a metal) include, but are not limited to, CDTA, DTPA, DOTA, TETA, and desferioximine, or chelator analogs thereof may be used in the NAPS as the only chelating agent or may be used in combination with another chelating agent. The amount or concentration of the chelating agent will depend upon its affinity for metal, which may need to be determined empirically. For CDTA, concentrations in the 1-20 mM range are sufficient, however other concentrations would work.

[0016] The NAPS includes one or more denaturing agents such as a detergent, chaotrope, or alcohol. Preferred denaturing agents can be selected from the group consisting of: urea, sodium dodecyl sulfate, dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate, perchlorate, and an alcohol. Preferably, the denaturing agent is urea, dodecyl sulfate, or an alcohol, wherein the alcohol is 10%, 20%, 30%, 40%, 50%, or even 60% of the total NAPS volume. Suitable alcohols include methanol, ethanol, n-propanol, isopropanol, n-butanol, trifluoroethanol, phenol, or 2,6-di-tert-butyl-4-methylphenol.

[0017] In certain embodiments, the NAPS further comprises one or more reducing agents. Suitable reducing agents include ascorbic acid, dithionite, erythiorbate, N-acetylcysteine, cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, dierythritol, a resin-supported thiol, a resin-supported phosphine, vitamin E, and trolox, or salts thereof. Preferably, the reducing agent is ascorbic acid, erythiorbate, N-acetylcysteine, dithiothreitol, or 2-mercaptoethanol, and more preferably, the reducing agent is ascorbic acid or 2-mercaptoethanol. In particular embodiments, the concentration of the reducing agent in the NAPS is greater than or equal to 50 millimolar. Antioxidant free-radical scavengers are also useful as reducing agents. Examples include antioxidant vitamins, antioxidant hormones, antioxidant enzymes, thiols, and phenols.

[0018] In some embodiments, the NAPS may further comprise an antimicrobial agent. “Antimicrobial agent” as used herein refers to a substance that reduces the rate of growth of a microbial organism compared to the rate of growth of the microbe in the absence of that substance. A reduction in the rate of growth of a microbe may be by at least 5%, preferably, by at least 10%, more preferably, by at least 20%, at least 50%, or at least 75%, and most preferably, by 90% or more. Antimicrobial agent includes substances that affect the viability, virulence, or pathogenicity of a microbe. An antimicrobial agent can be naturally occurring (e.g., derived or obtained from bacteria), synthetic, or recombinant. An antimicrobial agent may have static, cidal, or both properties. An antimicrobial agent is static if it inhibits cell division without affecting the viability of the inhibited cell. An antimicrobial agent is cidal if it causes cell death. Cell death is commonly detected by the absence of cell growth in liquid growth medium (e.g., absence of turbidity) or on a solid surface (e.g., absence of colony formation on agar). Those of skill in the art know that a substance or group of substances which is static at a given concentration may be cidal at a higher concentration. Certain static substances are not cidal at any concentration. Preferably, the solution includes an alcohol as an antimicrobial agent, preferably ethanol.

[0019] In still other embodiments, the NAPS further includes a ribonuclease inhibitor. Preferred ribonuclease inhibitors are selected from the group consisting of: heparin, heparan sulfate, oligo(vinylsulfonic acid), poly(vinylsulfonic acid), oligo(vinylphosphonic acid), and poly(vinylsulfuric acid), or salts thereof. The inclusion a ribonuclease inhibitor in the solution is particularly preferred when template nucleic acid in the sample is to be RNA, particularly mRNA, or when the template nucleic acid is from a microbe.

[0020] As used herein, the term “biological sample” refers to any liquid or solid material that is obtained from a biological source, preferably from an animal, most preferably from a human. A biological sample may be a cell sample or may be an acellular liquid sample. In preferred embodiments, the acellular liquid sample is an acellular body fluid sample.

[0021] As used herein, the term “cell sample” includes any source of cells containing nucleic acids that are desired to be used as a template in a nucleic acid amplification reaction. Cells may be prokaryotic or eukaryotic. Eukaryotic cell samples may be animal or plant cells. Preferred eukaryotic cell samples are mammalian cells, preferably human. In some embodiments, a cell sample can be cells in culture or a tissue sample from an animal, most preferably, a human. Tissue samples include, but are not limited to, blood, bone marrow, cell-containing body fluids such as cerebrospinal fluid, or tissue (e.g., biopsy material). Preferred samples include whole blood or white blood cells (WBC). Cell samples may be packed cells or cells suspended in liquid.

[0022] The terms “body fluid” or “bodily fluid” are used interchangeably herein and refer to a fluid sample from a human or other animal. Body fluids include, but are not limited to amniotic fluid, blood, cerebrospinal fluid, peritoneal fluid, plasma, pleural fluid, saliva, semen, serum, sputum, tears, and urine. Body fluids may be cell-containing or may be acellular.

[0023] As used herein “acellular body fluid” refers to a body fluid lacking cells. Such acellular body fluids are generally produced by processing a cell-containing body fluid by, for example, centrifugation or filtration, to remove the cells. Acellular body fluid, however, may contain cell fragments or cellular debris. Preferred acellular body fluids are plasma and serum.

[0024] As used herein “modified biological sample” or “modified sample” are used interchangeably and refers to a biological sample that has been processed so that the nucleic acids contained therein may be directly amplified by a standard nucleic acid amplification method. Nucleic acids in a modified biological sample need not be extracted from the sample. Preferably, modified biological samples are the result of contacting a biological sample with a NAPS comprising a chelating agent, a denaturing agent, and a buffering agent, as described herein. Modified samples include modified cell samples and modified acellular body fluid samples.

[0025] As used herein “modified cell sample” refers to a cell sample that has been processed so that the nucleic acids contained therein may be directly amplified by a standard nucleic acid amplification method. Nucleic acids in a modified cell sample need not be extracted from the sample. Preferably, modified cell samples are the result of contacting a cell sample with a NAPS comprising a chelating agent, a denaturing agent, and a buffering agent, as described herein.

[0026] As used herein “modified acellular body fluid sample” refers to an acellular body fluid sample that has been processed so that the nucleic acids contained therein may be directly amplified by a standard nucleic acid amplification method. Nucleic acids in a modified acellular body fluid sample need not be extracted from the sample. Preferably, modified acellular body fluid samples are the result of contacting an acellular body fluid sample with a NAPS comprising a chelating agent, a denaturing agent, and a buffering agent, as described herein.

[0027] As used herein, “nucleic acid” refers broadly to genomic DNA, segments of a chromosome, segments or portions of DNA, cDNA, and/or RNA. Nucleic acid may be derived or obtained from an originally isolated nucleic acid sample from any source (e.g., isolated from, purified from, amplified from, cloned from, reverse transcribed from sample DNA or RNA). Nucleic acids include those resident in a biological sample, preferably a cell sample or an acellular body fluid sample.

[0028] As used herein the term “extracted” used in reference to nucleic acids in a cell sample means that the nucleic acids have been physically separated from cells containing the nucleic acid by addition of a sufficient volume of organic solvent to lyse the cells and separate the protein from the nucleic acids, wherein the nucleic acid in the aqueous phase is separated from the protein. The nucleic acids in the aqueous phase can be concentrated by addition of a sufficient volume of ethanol to precipitate the nucleic acids. Other methods of extracting nucleic acids include the capture of nucleic acids on solid phase using, for example, an oligonucleotide-coupled bead (e.g., oligo-dT). When used in reference to nucleic acids in an acellular body fluid, extracted means that the nucleic acids have not been separated from the sample by, for example, alcohol precipitation.

