WO2005117514A2 - Method of detecting cystic fibrosis associated mutations - Google Patents

Abstract

The present invention describes a method for the simultaneous identification of two or more single base changes, insertions, deletions or translocations in a plurality of target nucleotide sequences that are markers associated with cystic fibrosis. Multiplex detection is accomplished using multiplexed tagged allele specific primer extension (ASPE) and hybridization of such extended primers to a probe, preferably an addressable anti-tagged support.

Description

Method of Detecting Cystic Fibrosis Associated Mutations

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to methods and kits for the detection of mutations associated with cystic fibrosis.

DESCRIPTION OF THE PRIOR ART

[0002] Cystic Fibrosis (CF) is the most common autosomal recessive disorder in the Caucasian population, with an incidence of approximately 1 in 3200 live births. The incidence of CF in additional ethnic groups is summarized in Table 1. Ethnic Group Incidence of Cystic Fibrosis North American Caucasian 1 in 3200 Ashkenazi Jewish 1 in 3300 Hispanic 1 in 9500 African American 1 in 15 300 Asian American 1 in 32 100 Native American (Pueblo) 1 in 3970 Native American (Zuni) 1 in 1347

Table 1 : Incidence of CF in Various Ethnic Groups

[0003] CF affects many functions of the body including breathing, digestion and reproduction. Common symptoms of CF include; coughing, wheezing, susceptibility to infections, pneumonia, nasal polyps, digestive problems, inhibited growth, and infertility. [0004] The gene for CF was isolated by positional cloning in 1989 (Rommens, Iannuzzi et al. 1989) and was found to encode a 1480 amino acid transmembrane protein, which was named cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR protein functions as a chloride channel (Bear, Li et al. 1992), and also controls the regulation of other transport pathways (Gabriel, Clarke et al. 1993; Schwiebert, Egan et al. 1995). Mutations in CFTR result in defective chloride ion transport and defective electrolyte transport (Ratjen and Doring 2003). [0005] Over 1200 mutations have been found in the CFTR gene; however many of these mutations have only been found in either single cases, or in a small number of cases. The most common mutation is a three base pair deletion that results in the loss of a phenylalanine at amino acid 508 (ΔF508) — this mutation accounts for 30 to 88 percent of all CF mutations depending on the ethnic group (Gibson, Moskowitz et al. 2001). ΔF508 is the most common mutation in most ethnicities.

[0006] The American College of Medical Geneticists (ACMG) and the American College of Obstetricians and Gynecologists (ACOG) has recommended a specific panel of 25 mutations for CF genetic testing, plus reflex testing of four variants. The 25 mutations included in the panel occur at or greater than a frequency of 0.1 percent in the U.S. population as a whole.

[0007] Four CFTR variants are recommended for reflex testing: 5T/7T/9T, I506N, I507N, and F508C. Reflex testing is done when positive results are obtained for certain mutations in order to clarify, confirm or expand the positive results.

[0008] Table 2 lists the 40 most common mutations which have been associated with CF, as well as the four CFTR variants. The 25 mutations identified by the ACMG and the ACOG are highlighted in bold, and the four CFTR variants are indicated in italics.

Table 2: Common Mutations Associated with Cystic Fibrosis

[0009] The mutations recited in Table 2 are described in further detail in, for example, the articles listed in the reference section below.

[0010] Several kits are commercially available for identification of the 25 mutations identified by the ACMG and the ACOG, each of which utilizes a different technology to detect mutations. Such kits have been produced by, for example, Ambion, Celera Diagnostics/ Abbott,

Roche Diagnostics and Innogenetics, Orchid, Nanogen, Third Wave, and Genzyme.

[0011] Multiplex Allele Specific Primer Extension and Solid Support Detection of

Mutations [0012] Multiplex allele specific primer extension, and hybridization of extended primers to a solid support is described generally in the prior art. ASPE technology has been generally described in U.S. Patent No. 4,851,331. The technology is designed to identify the presence or absence of specific polymorphic sites in the genome.

[0013] Multiplex ASPE in conjunction with hybridization to a support for mutation detection can be described generally as follows:

[0014] 1) Amplifying regions of DNA comprising polymorphic loci utilizing a multiplexed,

PCR.

[0015] 2) Allele specific extension of primers wherein the amplified regions of DNA serve as target sequences for the allele specific extension. Extension primers that possess a 3' terminal nucleotide which form a perfect match with the target sequence are extended to form extension products. Modified nucleotides are incorporated into the extension product, such nucleotides effectively labelling the extension products for detection purposes. Alternatively, an extension primer may instead comprise a 3' terminal nucleotide which forms a mismatch with the target sequence. In this instance, primer extension does not occur unless the polymerase used for extension possesses exonuclease activity.

[0016] 3) Hybridizing the extension product to a probe on a solid support, such as a microanay, wherein the probe is complementary to the 5' end of the extension product.

[0017] The extension primers used in a methodology as described above, possess unique sequence tags at their 5' ends. For example, the sequence tags may allow the extension products to be captured on a solid support.

[0018] Variations of the above technology have been described, for example, in U.S. Patent

No. 6,287,778 and PCT Application (WO 00/47766).

[0019] ASPE technology may be used to identify numerous types of mutations including,, deletions, single nucleotide polymorphisms (SNPs), and insertions. [0020] It is an object of the present invention to provide a convenient and rapid multiplex

ASPE/microanay approach for the detection of at least two mutations that have been associated with cystic fibrosis. SUMMARY OF THE INVENTION

[0021] In one embodiment, the present invention provides a method for detecting the presence or absence of mutations in a sample selected from the group of mutations identified in Table 2, the method comprising the steps of:

Amplifying regions of DNA which may contain the above mentioned mutations using at least two PCR primers pairs selected from the group consisting of SEQ LD NO.: 11 and SEQ ID NO. 12, SEQ ID NO.: 13 and SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ TD NO.: 16, SEQ ID NO. 1 and SEQ TD NO.: 2, SEQ ID NO.: 17 and SEQ ID NO.: 18, SEQ ID NO.: 3 and SEQ TD NO. 4, SEQ ID NO.: 19 and SEQ TD NO.: 20, SEQ ID NO.: 21 and SEQ TD NO.: 22, SEQ ID NO. 25 and SEQ ID NO.: 26, SEQ LD NO.: 5 and SEQ ID NO.: 6, SEQ ID NO.: 7 and SEQ JJD NO. 8, SEQ ID NO.: 27 and SEQ ID NO.: 28, SEQ ID NO.: 29 and SEQ ID NO.: 30, SEQ ID NO. 31 and SEQ ID NO.: 32, SEQ LD NO.: 9 and SEQ ID NO.: 10, and SEQ ID NO.: 23 and SEQ ID NO.: 24. [0022] Hybridizing at least two tagged allele specific extension primers, the allele specific extension primers selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 118, to a complementary region of amplified DNA, each tagged allele specific primer having a 3' portion complementary to a region of the amplified DNA, a 3' terminal nucleotide complementary to one allele of one of the mutation sites (wild type or mutant) mentioned above, and a 5' portion complementary to a probe sequence.

[0023] Extending tagged ASPE primers, whereby a labelled extension product of the primer is synthesised when the 3' terminal nucleotide of the primer is complementary to a conesponding nucleotide in the target sequence; no extension product is synthesised when the terminal nucleotide of the primer is not complementary to the conesponding nucleotide in the target sequence.