[0029] In some embodiments, the method may include a centrifugation step. For example, a centrifugation step may be used to concentrate the cells of the cell sample prior to the addition of NAPS. In another example, a centrifugation step may be used to remove cellular debris from a modified cell sample. Such a centrifugation step is not considered an extraction of nucleic acids.

[0030] “Genomic nucleic acid” or “genomic DNA” refers to some or all of the DNA from the nucleus of a cell. Genomic DNA may be intact or fragmented (e.g., digested with restriction endonucleases by methods known in the art). In some embodiments, genomic DNA may include sequence from all or a portion of a single gene or from multiple genes, sequence from one or more chromosomes, or sequence from all chromosomes of a cell. In contrast, the term “total genomic nucleic acid” is used herein to refer to the full complement of DNA contained in the genome of a cell. As is well known, genomic nucleic acid includes gene coding regions, introns, 5′ and 3′ untranslated regions, 5′ and 3′ flanking DNA and structural segments such as telomeric and centromeric DNA, replication origins, and intergenic DNA.

[0031] As used herein, the term “hybridize” or “specifically hybridize” refers to a process where two complementary nucleic acid strands anneal to each other under appropriately stringent conditions. Hybridizations are typically and preferably conducted with probe-length nucleic acid molecules, preferably 20-100 nucleotides in length. Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, N.J.

[0032] The term “amplification” or “amplify” as used herein means one or more methods known in the art for copying a target or template nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. A target or template nucleic acid may be either DNA or RNA. The sequences amplified in this manner form an “amplified region” or “amplicon.” While the exemplary methods described hereinafter relate to amplification using the polymerase chain reaction (“PCR”), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.). The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, Calif. 1990, pp 13-20; Wharam et al., Nucleic Acids Res. 2001 Jun. 1; 29(11):E54-E54; Hafner et al., Biotechniques 2001 April; 30(4):852-6, 858, 860 passim. Further amplification methods suitable for use with the present methods include, for example, reverse transcription PCR (RT-PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence based amplification (NASBA) reaction, self-sustained sequence replication (3SR), strand displacement amplification (SDA) reaction, boomerang DNA amplification (BDA), Q-beta replication, or isothermal nucleic acid sequence based amplification.

[0033] As used herein, the phrase “using the modified cell sample as a template in a nucleic acid amplification reaction” means that the nucleic acids have not been extracted from the modified cell sample prior to amplifying the template nucleic acids contained therein. Similarly, the phrase “using the modified acellular body fluid sample as a template in a nucleic acid amplification reaction” means that the nucleic acids have not been extracted from the modified acellular body fluid sample prior to amplifying the template nucleic acids contained therein.

[0034] “Sense strand” means the strand of double-stranded DNA (dsDNA) that includes at least a portion of a coding sequence of a functional protein. “Anti-sense strand” means the strand of dsDNA that is the reverse complement of the sense strand.

[0035] As used herein, a “forward primer” is a primer that anneals to the anti-sense strand of dsDNA. A “reverse primer” anneals to the sense-strand of dsDNA.

[0036] As used herein, the term “about” when used in reference to a numerical value, means plus or minus 10%.

[0037] The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.

[0038] The methods of the present invention can be used to prepare biological samples for use in nucleic acid amplification reactions without extracting the nucleic acids from the rest of the sample. This method is achieved by mixing the biological sample with NAPS to form a modified biological sample, which can be used directly as template in an amplification reaction. In preferred embodiments, the biological sample is a cell sample or an acellular body fluid sample.

[0039] In some embodiments, NAPS may be added directly to cell samples or may be added to packed cells obtained from cell samples in which the cells have been pelleted by centrifugation. NAPS is added to the cell sample at a ratio of, for example, 1:1, 1:2, 1:3, 1:4, 1:5 or higher of cell sample to NAPS. The actual ratio depends on the cell concentration. In preferred embodiments, the cell sample is whole blood and is mixed with NAPS at a ratio of, for example, 1:1, 1:2, 1:3, 1:4, 1:5, or higher of blood to NAPS. Cell samples in which the cells have been concentrated and thus have a higher density of cells (e.g., a packed cell sample) may require more NAPS than those cell samples in which the cells have not been concentrated. For example, in processing a sample of packed cells, a volume of NAPS equivalent to 2-3 times (or higher) the volume of the packed cells may be used.

[0040] In one example, a NAPS of 2% SDS, 10 mM EDTA, and 50 mM Tris-HCl pH 8.0 is added to a sample of blood to prepare the sample for direct use in PCR. In another example, a NAPS of 0.5M NaOAc, 0.2M Tris-HCl, 0.15M sodium ascorbate, 10 mM CDTA, 1% SDS, 30% (v/v) ethanol, pH 9.5 (see PCT International Publication No. WO03104251 at Table 3, lane 1) is added to a sample of blood to prepare the sample for direct use in PCR. In a further example, the NAPS can be the DNA preserving solution contained in the Oragene™ DNA Self-Collection Kit (DNA Genotek, Ottawa, Ontario, Canada). In this case, a blood sample is applied to the sample reservoir, the reservoir is capped, releasing the DNA preserving solution into the chamber containing the blood, thus stabilizing the DNA that is present. The composition of additional NAPS are described in PCT International Publication No. WO03104251 or U.S. Patent Application Publication No. 2003-104251 (see “compositions”, at, for example, Table 3).

[0041] In other embodiments, NAPS may be added directly to acellular body fluid samples. NAPS is added to the acellular body fluid sample at a ratio of, for example, 1:1, 1:2, 1:3, 1:4, 1:5 or higher of acellular body fluid sample to NAPS. The actual ratio may depend on the type of acellular body fluid and the concentration of nucleic acids contained therein. In preferred embodiments, the acellular body fluid sample is plasma and is mixed with NAPS at a ratio of, for example, 1:1, 1:2, 1:3, 1:4, 1:5, or higher of plasma to NAPS.

[0042] In one example, a NAPS of 2% SDS, 10 mM EDTA, and 50 mM Tris-HCl pH 8.0 is added to a sample of plasma to prepare the sample for direct use in PCR. In another example, a NAPS of 0.5M NaOAc, 0.2M Tris-HCl, 0.15M sodium ascorbate, 10 mM CDTA, 1% SDS, 30% (v/v) ethanol, pH 9.5 (see PCT International Publication No. WO03104251 at Table 3, lane 1) is added to a sample of plasma to prepare the sample for direct use in PCR. In a further example, the NAPS can be the DNA preserving solution contained in the Oragene™ DNA Self-Collection Kit (DNA Genotek, Ottawa, Ontario, Canada). In this case, a plasma sample is applied to the sample reservoir, the reservoir is capped, releasing the DNA preserving solution into the chamber containing the plasma, thus stabilizing the DNA that is present. The composition of additional NAPS are described in PCT International Publication No. WO03104251 or U.S. Patent Application Publication No. 2003-104251 (see “compositions”, at, for example, Table 3).

[0043] Following addition of the NAPS, the resulting modified sample may be further treated with an optional Purifier solution (DNA Genotek, Ottawa, Ontario, Canada).

[0044] The modified sample may be optionally centrifuged and the supernatant used as template in amplification methods.

[0045] The modified samples processed as described above may desirably be diluted in water or an appropriate buffer for optimal use as a template in an amplification reaction. The skilled artisan will recognize that the dilution factor may vary with sample type and amplification method. For example, undiluted modified samples may be too concentrated for optimal amplification of nucleic acid. Similarly, modified samples that are highly diluted may have too low of a concentration of template for optimal amplification. Preferred dilutions include, but are not limited to, ratios of 1:5, 1:10, 1:25, 1:50, 1:100, 1:125, or higher of sample to water or dilution buffer. The skilled artisan will further understand that a minimal amount of testing may be needed to determine the optimal dilution factor for the specific sample used.