[0024] Hybridizing extension products to a probe and detection of labelled extension products. Detection of a labelled extension product is indicative of the presence of the allele complementary to the 3 '-terminal nucleotide of the ASPE primer. In the absence of a labelled extension product, it is determined that the allele conesponding to the 3' end of the ASPE primer is not present in the sample.

[0025] In another embodiment, the present invention provides a kit for use in detecting the presence or absence of at least two mutations identified in Table 2, the kit including at least two tagged allele specific extension primers selected from the group consisting of SEQ LD NO: 33 to SEQ ID NO: 118, and per primers pairs selected from the group consisting of SEQ ID NO.: 11 and SEQ ID NO.: 12, SEQ ED NO.: 13 and SEQ LD NO.: 14, SEQ TD NO.: 15 and SEQ ID NO.: 16, SEQ LD NO.: 1 and SEQ LD NO.: 2, SEQ ID NO.: 17 and SEQ LD NO.: 18, SEQ J_D NO.: 3 and SEQ TD NO.: 4, SEQ TD NO.: 19 and SEQ LD NO.: 20, SEQ ID NO.: 21 and SEQ LD NO.: 22, SEQ LD NO.: 25 and SEQ TD NO.: 26, SEQ ID NO.: 5 and SEQ LD NO.: 6, SEQ LD NO.: 7 and SEQ J_D NO.: 8, SEQ TD NO.: 27 and SEQ TD NO.: 28, SEQ TD NO.: 29 and SEQ ID NO.: 30, SEQ TD NO.: 31 and SEQ LD NO.: 32, SEQ ID NO.: 9 and SEQ LD NO.:10, and SEQ LD NO.: 23 and SEQ TD NO.: 24. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] These and other features of the prefened embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein: [0027] Figure 1 depicts an example of steps of the present invention. [0028] Figure 2 is a photograph of a gel presenting results of a genotyping test using the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Definitions: [0030] The following terms used in the present application will be understood to have the meanings defined below.

[0031] The term "mutations" as used herein refers to a number of classes of alteration in a nucleotide sequence including but not limited to, deletions, single nucleotide polymorphisms (SNP), and insertions. An example of a deletion is the ΔF508 nucleotide deletion of the CFTR gene associated with CF. An example of an insertion is the 3905 insT mutation associated with CF. An example of an SNP is the 3120+1 G→ A mutation associated with CF. [0032] The terms "oligonucleotide" and "polynucleotide" as used in the present application refer to DNA sequences being of greater than one nucleotide in length. Such sequences may exist in either single or double-stranded form. Examples of oligonucleotides described herein include PCR primers, ASPE primers, and anti-tags.

[0033] The term "allele" is used herein to refer to different versions of a nucleotide sequence. [0034] The expression "allele specific primer extension (ASPE)", as used herein, refers to a mutation detection method utilizing primers which hybridize to a conesponding DNA sequence and which are extended depending on the successful hybridization of the 3' terminal nucleotide of such primer. Amplified regions of DNA serve as target sequences for ASPE primers. Extension primers that possess a 3' terminal nucleotide which form a perfect match with the target sequence are extended to form extension products. Modified nucleotides can be incorporated into the extension product, such nucleotides effectively labelling the extension products for detection purposes. Alternatively, an extension primer may instead comprise a 3' terminal nucleotide which forms a mismatch with the target sequence. In this instance, primer extension does not occur unless the polymerase used for extension inadvertently possesses exonuclease activity.

[0035] The term "genotype" refers to the genetic constitution of an organism. More specifically, the term refers to the identity of alleles present in an individual. "Genotyping" of an individual or a DNA sample refers to identifying the nature, in terms of nucleotide base, of the two alleles possessed by an individual at a known polymorphic site.

[0036] The term "polymorphism", as used herein, refers to the coexistence of more than one form of a gene or portion thereof.

[0037] The term "PCR", as used herein, refers to the polymerase chain reaction. PCR is a method of amplifying a DNA base sequence using a heat stable polymerase and a pair of primers, one primer complementary to the (+)-strand at one end of the sequence to be amplified and the other primer complementary to the (-) strand at the other end of the sequence to be amplified. Newly synthesized DNA strands can subsequently serve as templates for the same primer sequences and successive rounds of heat denaturation, primer annealing and strand elongation results in rapid and highly specific amplification of the desired sequence. PCR can be used to detect the existence of a defined sequence in a DNA sample.

[0038] The term "primer", as used herein, refers to a short single-stranded oligonucleotide capable of hybridizing to a complementary sequence in a DNA sample. A primer serves as an initiation point for template dependent DNA synthesis. Deoxyribonucleotides can be joined to a primer by a DNA polymerase. A "primer pair" or "primer set" refers to a set of primers including a 5 'upstream primer that hybridizes with the complement of the 5' end of the DNA sequence to be amplified and a 3' downstream primer that hybridizes with the 3' end of the DNA sequence to be amplified. The term "PCR primer" as used herein refers to a primer used for a PCR reaction. The term "ASPE primer" as used herein refers to a primer used for an ASPE reaction.

[0039] The term "tag" as used herein refers to an oligonucleotide sequence that is coupled to an ASPE primer. The sequence is generally unique and non-complementary to the human genome while being substantially complementary to a probe sequence. The probe sequence may be, for example, attached to a solid support. Tags serve to bind the ASPE primers to a probe.

[0040] The term "tagged ASPE primer" as used herein refers to an ASPE primer that is coupled to a tag.

[0041] The term "anti-tag" or "probe" as used herein refers to an oligonucleotide sequence having a sequence complementary to, and capable of hybridizing to, the tag sequence of an

ASPE primer. The "anti-tag" may be coupled to a support.

[0042] The term "wild type" or "wt" as used herein refers to the normal, or non-mutated, or functional form of a gene.

[0043] The term "homozygous wild-type" as used herein refers to an individual possessing two copies of the same allele, such allele characterized as being the normal and functional form of a gene.

[0044] The term "heterozygous" or "HET" as used herein refers to an individual possessing two different alleles of the same gene.

[0045] The term "homozygous mutant" as used herein refers to an individual possessing two copies of the same allele, such allele characterized as the mutant form of a gene.

[0046] The term "mutant" or "mut" as used herein refers to a mutated, or potentially nonfunctional form of a gene.

[0047] The term "call" as used herein refers to the assigned genotype for a particular mutation or variant of the CFTR gene. [0048] The expression "sample failure" as used herein refers to a failure to provide any genotype using a testing method.

DESCRIPTION OF THE INVENTION

[0049] The present invention was developed in response to a need for a rapid, highly specific, and cost-effective method to simultaneously identify multiple genetic risk factors associated with cystic fibrosis. Such identification of risk factors can enhance both treatment and prevention of serious health problems associated with the disease. [0050] The present invention provides a novel, multiplex method of detecting multiple mutations associated with cystic fibrosis. Specifically, the methodology can be used for the detection of the presence or absence of two or more mutations selected from the group consisting of the mutations identified in Table 2. In a preferred embodiment, the present invention provides a method of detecting the presence or absence of all the mutations identified in Table 2.

[0051] The positive detection of one or more of the mutations identified in Table 2 may be indicative of an individual having a predisposition for cystic fibrosis.