[0046] Amplification of Nucleic Acids

[0047] As detailed herein, the addition of NAPS to a biological sample results in a modified biological sample, which can be directly used as a source of template in amplification methods. In preferred embodiments, template nucleic acids may be amplified from modified biological samples using PCR or RT-PCR, however, the skilled artisan will understand that numerous methods are known in the art for amplification of nucleic acids, and that these methods may be used either in place of, or together with, PCR. Nucleic acid amplification methods, such as PCR, RT-PCR, isothermal methods, rolling circle methods, etc., are well known to the skilled artisan. See, e.g., Saiki, “Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, Calif. 1990, pp 13-20; Wharam et al., Nucleic Acids Res. 2001 Jun. 1; 29(11):E54-E54; Hafner et al., Biotechniques 2001 April; 30(4):852-6, 858, 860 passim; Zhong et al., Biotechniques 2001 April; 30(4):852-6, 858, 860 passim.

[0048] Amplification methods suitable for use with the present methods include, for example, polymerase chain reaction (PCR), reverse transcription PCR(RT-PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence based amplification (NASBA) reaction, self-sustained sequence replication (3SR), strand displacement amplification (SDA) reaction, boomerang DNA amplification (BDA), Q-beta replication: or isothermal nucleic acid sequence based amplification. These methods of amplification each described briefly below and are well-known in the art.

[0049] PCR is a technique for making many copies of a specific template DNA sequence. The reaction consists of multiple amplification cycles and is initiated using a pair of primer oligonucleotides that hybridize to the 5′ and 3′ ends of the sequence to be copied. The amplification cycle includes an initial denaturation, and up to 50 cycles of annealing, strand elongation (or extension) and strand separation (denaturation). In each cycle of the reaction, the DNA sequence between the primers is copied. Primers can bind to the copied DNA as well as the original template sequence, so the total number of copies increases exponentially with time. PCR can be performed as according to Whelan, et al, Journal of Clinical Microbiology, 33(3):556-561 (1995). Briefly, a PCR reaction mixture includes two specific primers, dNTPs, approximately 0.25 U of Taq polymerase, and 1×PCR Buffer, which is amplified using a thermal cycler. Cycling parameters can be varied, depending on, for example, the melting temperature of the primer or the length of nucleic acids to be extended. The skilled artisan is capable of designing and preparing primers that are appropriate for amplifying a target sequence. The length of the amplification primers for use in the present invention depends on several factors including the nucleotide sequence identity and the temperature at which these nucleic acids are hybridized or used during in vitro nucleic acid amplification. The considerations necessary to determine a preferred length for an amplification primer of a particular sequence identity are well-known to a person of ordinary skill and include considerations described herein. For example, the length of a short nucleic acid or oligonucleotide can relate to its hybridization specificity or selectivity.

[0050] Real time PCR is PCR-based amplification method in which PCR products are detected in real time, that is, the accumulation of PCR products can be determined at each cycle. Real Time PCR is performed using TaqMan® probes in combination with a suitable amplification/analyzer such as Applied Biosystems (ABI) Prism 7900HT Sequence Detection System, which is a high-throughput real-time PCR system. Briefly, TaqMan™ probes specific for the amplified target sequence are included in the PCR amplification reaction. These probes contain a reporter dye at the 5′ end and a quencher dye at the 3′ end. Probes hybridizing to different target sequences are conjugated with a different fluorescent reporter dye. In this way, more than one target sequence can be assayed for in the same reaction vessel. During PCR, the fluorescently labeled probes bind specifically to their respective target sequences; the 5′ nuclease activity of Taq polymerase cleaves the reporter dye from the probe and a fluorescent signal is generated. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. A mismatch between probe and target greatly reduces the efficiency of probe hybridization and cleavage. The ABI Prism 7700HT or 7900HT Sequence detection System measures the increase in fluorescence during PCR thermal cycling, providing “real time” detection of PCR product accumulation. Real Time detection on the ABI Prism 7900HT or 7900HT Sequence Detector monitors fluorescence and calculates Rn during each PCR cycle. The threshold cycle, or Ct value, is the cycle at which fluorescence intersects the threshold value. The threshold value is determined by the sequence detection system software or manually.

[0051] “RT-PCR” as used herein refers to the combination of reverse transcription and PCR in a single assay. “Reverse transcription” is a process whereby an RNA template is transcribed into a DNA molecule by a reverse transcriptase enzyme. Thus, “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases, that is, such polymerases use an RNA template to synthesize a DNA molecule. Historically, reverse transcriptases have been used to reverse-transcribe mRNA into cDNA. However, reverse transcriptases can be used to reverse-transcribe other types of RNAs such as viral genomic RNA or viral sub-genomic RNA. Standard reverse transcriptases include Maloney Murine Leukemia Virus Reverse Transcriptase (MoMuLV RT) and Avian myoblastosis virus (AMV). These enzymes have 5′->3′ RNA-dependent DNA polymerase activity, 5′->3′ DNA-dependent DNA polymerase activity, and RNase H activity. However, unlike many DNA-dependent DNA polymerases, these enzymes lack 3′->5′ exonuclease activity necessary for “proofreading,” (i.e., correcting errors made during transcription). After a DNA copy of an RNA has been prepared, the DNA copy may be subjected to various DNA amplification methods such as PCR.

[0052] LCR is a method of DNA amplification similar to PCR, except that it uses four primers instead of two and uses the enzyme ligase to ligate or join two segments of DNA. LCR can be performed as according to Moore et al., Journal of Clinical Microbiology 36(4):1028-1031 (1998). Briefly, an LCR reaction mixture contains two pair of primers, dNTP, DNA ligase and DNA polymerase representing about 90 μl, to which is added 100 μl of isolated nucleic acid from the target organism. Amplification is performed in a thermal cycler (e.g., LCx of Abbott Labs, North Chicago, Ill.).

[0053] TAS is a system of nucleic acid amplification in which each cycle is comprised of a cDNA synthesis step and an RNA transcription step. In the cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase (i.e., a polymerase-binding sequence or PBS) is inserted into the cDNA copy downstream of the target or marker sequence to be amplified using a two-domain oligonucleotide primer. In the second step, an RNA polymerase is used to synthesize multiple copies of RNA from the cDNA template. Amplification using TAS requires only a few cycles because DNA-dependent RNA transcription can result in 10-1000 copies for each copy of cDNA template. TAS can be performed according to Kwoh et al., PNAS 86:1173-7 (1989). Briefly, extracted RNA is combined with TAS amplification buffer and bovine serum albumin, dNTPs, NTPs, and two oligonucleotide primers, one of which contains a PBS. The sample is heated to denature the RNA template and cooled to the primer annealing temperature. Reverse transcriptase (RT) is added the sample incubated at the appropriate temperature to allow cDNA elongation. Subsequently T7 RNA polymerase is added and the sample is incubated at 37° C. for approximately 25 minutes for the synthesis of RNA. The above steps are then repeated. Alternatively, after the initial cDNA synthesis, both RT and RNA polymerase are added following a 1 minute 100° C. denaturation followed by an RNA elongation of approximately 30 minutes at 37° C. TAS can be also be performed on solid phase as according to Wylie et al., Journal of Clinical Microbiology, 36(12):3488-3491 (1998). In this method, nucleic acid targets are captured with magnetic beads containing specific capture primers. The beads with captured targets are washed and pelleted before adding amplification reagents which contains amplification primers, dNTP, NTP, 2500 U of reverse transcriptase and 2500 U of T7 RNA polymerase. A 100 μl TMA reaction mixture is placed in a tube, 200 μl oil reagent is added and amplification is accomplished by incubation at 42° C. in a waterbath for one hour.