[0052] The present invention is further characterized by a high level of specificity. Such specificity is required in order to ensure that any result generated is a true representation of the genomic target and not simply the result of non-specific interactions occurring between reagents present in reactions. This is especially important for multiplexed DNA-based tests where the numerous sequences present in the reaction mixture, most of which are non-complementary, may interact non-specifically depending on the reaction conditions. The high specificity of the present invention is described by example further below. [0053] The present invention is also characterized by its high level of accuracy when compared to existing methodologies for the detection of mutations associated with cystic fibrosis. An example illustrating the accuracy of the present method is provided further below. [0054] The methodology of the present invention utilizes the combination of multiplex ASPE technology with hybridization of tagged and labelled extension products to probes in order to facilitate detection. Such methodology is suitable for high-throughput clinical genotyping applications.

[0055] In one embodiment, the present invention provides a method for detecting the presence or absence of mutations in a sample selected from the group of mutations identified in Table 2, the method comprising the steps of: [0056] Amplifying regions of DNA which may contain the above mentioned mutations. [0057] Hybridizing at least two tagged allele specific extension primers to a complementary region of amplified DNA, each tagged allele specific primer having a 3' portion complementary to a region of the amplified DNA, a 3' terminal nucleotide complementary to one allele of one of the mutation sites (wild type or mutant) mentioned above, and a 5' portion complementary to a probe sequence.

[0058] Extending tagged ASPE primers, whereby a labelled extension product of the primer is synthesised when the 3' terminal nucleotide of the primer is complementary to a conesponding nucleotide in the target sequence; no extension product is synthesised when the terminal nucleotide of the primer is not complementary to the conesponding nucleotide in the target sequence.

[0059] Hybridizing extension products to a probe and detection of labelled extension products. Detection of a labelled extension product is indicative of the presence of the allele complementary to the 3 '-terminal nucleotide of the ASPE primer. In the absence of a labelled extension product, it is determined that the allele conesponding to the 3' end of the ASPE primer is not present in the sample. [0060] A general overview of one example of the above-mentioned method is presented in figure 1. The present invention should not be limited to the example provided in figure 1. A DNA sample is first prepared 10 using methods known in the art. Multiplex PCR amplification 20 is conducted in order amplify regions of DNA containing SNP sites that are associated with cystic fibrosis. A multiplex ASPE reaction 30 is then conducted. By example only, 33 illustrates a wild type and a mutant allele of a gene. At step 36 ASPE primers are hybridized to amplified regions of DNA. If the 3' terminal nucleotide of an ASPE primer is complementary to a conesponding nucleotide in the target sequence, a labelled extension product is formed 39 as will be described further below. The ASPE may be sorted on an addressable universal sorting anay 40 wherein the presence of a labelled extension product may be detected using, for example, xMAP detection 50. DNA Sample Preparation

[0061] Patient samples can be extracted with a variety of methods known in the art to provide nucleic acid (most preferably genomic DNA) for use in the following method.

Amplification

[0062] In a first step at least two regions of DNA containing mutation sites associated with cystic fibrosis are amplified.

[0063] In a prefened embodiment of the present invention, PCR amplification of regions containing mutation sites associated with cystic fibrosis is initiated using at least two pairs of PCR primers selected from the group of primer pairs consisting of: SEQ ID NO.: 11 and SEQ ID NO.: 12, SEQ ID NO.: 13 and SEQ BD NO.: 14, SEQ TD NO.: 15 and SEQ ID NO.: 16, SEQ LD NO.: 1 and SEQ ID NO.: 2, SEQ ID NO.: 17 and SEQ TD NO.: 18, SEQ TD NO.: 3 and SEQ ID NO.: 4, SEQ TD NO.: 19 and SEQ ID NO.: 20, SEQ LD NO.: 21 and SEQ ID NO.: 22, SEQ ID NO.: 25 and SEQ ID NO.: 26, SEQ ID NO.: 5 and SEQ ID NO.: 6, SEQ ID NO.: 7 and SEQ ID NO.: 8, SEQ ID NO.: 27 and SEQ ED NO.: 28, SEQ TD NO.: 29 and SEQ TD NO.: 30, SEQ TD NO.: 31 and SEQ ID NO.: 32, SEQ ID NO.: 9 and SEQ ED NO.:10, and SEQ TD NO.: 23 and SEQ ID NO.: 24. [0064] The relationships of each pair of primers to the mutation sites listed in Table 2 is presented in Table 3. TABLE 3: Primer Pairs Used to Amplify Regions Containing Cystic Fibrosis Associated Mutations

[0065] An individual skilled in the art will recognize that alternate PCR primers could be used to amplify the target polymorphic regions, however, in a prefened embodiment the primers listed in Table 3 are selected due to their minimal non-specific interaction with other sequences in the reaction mixture. ASPE [0066] The ASPE step of the method of the present invention is conducted using at least two tagged ASPE primers selected from the group of ASPE primers consisting of SEQ ED NO: 33 to SEQ ID NO.: 118. [0067] The ASPE primer set of the present invention has been optimized, as described further below by example, to ensure high specificity and accuracy of diagnostic tests utilizing such allele specific primers. [0068] Table 4: ASPE Primers of the Present Invention

π

[0069] Table 4 presents a listing of the ASPE primers used in a preferred embodiment of the present invention. The suffix "wt" represents an ASPE primer used to detect the wild type form of the CFTR gene at a specific mutation site. The suffix "mut" represents an ASPE primer used to detect a mutant form of the CFTR gene at a specific mutation site. Bases 1 to 24 of each of SEQ ID NO.: 33 to SEQ ID NO: 118 are the 5' portions of the ASPE primers that are complementary to specific probe sequences. Although the specific sequences listed in table 4 are prefened, in alternate embodiments of the present invention, it is possible to combine different 5' portions of the sequences in Table 4 (bases 1 to 24 of SEQ ID NOs: 33 to 118) with different 3' end hybridizing portions of the sequences in Table 4 (bases 25 and up of SEQ ID NOs: 33 to 118).

[0070] The 3' end hybridizing portion of the extension primer is hybridized to the amplified material. Where the 3' terminal nucleotide of an ASPE primer is complementary to the polymorphic site, primer extension is canied out using a modified nucleotide. Where the 3' terminal nucleotide of the ASPE primer is not complementary to the polymorphic region, no primer extension occurs.

[0071] In one embodiment, labelling of the extension products is accomplished through the incorporation of biotinylated nucleotides into the extension product which may be identified using fluorescent (Streptavidin-Phycoerythrin) or chemiluminescent (Streptavidin-Horseradish Peroxidase) reactions. However, an individual skilled in the art will recognize that other labelling techniques may be utilized. Examples of labels useful for detection include but are not limited to radiolabels, fluorescent labels (e.g fluorescein and rhodamine), nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner, and chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. [0072] Each ASPE primer used in the methodology as described above, possess a unique sequence tag at their 5' ends. The sequence tags allow extension products to be detected with a high degree of specificity, for example, through capture on a solid support in order to facilitate detection. [0073] Detection [0074] The tagged 5' portions of the allele specific primers of the present invention are complementary to probe sequences. Upon hybridization of the allele specific primers to a conesponding probe sequence the presence of extension products can be detected. [0075] In a prefened embodiment, probes used in the methodology of the present invention are coupled to a solid support, for example a 'universal' bead-based microanay. [0076] Examples of supports that can be used in the present invention include, but are not limited to, bead based microanays and 2D glass microarrays. The preparation, use, and analysis of microarrays are well known to persons skilled in the art. (See, for example, Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena, et al. (1996) Proc. Natl. Acad. Sci. 93:10614- 10619; Baldeschweiler et al. (1995), PCT Application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M: J. et al. (1997) U.S. Pat. No. 5,605,662.). Detection can be achieved through anays using, for example, chemiluminescence or fluorescence technology for identifying the presence of the SNPs.

[0001] Universal anays function as sorting tools indirectly detecting the target of interest and are designed to be isothermal and minimally cross-hybridizing as a set. Examples of microarrays which can be used in the present invention include, but should not be limited to, Luminex's^® bead based microanay systems, and Metrigenix's™ Flow Thru chip technology.

[0078] In one embodiment, for example, Luminex's 100 xMAP fluorescence based solid support microanay system is utilized. Anti-tag sequences complementary to the tag regions of the ASPE primers/extension products, described above, are coupled to the surface of internally fluorochrome-color-coded microspheres. An array of anti-tag microspheres is produced, each set of microspheres having its own characteristic spectral address. The mixture of tagged, extended, biotinylated ASPE primers is combined with the anay of anti tagged microspheres and is allowed to hybridize under stringent conditions.

[0079] In a reaction mixture, a fluorescent reporter molecule (e.g. streptavidin- phycoerythrin) is used to detect labelled extension products which are synthesized when the terminal nucleotide of an ASPE primer is complementary to a conesponding nucleotide in the target sequence.

[0001] The reaction mixture, comprising microspheres, extension products etc. is injected into a reading instrument, for example Luminex's 100 xMAP , which uses microfluidics to align the microspheres in single file . Lasers are used to illuminate the colors both internal to the microspheres, and attached to the surface in the form of extension products hybridized to anti-tag sequences. The Lurninex 100 xMAP , interprets the signal received and identifies the presence of wild type and/or mutant alleles. The presence of the mutant allele of any one or more of the 44 mutations presented in Table 2 may be indicative of cystic fibrosis, or a pre-disposition to cystic fibrosis. Software can be provided which is designed to analyze data associated with the specific extension products and anti-tagged microspheres of the present invention. [0081] In another embodiment, the Metrigenix Flow-Thru three dimensional microchannel biochip (Cheek, B J., Steel A.B., Tones, M.P., Yu, Y., and Yang H. Anal. Chem. 2001, 73, 5777- 5783) is utilized for genotyping as known in the art. In this embodiment, each set of microchannels represents a different universal anti-tag population. Anti-tag sequences conesponding to. the tag regions of the ASPE primers/extension products, described above, are attached to the inner surface of multiple microchannels comprising a cell. Multiple cells make up a chip. The reaction mixture, including biotinylated extension products flows through the cells in the presence of a chemiluminescent reporter substrate such as streptavidin-horseradish peroxidase. Microanay chips can be imaged using technology known in the art, such as an ORCA-ER CCD (Hamamatsu Photonics K. K., Hamamatsu City, Japan), and imaging software, in order to identify the genotype of an individual. Kits

[0082] In an additional embodiment, the present invention provides kits for the multiplex detection of mutations associated with cystic fibrosis.

[0083] A kit that can be used for detection of the mutations of interest may contain the following components including: a PCR primer mix for amplifying regions containing mutation sites of interest (optionally including dNTPs), an ASPE primer mix for generation of labelled extension products (optionally including dNTPs) and a solid support, such as microarray beads, the beads having anti-tags complementary to the tagged regions of the ASPE primers. In addition, an individual skilled in the art would recognize other components which could be included in such kits including, for example, buffers and polymerases. [0084] Kits of the present invention may include PCR primer pairs, ASPE primers, and tagged supports for all the mutations to be detected, or may be customized to best suit the needs of an individual end user. For example, if an end user wishes to detect only 25 of the mutations associated with cystic fibrosis, a kit can be customized to include only the PCR primer pairs, ASPE primers, and support required for the detection of the desired mutations. As such, the end user of the product can design a kit to match their specific requirements. In addition, the end user can also control the tests to be conducted at the software level when using, for example, a universal bead based-microanay for detection. For example, software can be provided with a is kit, such software reading only the beads for the desired mutations or by reporting only the results from. the desired mutation data. Similar control of data reporting by software can be obtained when the assay is performed on alternate platforms.

[0085] An individual skilled in the art will recognize that although the present method has been described in relation to 44 specific cystic fibrosis associated mutations, PCR primers and ASPE primers used to detect additional mutations could be included in the above method and kits.

[0086] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety

[0087] The examples presented below are provided to illustrate the present invention and are not meant to limit the scope of the invention as will be apparent to persons skilled in the art. [0088] EXAMPLE #1 : ASPE/Microarray Detection of CFTR Mutations [0089] MATERIALS and METHODS [0090] 1) Oligonucleotides

[0091] All oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). PCR primers were unmodified and were purified by standard desalting procedures. Universal anti-tags (probes) were 3'-C7 amino-modified for coupling to carboxylated microspheres. All anti-tags were reverse phase HPLC -purified. Chimeric ASPE primers which consisted of a 24mer universal tag sequence 5' to the allele-specific sequence were also unmodified but were purified by polyacrylamide gel electrophoresis. Following reconstitution, exact oligo concentrations were determined spectrophotometrically using extinction coefficients provided by the supplier. Reconstituted oligos were scanned between 200 and 800 nm and absorbance was measured at 260 nm to calculate oligo concentration. [0092] 2) Reagents

[0093] Platinum Taq, Platinum Tsp, individual dNTPs and biotin-dCTP were purchased from Invitrogen Corporation (Carlsbad, CA). Shrimp alkaline phosphatase and exonuclease I were purchased from USB Corporation (Cleveland, OH). Carboxylated fluorescent microspheres were provided by Luminex Corporation (Austin, TX). The EDC cross-linker (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride) was purchased from Pierce (Rockford, EL). OmniPur reagents including MES (2-(N-morpholino)ethane sulfonic acid), 10% SDS, NaCl, Tris, Triton X-100, Tween-20 and TE buffer were purchased from EM Science (Darmstadt, Germany). The streptavidin-conjugated phycoerythrin was obtained from Molecular Probes Inc. (Eugene, OR). [0094] 3) Genotyping

[0095] a) MULTIPLEX PCR (16-plex): Multiplex PCR was carried out using 25 ng genomic DNA in a 25 uL final volume. A 'no target' PCR negative control was included with each assay run. The reaction consisted of 30 mmol/L Tris-HCl, pH 8.4, 75 mmol/L KCl, 2 mmol/L MgC12, 200 umol/L each dNTP, 5 units Platinum Taq, with primers ranging from 0.15 to 0.6 umol/L. Samples were cycled in an MJ Research PTC-200 thermocycler (Reno, NV) with cycling parameters set at 95°C for 5 minutes followed by 30 cycles at 95°C for 30 seconds, 58°C for 30 seconds and 72°C for 30 seconds. Samples were then held at 72°C for 5 minutes and kept at 4°C until use.

[0096] b) ALLELE-SPECIFIC PRIMER EXTENSION: Prior to the ASPE reaction, each PCR reaction was treated with shrimp alkaline phosphatase (SAP) to inactivate any remaining nucleotides (particularly dCTP) so that biotin-dCTP could be efficiently incorporated during the primer extension reaction. Each PCR reaction was also treated with exonuclease I (EXO) to degrade remaining PCR primers in order to avoid any interference with the tagged ASPE primers and the extension reaction itself. To each 25 uL PCR reaction, 2.5 uL SAP (= 2.5 units) and 1.0 uL EXO (= 10 units) were added directly. Samples were then incubated at 37°C for 30 minutes followed by a 15 minute incubation at 99°C to inactivate the enzymes. Samples were then added directly to the ASPE reaction.