[0054] NASBA is a transcription-based amplification method which amplifies RNA from either an RNA or DNA target. NASBA is a method used for the continuous amplification of nucleic acids in a single mixture at one temperature. For example, for RNA amplification, avian myeloblastosis virus (AMV) reverse transcriptase, RNase H and T7 RNA polymerase are used. This method can be performed as according to Heim, et al., Nucleic Acids Res., 26(9):2250-2251 (1998). Briefly, an NASBA reaction mixture contains two specific primers, dNTP, NTP, 6.4 U of AMV reverse transcriptase, 0.08 U of Escherichia coli Rnase H, and 32 U of T7 RNA polymerase. The amplification is carried out for 120 min at 41° C. in a total volume of 201.

[0055] In a related method, self-sustained sequence-replication (3SR) reaction, isothermal amplification of target DNA or RNA sequences in vitro using three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H. This method may be modified from a 3-enzyme system to a 2-enzyme system by using human immunodeficiency virus (HIV)-1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E. coli ribonuclease H. In the 2-enzyme 3SR, the amplified RNA is obtained in a purer form compared with the 3-enzyme 3SR (Gebinoga & Oehlenschlager European Journal of Biochemistry, 235:256-261, 1996).

[0056] SDA is an isothermal nucleic acid amplification method. A primer containing a restriction site is annealed to the template. Amplification primers are then annealed to 5′ adjacent sequences (forming a nick) and amplification is started at a fixed temperature. Newly synthesized DNA strands are nicked by a restriction enzyme and the polymerase amplification begins again, displacing the newly synthesized strands. SDA can be performed as according to Walker, et al., PNAS, 89:392-6 (1992). Briefly, an SDA reaction mixture contains four SDA primers, dGTP, dCTP, TTP, dATP, 150 U of Hinc II, and 5 U of exonuclease-deficient of the large fragment of E. coli DNA polymerase I (exo⁻ Klenow polymerase). The sample mixture is heated 95° C. for 4 minutes to denature target DNA prior to addition of the enzymes. After addition of the two enzymes, amplification is carried out for 120 min. at 37° C. in a total volume of 50 μl. Then, the reaction is terminated by heating for 2 minutes at 95° C.

[0057] Boomerang DNA amplification (BDA) is a method in which the polymerase begins extension from a single primer-binding site and then makes a loop around to the other strand, eventually returning to the original priming site on the DNA. BDA is differs from PCR through its use of a single primer. This method involves an endonuclease digestion of a sample DNA, producing discrete DNA fragments with sticky ends, ligating the fragments to “adapter” polynucleotides (comprised of a ligatable end and first and second self-complementary sequences separated by a spacer sequence) thereby forming ligated duplexes. The ligated duplexes are denatured to form templates to which an oligonucleotide primer anneals at a specific sequence within the target or marker sequence of interest. The primer is extended with a DNA polymerase to form duplex products followed by denaturation of the duplex products. Subsequent multiple cycles of annealing, extending, and denaturing are performed to achieve the desired degree of amplification (U.S. Pat. No. 5,470,724).

[0058] The Q-beta replication system uses RNA as a template. Q-beta replicase synthesizes the single-stranded RNA genome of the coliphage Qβ. Cleaving the RNA and ligating in a nucleic acid of interest allows the replication of that sequence when the RNA is replicated by Q-beta replicase (Kramer & Lizardi Trends Biotechnol. 1991 9(2):53-8, 1991).

[0059] A variety of amplification enzymes are well known in the art and include, for example, DNA polymerase, RNA polymerase, reverse transcriptase, Q-beta replicase, thermostable DNA and RNA polymerases. Because these and other amplification reactions are catalyzed by enzymes, in a single step assay that the nucleic acid releasing reagents and the detection reagents should not be potential inhibitors of amplification enzymes if the ultimate detection is to be amplification based.

[0060] Amplified nucleic acid product may be detected by any of a variety of well-known methods, for example, electrophoresis (e.g., gel electrophoresis or capillary electrophoresis). Amplified fragments may be subjected to further methods of detecting, for example, variant sequences (e.g., single nucleotide polymorphisms (SNPs)). An exemplary method is single nucleotide primer extension (Lindblad-Toh et al., Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. Nature Genet. 2000 April; 24(4):381-6). In this reaction, an oligonucleotide primer is designed to have a 3′ end that is one nucleotide 5′ to a specific mutation site. In some embodiments, the extension primers are labeled with a tag or a member of a binding pair to allow the capture of the primer on solid phase. In particular embodiments, the primers may be tagged with varying lengths of nonspecific polynucleotides (e.g., poly-GACT) to allow multiplex detection of preferably 2 or more, more preferably 3 or more, 4 or more, 5 or more, even 10 or more different mutations (polymorphisms) in a single reaction. The primer hybridizes to the PCR amplicon in the presence of one or more labeled ddNTPs and a DNA polymerase. The polymerase extends the primer by one nucleotide, adding a single, labeled ddNTP to the 3′ end of the extension primer. The addition of a dideoxy nucleotide terminates chain elongation. If more than one dideoxynucleotide (e.g., ddATP, ddGTP, ddCTP, ddTTP, ddUTP, etc.) is used in a reaction, one or more can be labeled. If multiple labels are used, the labels can be distinguishable e.g., each is labeled with a different fluorescent colored dye. The products are labeled oligonucleotides, each one of which may be detected based on its label. Further methods of detecting variant sequences include the READIT SNP Genotyping System (Promega Corporation, Madison Wis.) and oligonucleotide ligation assays.

[0061] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.

[0062] Processed whole blood samples provided the template for a triplex PCR reaction used to amplify three regions of the TPMT gene each containing one of the three mutation sites of interest (i.e., G238C, G460A, and A719G). The amplification reaction was followed by two single nucleotide primer extension reactions to detect the presence of mutations.

[0063] Whole blood samples were prepared for use in the TPMT assay by the following two methods:

[0064] A) A 30 μL aliquot of a blood sample was mixed with 90 μL of NAPS consisting of 2% SDS, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA. The resulting solution was diluted 1:125 with water prior to use as template in PCR.

[0065] B) A 30 μL aliquot of a blood sample was mixed with 90 μL of DNA preserving solution from the Oragene™ DNA Self-Collection Kit (DNA Genotek, Ottawa, Ontario, Canada). The mixture was incubated at 65° C. for approximately 30 minutes. 60 μL H₂O was added and the solution mixed and centrifuged at 5000 rpm for 5 minutes. A 30 μL aliquot was removed from the upper portion of the liquid following centrifugation, transferred to a fresh plate, and diluted 1:25-1:125 prior use as template in PCR.

[0066] PCR was conducted using the following amplification primers.