[0097] Multiplex ASPE was carried out using 5 uL of treated PCR product in a final volume of 20 uL. Each reaction consisted of 20 mmol/L Tris-HCl pH 8.4, 50 mmol/L KCl, 1.25 mmol/L MgC12, 5 umol/L biotin-dCTP, 5 umol/L each of dATP, dGTP and dTTP, 4.5 units Platinum Tsp and 10 nmol/L ASPE primer pool (ie. each ASPE primer present at 200 fmol/reaction). The ASPE reactions were incubated at 96°C for 2 minutes and then subjected to 40 cycles at 94°C for 30 seconds, 52°C for 30 seconds and 74°C for 60 seconds. Reactions were then held at 4°C until use.

[0098] c) BEAD COUPLING: Amino-modified anti-tag sequences were coupled to carboxylated microspheres following Luminex's one-step carbodiimide coupling procedure. Briefly, 5 x 10⁶ microspheres were combined with 1 nmol NH₂-oligo in a final volume of 50 uL 0.1 mol/L MES, pH 4.5. A 10 mg/mL EDC working solution was prepared just prior to use and 2.5 uL was added to the bead mixture and incubated for 30 minutes. A second 2.5 uL aliquot of freshly prepared EDC was added followed by an additional 30 minute incubation. Following washes in 0.02% (v/v) Tween-20 and 0.1% (w/v) SDS, the anti-tag coupled beads were resuspended in 100 uL TE buffer (10 mmol/L Tris, pH 8.0, 1 mmol/L EDTA). Bead concentrations were determined using a Beckman Coulter Z2 Particle Count and Size Analyzer (Coulter Corp, Miami FL).

[0099] d) UNJNERSAL ARRAY HYBRIDIZATION: Each hybridization reaction was carried out using approximately 2500 beads of each of the 86 anti-tag bearing bead populations. The beads were combined in hybridization buffer (0.22 mol/L NaCl, 0.11 mol L Tris, pH 8.0 and 0.088% (v/v) Triton X-100) and 45 uL of the mix were added to each well of an MJ Research 96-well plate (Reno, NV). A 5 uL aliquot of each ASPE reaction was then added directly to each well. The samples were then heated to 96°C for 2 minutes in an MJ Research PTC-200 followed by a one hour incubation at 37°C. Following this incubation, samples were filtered through a 1.2 um Durapore Membrane (Millipore Corp, Bedford, MA) and washed once using wash buffer (0.2 mol/L NaCl, 0.1 mol/L Tris, pH 8.0 and 0.08% (v/v) Triton X-100). The beads were then resuspended in 150 uL reporter solution (1 ug/mL sfreptavidin-conjugated phycoerythrin in wash buffer) and incubated for 15 minutes at room temperature. The reactions were read on the Luminex xMAP. Acquisition parameters were set to measure 100 events per bead population and a 100 uL sample volume. A gate setting was established prior to ninning the samples and maintained throughout the course of the study. [00100] RESULTS

[00101] For optimal PCR, buffer composition, cycling parameters, annealing temperature, genomic DNA input as well as primer concentrations for each mutation were examined. PCR products generated under the final optimized conditions were analyzed by gel electrophoresis using the Helixx SuperGel350 system (Scarborough, ON) which is capable of resolving 2-5 basepair differences within products below 500 bp. A gel image of 5 patient samples amplified under optimal conditions is provided in Figure 2 and clearly demonstrates that the multiplex PCR reaction of the present invention was highly specific for the desired amplimers. The migration and number of bands seen at 271 bp (size of the amplimer contaimng the ΔF508 mutation) conesponds to the genotype of the sample for the ΔF508 mutation. [00102] EXAMPLE #2

[00103] This example illustrates both the accuracy and the specificity of the present invention. Accuracy, is a measure of concordance of the resultant genotyping calls on the 44 mutations/variants determined by the method of the present invention (from hereon in this example refened to as the CFTR 40+4 genotyping assay) to the genotyping calls from reference methods.

[00104] The reference methods used were (1) DNA sequencing employed by Genaissance Pharmaceuticals and (2) the Applied Biosystems, Inc. Cystic Fibrosis (ABI-CF) System.

[00105] The present invention was used to analyze 139 genomic DNA samples. All 139 genomic DNA samples analyzed with the CFTR 40+4 genotyping assay provided calls for all 44 mutations and variants detected by the CFTR 40+4 genotyping assay. Thus, >95% of the genomic DNA samples tested yielded genotyping calls over all 44 mutations and variants tested for by the CFTR 40+4 genotyping assay.

[00106] In this example, there were initially a maximum of 6116 genotypic calls possible (139 samples, each genotyped for 44 mutations/variants) by each of either the CFTR 40+4 genotyping assay or by the reference methods. This total number of comparable calls was reduced by 37 calls due. to the inability of the DNA sequencing to genotype 37 separate mutations/variants of 24 individual DNA samples. A further two genotyping calls were removed from analysis because of an allele that was not detectable by the CFTR 40+4 genotyping assay. Thus, there were ultimately a total of 6077 possible calls (i.e.: 6116 - 37 - 2) that could be made by, and compared between the CFTR 40+4 genotyping assay and the reference methods. [00107] Upon initial comparison of the calls obtained from the CFTR 40+4 genotyping assay and the conesponding calls obtained by the reference methods, eight discordant calls were identified, giving an initial overall percent concordance of 99.87% for the method of the present invention.

[00108] The DNA samples with discordant genotyping calls were reanalyzed by DNA sequencing. Upon reanalysis of the eight discordant calls made by the CFTR 40+4 genotyping assay, all mutations/variants were resolved and found to be concordant to the conesponding calls made by DNA sequencing. Therefore, after resolving the discordances the overall percent concordance of the CFTR 40+4 genotyping assay to the reference methods was 100%. [00109] Finally, each of the 44 mutations and variants detected by the CFTR 40+4 genotyping assay initially yielded overall percent concordances of >95%, when compared to the results obtained by the reference methods.

[00110] INTRODUCTION AND BACKGROUND INFORMATION [00111] An overview of the protocol used for this example is outlined below. [00112] For each sample, 25 ng of genomic DNA was amplified in a single multiplex (16- plex) polymerase chain reaction (PCR). The amplimer sizes ranged from 179 bp to 465 bp. To enable efficient incorporation of biotin-dCTP during the multiplex Allele Specific Primer Extension (ASPE) reaction, each PCR product was treated with Shrimp Alkaline Phosphatase (SAP) to inactivate any remaining nucleotides (especially dCTP), and with Exonuclease I (EXO) to degrade any primers left over from the PCR. A 5 μL aliquot of the treated PCR product was used in the ASPE reaction containing 86 universally-tagged primers. The ASPE products are then sorted by hybridization to the universal anay (Bead Mix) in the presence of a hybridization buffer, and then incubated with Streptavidin, R-Phycoerythrin conjugate (reporter solution). Samples were read on the Luminex® 100 xMAP™ instrument and a signal was generated for each of the 40 mutations and 4 variants (and/or their conesponding wild-type alleles). These fluorescence values were then analyzed to determine whether for each mutation or variant the samples were wild-type, heterozygous or that at least one mutant allele is present. [00113] OVERVIEW OF TESTING PROCEDURES [00114] One hundred and thirty nine (139) genomic DNA samples were obtained as solutions. These DNA samples were analyzed with the method of the present invention. The DNA samples were previously characterized for 30 of the 44 mutations/variants utilizing the Applied Biosystems, Inc. Cystic Fibrosis System. The remaining 14 mutations and variants not detected by the ABI-CF System were genotyped by DNA sequencing (the other reference method) which was performed by Genaissance Pharmaceuticals. Table 5 indicates the different methodologies and the mutations and/or variants that were genotyped by each method. [00115] ^' Table 5. Methods employed to genotype the 44 mutations and variants of the CFTR that are detected by the method of the present invention.