[0067]

TABLE 1
TPMT amplification primers containing a
universal linker sequence
Primer			Position
Location			in GenBank
in TPMT	Primer		Accession
Gene	Name	Primer Sequence	No. AB045146
Exon 5	TPMT-	5′GCGGTCCCAAAAGGGTCAGT	12089-12110
	E5F-	TGTCTTTGAAACCCTATGAACC
	Linker	TG-3′
		(SEQ ID NO:1)
Intron 5	TPMT-	5′GCGGTCCCAAAAGGGTCAGT	12435-12454
	5R-	TGTAGGAACCATCGGACACAT
	Linker	G-3′
		(SEQ ID NO:2)
Intron 6	TPMT-	5′GCGGTCCCAAAAGGGTCAGT	16753-16775
	I6F-	TGCTCCACACCCAGGTCCACAC
	Linker	ATT-3′
		(SEQ ID NO:3)
Intron 7	TPMT-	5′GCGGTCCCAAAAGGGTCAGT	17010-17042
	I7R-	TGGTATAGTATACTAAAAAATT
	Linker	AAGACAGCTAAAC-3′
		(SEQ ID NO:4)
Intron 9	TPMT-	5′GCGGTCCCAAAAGGGTCAGT	25122-25150
	I9F-	TGAATCCCTGATGTCATTCTTC
	Linker	ATAGTATTT-3′
		(SEQ ID NO:5)
Exon 10	TPMT-	5′GCGGTCCCAAAAGGGTCAGT	25342-25372
	R971-	TGCATCCATTACATTTTCAGGC
	Linker	TTTAGCATAAT-3′
		(SEQ ID NO:6)
Universal linker portion of each primer shown in bold

[0068] A 5× solution of TPMT primer mix was prepared according to table 2 below.

[0069]

TABLE 2
5X solution of TPMT primer mix
		Concentration	Concentration
		5X solution	Final solution
Primer (50 μM)	Volume (μL)	(μM)	(μM)
TPMT-E5F-linker	144	1.25	0.25
(SEQ ID NO:1)
TPMT-5R-linker	144	1.25	0.25
(SEQ ID NO:2)
TPMT-I6F-linker	230	2.00	0.40
(SEQ ID NO:3)
TPMT-I7R-linker	230	2.00	0.40
(SEQ ID NO:4)
TPMT-I9F-linker	173	1.50	0.30
(SEQ ID NO:5)
TPMT-R971-linker	173	1.50	0.30
(SEQ ID NO:6)
dH2O	4,666
Total	5,760

[0070] The TPMT PCR master mix was prepared according to table 3 below.

[0071]

TABLE 3
TPMT PCR master mix
	Components	Per Rxn (μL)	x3000 (μL)
	10X Roche PCR Buffer	2.5	7,500
	25 mM dNTP mix	0.25	750
	5X TPMT primer mix	5.0	15,000
	25 mM MgCl2	1.0	3,000
	Sterile H2O	14.0	42,000
	Total	22.75	68,250

[0072] A 25 μL reaction containing 22.75 μL TPMT master mix, 0.25 μL Taq polymerase (HotStarTaq) and 2 μL of the genomic DNA-containing test sample or control DNA was run under the following PCR conditions.

[0073]

TABLE 4
PCR amplification conditions
	Step	Temperature	Time
	1	95° C.	15 min.
	2	94° C.	30 sec
	3	60° C.	60 sec.
	4	70° C.	30 sec.
	5	go to step 2	34 cycles*
	6	70° C.	7 min.
	7	4° C.	Hold

[0074] After PCR was complete, the samples were treated with SAP (shrimp alkaline phosphatase) and Exo I (exonuclease I). A fresh SAP/ExoI cocktail was prepared prior to each use according to table 5 below.

[0075]

TABLE 5
SAP/ExoI cocktail
		Volume (μL) for	Volume (μL) for 120
	Reagent	1 reaction	rxns (full plate)
	SAP (1 unit/μL)	7.5	900
	Exo I (10 unit/μL)	0.3	36
	Sterile H20	14.7	1,764
	Total	22.5	2,700

[0076] 22.5 μL of the SAP/ExoI cocktail was dispensed into each well of a 96-well plate. 7.5 μL of PCR product was added to each well. The samples were incubated at 37° C. for 2 hours, 75° C. for 15 minutes then chilled to 4° C. and stored at 2-8° C. until use.

Single Nucleotide Primer Extension

[0077] Two single nucleotide primer extension reactions (termed “the A reaction” and “the G reaction”) were performed on the SAP/ExoI treated samples. The A reaction contains extension primers and labeled ddATP to allow the detection of mutation at position 460 (i.e., G460A) and the wild-type nucleotide at position 719. The G reaction contains extension primers and labeled ddGTP to allow the detection of mutation at position 238 of the antisense strand and the wild-type at position 238 of the sense strand. The G reaction further permits the detection of the wild-type nucleotide at position 460 and the mutation at position 460. The extension primers used in both the A reaction and the G reaction are as follows.

[0078]

TABLE 6
Exemplary extension primers containing an
oligonucleotide tag
			Position
			in GenBank
	Primer		Accession
Mutation	name	Primer Sequence	No. AB045146
G238C	2AF	5′TTCACTTTTCAATCAACTT	12194-12224
Sense	Tag	AAGTGTAAATGTATGATTTTA
strand		TGCAGGTTT-3′
		(SEQ ID NO:7)
G238C	2AR	5′CTTTTCATCAATAATCTTA	12226-12248
Antisense	Tag	CCTTTAACTACACTGTGTCCC
strand		CGGTCTG-3′
		(SEQ ID NO:8)
G460A	3A1F	5′TACACTTTAAACTTACTAC	16928-16953
	Tag	ACTAAAATTTGACATGATTTG
		GGATAGAGGA-3′
		(SEQ ID NO:9)
A719G	3A2F	5′TACACTTTCTTTCTTTCTT	25241-25269
	Tag	TCTTTGGGAATTGACTCGTCT
		TTTTGAAAAGTTAT-3′
		(SEQ ID NO:1)
Oligonucleotide tag portion of the primer shown in bold

[0079] A Reaction

[0080] The primer extension (PE) primer master mix for the A reaction was prepared according to table 7 below.

[0081]

TABLE 7
Primer Extension (PE) Primer Mix for A reaction
				Volume
		Concentration	Volume (μL)	(μL) for
Mutation	Primer	(μM)	for 1 rxn	4000 rxns
TPMT3B	3A1F Tag	100 uM	0.1	400
(G460A)	(SEQ ID NO:9)
TPMT3C	3A2F Tag	100 uM	0.1	400
(A719G)	(SEQ ID NO:10)
H2O			0.2	800

[0082] The above extension primers are tagged with unique Tag-It DNA Sequence™ (Luminex).

[0083] The biotin-ddATP/ddNTP mix for the A reaction was prepared according to table 8 below.

[0084]

TABLE 8
Biotin-ddATP/ddNTP mix for A reaction
		Volume (μL)	Volume (μL) for 120
Reagent	Concentration	for 1 rxn	rxns (full plate)
Biotin-	1 mM	0.1	12
ddATP
ddCTP	1 mM	0.5	60
ddGTP	1 mM	0.5	60
ddTTP	1 mM	0.1	12
Total		1.2	144

[0085] The primer extension master mix for the A reaction was prepared according to table 9 below.

[0086]

TABLE 9
Primer Extension Master Mix for A reaction
		1 Rxn	Full Plate
	Reagent	(μL)	112 Rxns (μL)
	10X PCR Buffer (20 mM	2.0	224
	Mg++)
	Biotin-ddATP/ddNTP mix	1.2	134.4
	PE Primer Mix for A reaction	0.4	44.8
	H2O	8.15	912.8
	Mixture Total	11.75	1,316

[0087] 7.5 μL of SAP/ExoI treated sample was added to 11.5 μL PE Master Mix for A Reaction and 0.75 μL Fast Start Taq (Roche). The thermal cycler conditions for the primer extension reaction were as follows:

[0088]

TABLE 10
Primer extension reaction conditions
	Step	Temperature	Time
	1	96° C.	2 min.
	2	95° C.	30 sec
	3	50° C.	30 sec
	4	60° C.	30 sec
	5	Go to Step 2	50 Cycles
	6	4° C.	5 min
	7	4° C.	Hold

[0089] G Reaction

[0090] The primer extension primer mix for the G reaction was prepared according to table 11 below.