Some of the mutations and variants, that were not detected by the ABI-CF sjstem, although not initially sequenced may be and in some cases were analyzed by DN. A sequencing since these mutations and variants were located within the DNA hcquencβ of exons that wero shipped to Genaissance Pharmaceuticals specifically for DNA sequencing.

[00116] The resulting genotype calls for the 44 mutations and variants obtamed from the CFTR 40+4 genotyping assay were then compared to the corresponding calls obtained by the reference methods (DNA sequencing and the ABI-CF System). These comparisons were used to determine the degree of concordance between the calls made by the CFTR 40+4 genotyping assay and the corresponding calls made by the reference methods. This accuracy measure of the CFTR 40+4 genotyping assay was determined for the calling of each particular variant and for the CFTR 40+4 genotyping assay as a whole. [00117] CALCULATION OF ACCURACY

[00118] The concordance of the genotypic calls obtained by the CFTR 40+4 genotyping assay to the reference methods was determined for each of the CFTR 40+4 mutation and variants per DNA sample. The overall percent concordance was then determined for each mutation and variant over the full genomic DNA sample set. Finally, the overall percent concordance of the method of the present invention was determined from the percentage of genotyping calls determined by the CFTR 40+4 genotyping assay that matched those determined by the reference methods. The formulas used in the calculation of percent concordance are presented in Appendix

1.

[00119] Results that yielded a: (1) variant failure, or (2) sample failure were not included in the accuracy calculations since these events do not report a call, but instead indicated a failure of the method or assay used to genotype the mutation or variant.

[00120] ACCEPTANCE CRITERIA

[00121] The acceptance criteria for the method of the present invention were as follows: • No more than 5% of the samples were allowed to fail (i.e.: not providing any genotype at all).

• Of the samples which do not fail, each variant must have an overall % concordance of at least 95% (i.e.: C_overan(m) >95% for all 44 mutations and variants).

• A minimum overall assay % concordance, for the method of the present invention, of at least 98% must be achieved (i.e.: Cτ_agn≥9c %).

[00122] RESULTS

[00123] Results from the ABI-CF System reference method

Table 6

Genotyping calls obtained by the reference methods and the CFTR 40*4 genotyping assay for the mutations/variants and T-tract of DNA Samples 1 -18

assay _t T e ABI-CF System etecte a tir mutatou 15S&K. an genotype tas a HET_> tns mutatons not etecte y te CFTR 40+4 gaiolypmg t

Table 7 Genotyping calls obtained by the reference methods and the CFTR 40+4 genol assay for the mutøtlonstariarrts and T-tract of DNA Samples 19 - 36

Table 8 Genotyping calls obtained the reference methods and the CFTR 40+4 genotyping assay for the mut s/variants and T-tract of DNA Samples 37 - 54

a. For the 1898+1 G> 4 mutation, DNA sample 37 was genotyped as WT by th& ABI-CF System and HET by both DN sequencing and the CFTR 40+4 genoljping assay. b. For the 1 S9& I-IΘ A mutation, DNA sample 38 was genotyped as HET by the ABI-CF Systsmi and WT by both DNA sequencing and the CFTR 40+4 genotyprag assay.

Table 9 Genotyping calls obtained by the reference methods d the CFTR 40+4 genot in* assay for the mutations/variants and T-tract of DNA Samples 55 - 72

a. DNA sample 57 was called WT foi all mutations and variants by bath the ABI-CF System and by the CFTR 4S+4 genotyping assay. h. FortheΔFSD! 3 variant, DNA sampL 5 63 was genotyped as WT hy the ABI ■CF System and HET by both DNA sequencing and the CFT 40+4 genotyping assay.

Table 10 Genotyping calls obtained by the reference methods and the CFTR 40+4 gen assay for the mutations/variants and T-tract of DNA Samples 73 - 92

Table 11 Genotyping calls obtained by the referenc methods and the CFTR 40+4 genotyping assay for the mutations/variants and T-tract of DNA Samples 93 - 112

Table 12 Genotyping calls obtained by the reference methods and the CFTR 40+4 genotyping assay for the mutations/variants mnd T-tract of DNA Samples 113 - 131

a. FDrtlιe 621+lQ Amutahoπ₄ DNA samp e 131 was genotyped as HET by the ABI-CF S slettt and WT by both DNA se uencing and the CFTR 4D+4 genølyping assay. For the ΔF50 S vai mat, the same TM A samp .e-was genotyped as WT by the ABI-CF System and HET by both DNA sequencing and the CFTR 40+4 genotyping assay.

Table 13

Genotyping calls obtained by the reference methods and the CFTR 40+4 genotyping assay for the mutations/variants and T ract of DNA Samples 132 - 141

Fαrthe 334W πiutatøn, DNA sample 137 vm$ genotyped as WThy the ABI-CF System an HBT by both DNA seq iencing and the CFTR 40+4 genotyping assay

For Tables 6 to 13, unless indicated, all other mutations and variants were called WT (i.e.: the mutant allele was not detected). For all samples (except for sample 15), no more than two mutation or variants in each were non-WT.

[00124] The genotyping calls obtained from the reference methods (DNA sequencing and ABI-CF System) are found in Tables 6-13. Of the 44 mutations and variants detected and simultaneously genotyped by the CFTR 40+4 genotyping assay, the ABI-CF System can genotype 30 of these mutations and variants. The remaining 14 mutations and variants were genotyped by Genaissance Pharmaceuticals via DNA sequencing.The genotyping calls obtained from the ABI-CF System are summarized in Table 14.

The genotyping calls were provided by Dr. Peter Ray's laboratory and ware ased απ those mutations and variants detected by the ABI-CF System. IF the ABI-CF System did not detect either the heterozygous (HET) or mutant (MTJT) genotype, the mutation or variant was Table 14 considered to be wild-type (WT) Far that DNA sample..

[00125] Results from the reference method of DNA sequencing [00126] The initial DNA sequencing reactions performed by Genaissance Pharmaceuticals produced a requirement for several sequencing repeats (Table 15) due to the absence of successful sequencing either in one or in both DNA sequencing directions. Table 15

a. For the exons containing two mutations (ie.: exons 10, 13 and 20), it was possible that either one of the pair or both of the mutations could not be genotyped from the same exon, ln these cases the total number of actual genotjping call failures was indicated as the total number of call failures (e.g. if mutation Y1892X and Ml 10 IK of sample 1 could not be genotyped, both mutations in the same exon were counted as individual call failures).

[00127] Table 15 presents the number of genotyping call failures observed from the initial

DNA sequencing run and in the DNA sequencing repeats (the 14 mutations and variants are indicated) for each of the 139 DNA samples tested. [00128] DNA samples which failed to give unambiguous genotyping calls for a particular mutation or variant in both directions were resequenced in both directions. Repeat sequencing was performed on the same amplimers. The only amplimers that required reamplification were for exon 13 (see below). [00129] Since there were many initial genotyping failures observed for some of the exons, it was more convenient for Genaissance Pharmaceuticals to repeat the sequencing for all samples for that exon.

[00130] Exons 11 (mutation A559T) and 12 (mutation 1898+5G>T) required a small number repeat DNA sequencing (5 and 3 respectively) and thus only those DNA samples which failed to be genotyped for their mutation were resequenced (samples 77, 78, 89, 91 and 114 for exon 11 and samples 13, 62 and 134 for exon 12). In exons 3 (mutation 394delTT) and 4 (mutation I148T), the DNA samples which required a sequencing repeat were spread throughout the 139 DNA samples so much so that it was more convenient to repeat the sequencing of the samples, rather than repeat the sequencing for those specific samples. When analyzing the DNA sequencing data from the repeats, it was only the samples which failed that were investigated. The DNA sequencing electropherograms of exon 13 indicated contamination of the opposite strand in the- sequencing data of both the forward and reverse DNA sequencing reactions. Because of this, it was decided to prepare fresh amplimers of exon 13 for each DNA sample. These new amplimers were shipped to Genaissance Pharmaceuticals for sequencing. The DNA sequencing did not indicate any contamination; moreover, most of genotyping calls could be made from this reaction. A small number did require resequencing but genotyping calls were made based on the DNA sequencing results from a single direction. DNA sample 14 was genotyped as WT for the 2307insA mutation from the reverse sequencing direction only and DNA sample 74 was genotyped as WT for the 2184delA mutation from the forward sequencing direction only.

[00131] For the case of the T-tract variants (detected by the CFTR 40+4 genotyping assay as the 5T, 7T and 9T alleles), the final genotyping calls obtained from DNA sequencing are summarized in Tables 6 to 13. The DNA sequencing called samples 97 and 101 each as a 7T/1 IT genotype. Since the CFTR 40+4 genotyping assay cannot detect the 1 IT allele, these two calls were eliminated from further comparison. [00132] Of the genomic DNA samples that required the DNA sequencing to be repeated, 37 genotyping calls could not be genotyped in the end. The 37 failed genotyping calls were therefore removed from further analysis. Two calls from the T -tract were also removed from the set of genotyping calls obtained from the reference methods that were compared against the CFTR 40+4 genotyping assay. A total of 6077 genotyping calls (i.e.: 6116 total expected genotypic calls - 37 mutations/variants that could not be called - 2 calls that could not be detected by the CFTR 40+4 genotyping assay) were thus compared against the corresponding genotyping calls obtained by the CFTR 40+4 genotyping assay. [00133] Table 16 summarizes the total number of samples that, through DNA sequencing, were called as either WT, FIET, or MUT for each mutation/variant tested for. This table accounts for the removal of all the individual genotyping call failures from DNA sequencing (see above). [00134] In general, the DNA sequencing results showed that only DNA sample 82 was called as heterozygous (HET) for mutation 3120+1G>A (found in exon 16), all other mutations and variants that were successfully sequenced were called WT. [00135] CFTR 40+4 genotyping assay results

[00136] The calls made by the software used in the method of the present invention are found in Tables 6 to 13. The genomic DNA samples were initially divided into seven batches, each contained up to 23 samples and one negative control. All 139 DNA samples analyzed with the CFTR 40+4 genotyping assay successfully provided genotypes without the need for reruns. [00137] DNA sample reanalysis due to discordance

[00138] Up until this point, any assay repeats performed were for obtaining unambiguous genotyping calls for all mutations or variants of each DNA sample. After all required DNA sequencing repeats, and the removal of mutations or variants that could not be genotyped, the remaining unambiguous calls were analyzed. The genotyping calls determined by the CFTR 40+4 genotyping assay were then compared to the corresponding genotyping calls obtained by the reference methods. When these calls were initially compared, eight discordant calls in seven DNA samples were identified (see below). The DNA samples were reanalyzed by, DNA sequencing in order to resolve the discordances. Upon the reanalysis, seven of these eight discordant calls from the CFTR 40+4 genotyping assay were resolved and found to be concordant to the corresponding calls obtained by DNA sequencing. Table 16

[00139] - DNA sample 15 was genotyped by the ABI-CF System as heterozygous (HET) for the R560T mutation (found in exon 11). The CFTR 40+4 genotyping assay called this sample as wild-type (WT). Results from the DNA sequencing of exon 11 indicated a wild-type (WT) genotype for this sample, which was concordant to the CFTR 40+4 genotyping assay. [00140] - DNA sample 36 was genotyped by the ABI-CF System as WT (i.e.: a mutant allele was not detected) for the 1898+1G>A mutation (found in exon 12). The CFTR 40+4 genotyping assay called this sample as HET. Results from the DNA sequencing of exon 12 indicated a HET genotype for this sample, which was concordant to the CFTR 40+4 genotyping assay. [00141] - DNA sample 37 was genotyped by the ABI-CF System as WT (i.e.: a mutant allele was not detected) for the 1898+1G>A mutation. The CFTR 40+4 genotyping assay called this sample as HET. The DNA sequencing results from exon 12 indicated a HET genotype for sample 37, which was concordant to the CFTR 40+4 genotyping assay.

[00142] - DNA sample 38 was genotyped by the ABI-CF System as HET for the ΔF508 mutation (found in exon 10). The CFTR 40+4 genotyping assay called this sample as WT, a result which was concordant to the DNA sequencing results of exon 10. [00143] - DNA sample 72 was genotyped by the ABI-CF System as WT (i.e.: a mutant allele was not detected) for the ΔF508 mutation. The CFTR 40+4 genotyping assay called this sample as HET. Analysis of the DNA sequencing results for exon 10 indicated a HET genotype for this sample, a result which was concordant to the CFTR 40+4 genotyping assay. [00144] - DNA sample 131 was genotyped by the ABI-CF System as WT (i.e.: a mutant allele was not detected) for the 621+lOT mutation (found in exon 4). The CFTR 40+4 genotyping assay called this sample as HET. Analysis of the DNA sequencing of exon 4 indicated a HET genotype for this sample, a result which was concordant to the CFTR 40+4 genotyping assay. This sample was also genotyped by the ABI-CF System as WT for the ΔF508 mutation. The CFTR 40+4 genotyping assay called this sample as HET and DNA sequencing analysis of exon 10 of this sample indicated a HET genotype, a result which was concordant to the CFTR 40+4 genotyping assay.