[0091]

TABLE 11
Primer extension (PE) primer mix for G reaction
			Volume	Volume
		Concentration	(μL)	(μL) for
Mutation	Primer	(μM)	for 1 rxn	4000 rxns
TPMT*2	2F Tag	100 uM	0.1	400
	(SEQ ID NO:7)
TPMT*2	2R Tag	100 uM	0.1	400
	(SEQ ID NO:8)
TPMP*3B	3A1F Tag	100 uM	0.1	400
(460G > A)	(SEQ ID NO:9)
TPMT*3C	3A2F Tag	100 uM	0.1	400
(719A > G)	(SEQ ID NO:10)
H2O			0.4	1,600

[0092] The above extension primers are tagged with unique Tag-It DNA Sequence™ (Luminex).

[0093] The Biotin-ddATP/ddNTP mix for G reaction was prepared according to table 12 below.

[0094]

TABLE 12
Biotin-ddATP/ddNTP mix for G reaction
		Volume (μL)	Volume (μL) for 120
Reagent	Concentration	for 1 rxn	rxns (full plate)
Biotin-	1 mM	0.1	12
ddGTP
ddCTP	1 mM	0.5	60
ddATP	1 mM	0.5	60
ddTTP	1 mM	0.1	12
Total		1.2	144

[0095] The primer extension (PE) master mix for the G reaction was prepared according to table 13 below.

[0096]

TABLE 13
Primer Extension Master Mix for G reaction
		1 Rxn	Full Plate
	Reagent	(μL)	112 Rxns (μL)
	10X PCR Buffer (20 mM Mg++)	2.0	224
	Biotin-ddGTP/ddNTP mix	1.2	134.4
	PE Primer Mix for G reaction	0.8	89.6
	H2O	7.75	868
	Mixture Total	11.75	1,316

[0097] 7.5 μL of SAP/ExoI treated sample was added to 11.5 μL PE Master Mix for G Reaction and 0.75 μL Fast Start Taq (Roche). The thermal cycler conditions for the primer extension reaction for the G reaction were the same as the conditions recited above for the A reaction.

Detection

[0098] The extension primers were captured on solid phase by hybridization of a the Tag-It DNA sequence to the corresponding Anti-Tag sequence (i.e., the complementary sequence to the tag sequence contained in the extension primer) coupled to the surface of a fluorescent-labeled microsphere. The microspheres were then subjected to flow cytometry in which the presence of labeled extension primer was detected.

[0099] 5× hybridization buffer is prepared according to table 14.

[0100]

TABLE 14
5X hybridization buffer
		Concentration	Volume (mL) or
	Regents	(M)	Weight (g)
	Tris-HCl (pH 8.0)	1 M	550	mL
	Triton-100	100%	4.4	mL
	NaCl		64.25	g
	H2O		~440	mL*
	*The H2O volume is an estimate, add H2O last to the final volume of 1 L.

[0101] The Luminex 4 bead mix was prepared according to the following table. All beads were obtained from Luminex Corp. (Austin, Tex.). Each bead bottle was vortexed for 20 seconds followed by sonication for 20 seconds. Vortexing and sonication were repeated once more. The beads are light-sensitive and thus the bead solution needs to be kept away from light.

[0102]

TABLE 15
Luminex 4 bead mix
Bead Stock
Solution			Full
2.5 × 105/mL			Plate
(Luminex FlexMAP	Anti-Tag-	1 Rxn	X112 Rxn
microsphere)	it Sequence	(μL)	(μL)
031	5′AGTTGATTGAAAAGT	9	1,000
(Cat. No. L-100-	GAA-3′
031)	(SEQ ID NO:25)
065	5′AAAGGTAAGATTATT	9	1,000
(Cat. No. L-100-	GATGAAAAG-3′
065)	(SEQ ID NO:26)
095	5′TTAGTGTAGTAAGTT	9	1,000
(Cat. No. L-100-	TAAAGTGTA-3′
095)	(SEQ ID NO:27)
012	5′AAAGAAAGAAAGAAA	9	1,000
(Cat. No. L-100-	GAAAGTGTA-3′
012)	(SEQ ID NO:28)
ddH2O		4	480
Total		40	4,480

[0103] The bead hybridization was performed as follows. A bead hybridization solution was prepared according to the following table.

[0104]

TABLE 16
Bead hybridization solution
			112 Rxns
	Reagent	1 Rxn (μL)	Per Plate(μL)
	4 Beads Mix	40	4,480
	5X Hyb.	10	1,120
	Buffer
	Total	50	5,600

[0105] 50 μL of the bead hybridization solution was added to each appropriate well of an ABI Optical 96-well plate. 5 μL of the Primer Extension product (from Example 3) was added. The plate was sealed but not vortexed or centrifuged. (Centrifugation of the bead hybridization plate will cause the beads to form a pellet at the bottom of the plate and not hybridize to the primer extension product.) The plate was placed in a thermal cycler and subjected to the conditions according to table 17 below.

[0106]

TABLE 17
Bead hybridization conditions
	Step	Temp.	Time
	1	96° C.	2 min.
	2	37° C.	1 hour
	3	37° C.	Hold

[0107] The wash solution was prepared according to table 18.

[0108]

TABLE 18
Wash solution (1X Hybridization Buffer)
			112 Rxn
	Reagent	1 Rxn (μL)	Full Plate (μL)
	ddH2O	120	13,440
	5X Hyb.	30	3,360
	Buffer
	Total	150	16,800

[0109] The reporter solution of streptavidin R-phycoerythrin (SA-PE) conjugate was prepared by vortexing the tube of SA-PE for 2-5 seconds. The appropriate amount of SA-PE was added to appropriate 1× hybridization buffer. SA-PE is light sensitive so exposure to light should be limited and the solution stored in the dark until ready to use. Prepare 5-10 minutes before use.

[0110]

TABLE 19
Reporter solution
			Full Plate x112
	Loading Mix	1 Rxn (μL)	Rxn
	1X Hybridization	150	16800
	Buffer
	SA-PE	0.4	44.8
	Total	150.4	16,844.8

[0111] The bead hybridization plate was removed from the thermal cycler. The plate was sealed and spun at 3000 RCF (not rpm) for 5 minutes in a microplate centrifuge. The plate was inverted over paper towels and tapped to remove excess liquid. 150 μL of wash solution was added to each well. The plate was sealed and vortexed for 10 seconds. Vortexing was repeated two more times. The plate was sealed and spun at 3000 RCF (not rpm) for 5 minutes in a microplate centrifuge. The plate was inverted over paper towels and tapped to remove excess liquid. 150 μL of reporter solution was added to each well. The 150 μL solution was carefully pipetted up and down 8 times. The solution was transferred to a new Falcon DB 96-well plate. The plate was placed in the dark for 15 minutes then placed on the Luminex 100 IS instrument. The results of the detection by the Luminex 100 IS instrument were correlated to the genotype at each mutation position as follows.

[0112]

TABLE 20
Detection results for the A Reaction
			Wild-type (Wt) or
	Primer	ΔMFI*	mutation	Genotypes
	3A1F Tag	>400	Mutation	460A
	3A2F Tag	>800	Wt	719A
	*Δ MFI is the MFI of samples with DNA minus the MFI of samples without DNA control.

[0113]

TABLE 21
Detection results for the G Reaction
			Wild-type (Wt) or
	Primer	ΔMFI*	mutation	Genotypes
	2AF Tag	>800	Wt	238G
	2AR Tag	>800	Mutation	238C
	3A1F Tag	>800	Wt	460G
	3A2F Tag	>800	Mutation	719G

[0114] Cell samples processed by method “A” above and diluted 1:125 with water prior to use in PCR, showed robust amplification (as determined by gel electrophoresis) and worked well in the single nucleotide primer extension step.

[0115] Cell samples processed by method “B” above and diluted 1:5 prior to PCR showed amplification was not robust (as determined by gel electrophoresis) and the single nucleotide primer extension step did not work. Cell samples processed by method “B” above and diluted 1:25 and 1:125 prior to PCR showed good amplification (as determined by gel electrophoresis) and worked well in the subsequent single nucleotide primer extension step.

[0116] Processed whole blood samples provided the template for a triplex PCR reaction used to amplify fragments of DNA containing Type I and IV mutations, the Type II mutation, and the Type III mutation in the Factor XI gene. The amplification reaction was followed by a single nucleotide primer extension reaction to detect the presence of mutations.

[0117] Whole blood samples were prepared for use in the Factor XI assay by the following two methods:

[0118] A) Whole blood samples were processed by adding 100 μL of DNA preserving solution from the Oragene™ DNA Self-Collection Kit (DNA Genotek, Ottawa, Ontario, Canada) to 50 μL whole blood. The samples were mixed and 6 μL Purifier solution (DNA Genotek, Inc.) was added. Samples were centrifuged and the supernatant collected. The supernatant was serial diluted in water the following ratios 1:5, 1:25, 1:125, 1:625, and 1:3125.

[0119] B) Whole blood samples were processed by adding 80 μL of DNA preserving solution from the Oragene™ DNA Self-Collection Kit (DNA Genotek, Ottawa, Ontario, Canada) to 40 μL whole blood. Samples were centrifuged and the supernatant collected. The supernatant was diluted 1:5 in water.

[0120] The following primers were used in the triplex PCR reaction.

[0121]

TABLE 22
Factor XI PCR primers
Location in
GenBank Acc.
No. AY191837	Primer Name	Sequence
10660-10682	F11F1	5′-TGT AAA ACG ACG GCC AGT
		GCT TGC AAC AAA GAC ATT
		TAT GT-3′
		(SEQ ID NO:11)
10856-10835	F11R1	5′-
		TTC ATC GAC CAC TCG AAT
		GTC C-3′
		(SEQ ID NO:12)
16730-16747	F11F2	5′-
		AGG GAG GGT CTC ACT CTG-3′
		(SEQ ID NO:13)
16879-16862	F11R2	5′-
		CGG ACG GCA TTG GTG CAC-3′
		(SEQ ID NO:14)
24311-24335	F11F3	5′-ATA ACC CAT AAG ATG ATC
		TGT GCC G-3′
		(SEQ ID NO:15)
24426-24405	F11R3	5′-CCC CAA CGC ATT AAG CAT
		TCC AA-3′
		(SEQ ID NO:16)
24351-24400	Control IVS	5′-GGA AGG ACG CTT GCA AGA
	V5 Mutant	TAA CAG AGT GTT CTT AGC
		CAA TGG AAT ATA TG-3′
		(SEQ ID NO:17)
24400-24379	Control Del	5′-CAT ATA TTC CAT TGG CTA
24364-24341	V5	AGA A GCA AGC GTC CTT CCC
		TCC TTC CCT-3′
		(SEQ ID NO:18)
Italicized bold sequences are linkers and not part of GenBank sequence.

[0122] A Master mix of Factor XI was prepared according to table 23 below.

[0123]

TABLE 23
5X solution of Factor XI primer mix
			Concentration
		Volume per	Final solution
	Primer (100 μM)	reaction (μL)	(μM)
	10X buffer with	1.5	1X
	MgCl2 (Roche)
	25 mM dNTP	0.1	159.2
	F11F1	0.03	0.2
	(SEQ ID NO:11)
	F11R1	0.03	0.2
	(SEQ ID NO:12)
	F11F2	0.03	0.2
	(SEQ ID NO:13)
	F11R2	0.03	0.2
	(SEQ ID NO:14)
	F11F3	0.06	0.40
	(SEQ ID NO:15)
	F11R3	0.06	0.40
	(SEQ ID NO:16)
	5X GC Rich solution	0.75
	Roche)
	dH2O	9.71
	Total	12.3

[0124] A 15 μL reaction containing 12.3 μL Factor XI master mix, 1.2 μL MgCl₂, 0.2 μL Taq polymerase (FastStarTaq, 5 U/μL) and 2 μL of diluted, processed DNA-containing sample or control DNA was run under the following PCR conditions.

[0125]

TABLE 24
PCR amplification conditions
Step	Temperature	Time
1	95° C.	5 min.
2	94° C.	15 sec
3	−0.5° C./sec	to 58° C.
4	58° C.	20 sec.
5	+0.3° C./sec	to 72° C.
6	72° C.	30 sec.
7	+0.5° C./sec	to 94° C.
8	go to step 2	33 times*
9	72° C.	5 min.
10	4° C.	Hold

[0126] The expected sizes of the amplicon for each primer pair are as reported in Table 25.

[0127]

TABLE 25
Factor XI gene amplicons.
	Target region		Amplicon
Primer pair	of gene	Mutation(s) Examined	Size (bp)
F11F1, F11R1	Exon 5	E117X	235
F11F2, F11R2	Exon 9	F283L	186
F11F3, F11R3	Exon14/Intron 14	IVS14N G > A;	117
		IVS14del14

[0128] After PCR was complete, the samples are treated with CIAP (calf intestinal alkaline phosphatase) and Exo I (exonuclease I). A fresh CIAP/ExoI cocktail is prepared prior to each use according to table 26 below.

[0129]

TABLE 26
CIAP/ExoI cocktail
		Volume (μL) for
	Reagent	1 reaction
	CIAP (1 unit/μL)	2.0
	Exo I (10 unit/μL)	0.2
	Sterile H20	12.8
	Total	15.0

[0130] 3 μL of PCR product was added to 15 μL of the CIAP/ExoI cocktail and then incubated at 37° C. for 2 hours, 75° C. for 15 minutes then chilled to 4° C. and stored at 2-10° C. until use.

[0131] The single nucleotide primer extension reaction was performed using the ABI PRISM® SNaPshot® Multiplex Kit (Applied Biosystems) and the following extension primers.

[0132]

TABLE 27
Factor XI extension primers.
Location in
GenBank Acc.	Primer
No. AY191837	Name	Sequence
10739-10764	E117X	5′-GTC ATC CGT GCA TCT TTC
(Rev)		TTG GCA TT-3′
		(SEQ ID NO:17)
16772-16802	F283L	5′-CTG CCA TTC TTC ATT TTA
		CCA TGA CAC TGA T-3′
		(SEQ ID NO:18)
24400-24369	IVS14N	5′-TTT TTT TTT TCA TAT ATT
	G > A	CCA TTG GCT AAG AAC ACT
		CTG TTA-3′
		(SEQ ID NO:19)
24345-24364	IVS14del14	5′-AAG GAG GGA AGG ACG CTT
		GC-3′
		(SEQ ID NO:20)
italicized bold sequences are linkers and not part of Genebank sequence.

[0133] The primer extension primer mix was prepared according to table 28 below.

[0134]

TABLE 28
Primer extension (PE) primer mix
	Concentration	Volume (μL)	Final conc.
Primer	(μM)	for 1 rxn	(μM)
E117X	100 uM	0.2	0.2
F283L	100 uM	0.2	0.2
IVS14N G > A	100 uM	0.5	0.5
IVS14del14	100 uM	0.2	0.2
Sterile dH2O		0.89
		1.0

[0135] The primer extension (PE) master mix was prepared according to table 29 below.

[0136]

TABLE 29
Primer Extension (PE) Master Mix
		Volume/Rxn
	Reagent	(μL)
	SNaPshot Ready Mix (contains	2.0
	AmpliTaq DNA Polymerase, fluorescent
	labeled ddNTPs) (Applied Biosystems)
	5X ABI sequencing buffer	2.0
	PE Primer Mix	1.0
	H2O	2.0
	Mixture Total	7.0

[0137] 3 μL of CIAP/ExoI digested PCR product was added to 7.0 μL PE Master Mix. The thermal cycler conditions for the primer extension reaction were as follows.

[0138]

TABLE 30
Primer extension reaction conditions
Step	Temperature	Time
1	95° C.	2 min
2	95° C.	20 sec
3	60° C.	20 sec
4	Go to Step 2	34 times
5	4° C.	Hold

[0139] 1 μL of CIAP (1 U/μL) was diluted with 1 μL of water and the resulting 2 μL of diluted CIAP added to the primer extension reaction product and incubated at 37° C. for 1 hour, 95° C. for 5 minutes then chilled to 4° C. and stored at 2-10° C. until use.

[0140] The CIAP digested products containing fluorescently labeled extension primers were run on an ABI PRISM 3100 Genetic Analyzer wherein the size of the oligonucleotide was determined and the incorporated fluorescent label detected and identified.

[0141] The expected peak size and color (for wild type and mutant alleles) for each labeled extension primer, as detected on the ABI 3100 genetic analyzer is as follows:

[0142]

TABLE 31
Expected peak size and color for wild type and mutant alleles
	Wild type allele	Mutant allele
	Primer		Extended		Size	Extended		Size
SNP	size (bp)	Location	nucleotide	Color	(bp)*	nucleotide	Color	(bp)*
IVS14del14	20	Exon14/Intron14	A	Green	24-26	T	Red	24.5-26.5
E117X	26	Exon 5	G	Black	30-32	T	Green	30.9-32.9
F283L	31	Exon 9	T	Red	33.5-35.5	C	Black	31.5-33
IVS14N	42	Intron 14	C	Black	42.5-44.5	T	Red	45-46.5
G > A
*The size of the primer extension product is affected by mobility shift caused by the incorporated fluorescent dye labeled ddNTP.

[0143] Cell samples processed by method “A” above and diluted 1:5, 1:25, and 1:125 worked in the Factor XI assay, whereas processed samples diluted 1:625 worked minimally (i.e., only one of four samples showed amplification, as determined by gel electrophoresis), and undiluted processed samples or those diluted 1:3125 did not work. Cell samples processed by method “B” above and diluted 1:5 prior to use in PCR, showed robust amplification (as determined by gel electrophoresis) and worked well in the SNaPshot assay.

[0144] Processed whole blood samples were assayed for the R506Q mutation in the Factor V (caused by a G to A transition at nucleotide position 1691 in the Factor V cDNA). The presence or absence of the mutation is determined using the READIT™ System (Promega Corp., Madison, Wis.) to interrogate PCR products encompassing nucleotide at position 1691.

[0145] Whole blood samples were processed by adding 80 μL of DNA preserving solution from the Oragene™ DNA Self-Collection Kit (DNA Genotek, Ottawa, Ontario, Canada) to 40 μL whole blood. Samples were centrifuged and the supernatant collected. The supernatant was diluted 1:5 in water. Diluted samples were assayed according to the Factor V assay as follows.

[0146] PCR was used to amplify a 234 bp region of the Factor V gene including the nucleotide at position 1691. A PCR Master Mix was prepared according to the following table.

[0147]

TABLE 32
Factor V PCR Master Mix
			Final
		Volume/	conc.
	Reagent	Rxn (μL)	(μM)
	2X Universal Master Mix	6.88	1X
	(Promega Corp.)
	FV-F Thioate primer	0.138	0.25
	(50 pm/uL)
	5′-G*A*T*GAA CCC ACA GAA
	AAT GAT GCC AG-3′
	(SEQ ID NO:21)
	FV-R primer (50 pm/uL)	0.138	0.25
	5′-GGA AAT GCC CCA TTA
	TTT AGC CAG GAG-3′
	(SEQ ID NO:22)
	Nuclease-free dIH2O	5.5
	Mixture Total	12.656	12.656
	“*” represents a phosphothioate linkage

[0148] 2 μL diluted processed blood sample was added to 11.5 μL PCR master mix, mixed and spun down. PCR was performed using the following conditions.

[0149]

TABLE 33
PCR conditions
Step	Temperature	Time
1	95° C.	1 min
2	95° C.	20 sec
3	60° C.	15 sec
4	72° C.	15 sec
5	Go to Step 2	39 times
6	72° C.	1 min
7	4° C.	Hold

[0150] PCR products were digested with calf intestinal alkaline phosphatase and exonuclease I. Genotyping of the PCR products was then performed using the following interrogation probes and the READIT SNP Genotyping System (Promega Corp.) in accordance with the manufacturer's protocol.

[0151]

TABLE 34
Factor V READIT System interrogation probes.
Location in
GenBank Acc.
No. Z99572	Primer Name	Sequence
62902-62931	FV WT	5′-CTT GAA GGA CAA AAT ACC
	(wild type	TGT ATT CCT CG-3′
	allele)	(SEQ ID NO:23)
62902-62931	FV MUT	5′--CTT GAA GGA CAA AAT
	(mutant	ACC TGT ATT CCT TG-3′
	allele)	(SEQ ID NO:24)
Nucleotide in bold and underlined represents the base at position 1691 in the wild type and mutant sequences.

[0152] The processed samples produced good results in the READIT system assay.

[0153] Processed blood samples were assayed for the presence of various mutations in the CF gene using PCR-oligonucleotide ligation assay (OLA) technology. Patient genomic DNA in a sample of whole blood is amplified in a 15-plex PCR. The amplicons are then interrogated using the Cystic Fibrosis OLA ASR assay version 3.0 (Applied Biosystems) as described by Grossman et al. (Nucleic Acids Research 22:4527-34, 1994) and Spitzer et al. (Eur. Respir. J 19:374-6, 2002).

[0154] Whole blood samples were processed for use in the CF assay by the following method: whole blood samples were processed by adding 80 μL of DNA preserving solution from the Oragene™ DNA Self-Collection Kit (DNA Genotek, Ottawa, Ontario, Canada) to 40 μL whole blood. Samples were centrifuged and the supernatant collected. The supernatant was diluted 1:5, 1:25, and 1:125 in water.

[0155] Diluted samples were assayed using the Cystic Fibrosis OLA ASR assay according to the manufacturer's protocol.

[0156] The samples processed according to the method above and diluted 1:5 produced the best results the CF OLA assay, whereas samples diluted 1:125 produced the worst results of the three dilution factors.

[0157] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All nucleotide sequences provided herein are presented in the 5′ to 3′ direction.

[0158] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0159] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0160] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0161] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0162] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0163] Other embodiments are set forth within the following claims.