[00145] - DNA sample 137 was genotyped by the ABI-CF System as WT (i.e.: a mutant allele was not detected) for the R334W mutation. The CFTR 40+4 genotyping assay called this sample as HET. This mutation was found in exon 7. In order to resolve this discordance sample 137 was amplified for exon 7 by the PCR and then sequenced. Subsequent DNA sequencing analysis of exon 7 of this sample indicated a HET genotype, a result which was concordant to the CFTR 40+4 genotyping assay. [00146] Analysis of the T-tract variant (5T/7T/9T) in exon 9

[00147] Samples 97 and 101 were called by DNA sequencing as a 7T/1 IT genotype for the T-tract variant found in exon 9. The CFTR 40+4 genotyping assay detected the 7T allele for these two samples. Though not necessarily discordant calls, what was evident was the ability of the CFTR 40+4 genotyping assay to successfully detect the 7T allele and inability of the CFTR 40+4 genotyping assay to detect the 11T allele. These two calls were thus mutually eliminated from comparison between the reference methods of genotyping and the CFTR 40+4 genotyping assay. All other DNA samples exhibited concordant genotyping calls between the CFTR 40+4 genotyping assay and DNA sequencing reference method for the T-tract variant. [00148] Mutation/Variant call percent concordance and kit overall percent concordance [00149] The call accuracy of the CFTR 40+4 genotyping assay, as measured by percent concordance to the reference methods of genotyping was determined for each of the 44 mutations and variants detected by the CFTR 40+4 genotyping assay. The final results indicated >97% concordance of the CFTR 40+4 genotyping assay to the reference methods except for those discordances indicated above. In these cases though, the percent concordances for the affected mutation or variant remained greater than 95%. The percent concordances for these affected mutations and variants are indicated in Table 9.

[00150] After the allowed reanalysis of the ABI-CF System results via DNA sequencing by Genaissance Pharmaceuticals, the percent concordance increased for these mutations and variants (see Table 17).

[00151] Table 17 presents the percent concordance between the genotyping calls obtained by the CFTR 40+4 genotyping assay and the reference methods prior to and after reanalysis of available DNA sequencing data. Table 17

[00152] In summary, prior to and after the reanalysis of discordant DNA samples, the overall CFTR 40+4 genotyping assay percent concordance to the reference methods was greater than the minimal 98% acceptance criteria. The initial overall CFTR 40+4 genotyping assay percent concordance was 99.87% (eight discordances). After reanalysis, the CFTR 40+4 genotyping assay percent concordance was 100%. [00153] Call rate comparison

[00154] It was also useful to be able to compare the call rate of the CFTR 40+4 genotyping assay to the call rate of the reference methods. This comparison did not address whether there was concordance in the calls but specifically the ability of the genotyping methods to successfully yield calls. Since the rerun rate for the non-sequencing genotyping methods was not known, the call rate of the reference methods was derived from the call rate of the DNA sequencing. To determine the call rate of the CFTR 40+4 genotyping assay and the reference method of DNA sequencing, the number of expected calls was determined from the final number of DNA samples tested equally by both methods for the 14 mutations and variants indicated in Table 5 Therefore, there were 1946 possible genotyping calls to be made on the 139 DNA samples that were tested by both methods.

[00155] The reference method of DNA sequencing had a call rate of 75% from a single initial sequencing run (482 failed calls that required sequencing repeats). After the sequencing repeats, 37 genotypes still failed to be called. Thus, sequencing of 14 mutations and variants for each of 139 DNA samples produced a final call rate of 98%, (see Table 18) for DNA sequencing. The final call rate for DNA sequencing even allowed for some samples to be called from DNA sequencing data obtained confidently from only one sequencing direction. [00156] In the case of the CFTR 40+4 genotyping assay, the initial assay runs resulted in the successful calling of all 139 DNA samples yielding a call rate of 100% and no requirement for a repeat of the assay, (see Table 18). It should be noted that all 37 genotyping calls that could not be made unambiguously by DNA sequencing were called by the CFTR 40+4 genotyping assay as WT.

Table 18: Call Rates of the CFTR 40+4 Genotyping Assay and DNA Sequencing Based on 14 Mutations and Variants That Were Tested by Both Methods

[00157] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention as outlined in the claims appended hereto. [00158] References

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for detecting the presence or absence of a variant nucleotide in at least two mutation sites associated with cystic fibrosis, said mutations sites selected from the group of mutations listed in Table 2, the method comprising the steps of; a) amplifying regions of DNA containing the at least two mutation sites to form amplified DNA products; b) hybridizing at least two tagged allele specific extension primers to a complementary target sequence in the amplified DNA products, wherein each tagged allele specific extension primer has a 3 '-end hybridizing portion substantially complementary to an allele of one of the mutation sites associated with cystic fibrosis and a 5'-end tag portion complementary to one of a set probes, the terminal nucleotide of the 3' end hybridizing portion being either complementary to a suspected variant nucleotide or to the corresponding wild type nucleotide of the mutation site; c) extending the at least two tagged allele specific extension primers, using labelled nucleotides, if the terminal nucleotide of the 3' end hybridizing portion is a perfect match to an allele of one of the mutation sites in the amplified DNA products; d) hybridizing the at least two allele two tagged allele specific extension primers to the set of probes and detecting the presence of labelled extension products.

2. The method of claim 1 wherein the 3' end hybridizing portions of the at least two tagged allele specific extension primers comprise a sequence corresponding to bases 25 and up of any two of SEQ ID NO: 33 to SEQ ID NO: 118.

3. The method of claim 2 wherein the 5 '-end tag portions of the at least two tagged allele specific primers comprises a sequence corresponding to bases 1 to 24 of any two of SEQ JJD NO: 33 to SEQ ID NO: 118.

4. The method of claim 1 wherein the at least two tagged allele-specific extension primers are selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 118.

5. The method of claim 1 wherein the amplifying step is conducted by PCR.

6. The method of claim 1 wherein the probes are coupled to a solid support.

7. The method of claim 6 wherein the solid support is selected from the group consisting of beads, spectrally coded beads, and a chip based microarray.

8. The method of claim 5 wherein the step of PCR amplifying is conducted using a set of PCR amplification primers, said set comprising at least two pairs of PCR primers selected from the group of pairs consisting of:

SEQ ID NO.: 11 and SEQ ID NO.: 12, SEQ ID NO.: 13 and SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16, SEQ ID NO.: 1 and SEQ ID NO.: 2, SEQ ID NO.: 17 and SEQ ID NO.: 18, SEQ ID NO.: 3 and SEQ ID NO.: 4, SEQ ID NO.: 19 and SEQ ID NO.: 20, SEQ ID NO.: 21 and SEQ ID NO.: 22, SEQ ID NO.: 25 and SEQ ID NO.: 26, SEQ ID NO.: 5 and SEQ ID NO.: 6, SEQ ID NO.: 7 and SEQ LD NO.: 8, SEQ ID NO.: 27 and SEQ ID NO.: 28, SEQ ID NO.: 29 and SEQ ID NO.: 30, SEQ LD NO.: 31 and SEQ ID NO.: 32, SEQ ID NO.: 9 and SEQ ID NO.: 10, and SEQ ID NO.: 23 and SEQ ID NO.: 24.

9. A kit for use in detecting the presence or absence of a variant nucleotide in at least two mutation sites associated with cystic fibrosis, said mutation sites selected the group of mutations listed in Table 2, said kit comprising a set of at least two tagged allele specific extension primers wherein each tagged allele specific extension primer has a 3 '-end hybridizing portion having a 3' terminal nucleotide substantially complementary to a first allele of one of the mutation sites associated with cystic fibrosis and a 5 '-end tag portion complementary to one of a set probes.

10. The kit of claim 9 wherein the 3' end hybridizing portion of the at least two tagged allele specific extension primers comprises a sequence corresponding to bases 25 and up of any two of SEQ ID NO: 33 to SEQ ID NO: 118.

11. The kit of claim 9 wherein the 5 '-end tag portion of the at least two tagged allele specific primers comprises a sequence corresponding to bases 1 to 24 of any two of SEQ ID NO: 33 to SEQ ID NO: 118.

12. The kit of claim 9 wherein the at least two tagged allele-specific extension primers are selected from the group consisting of SEQ TD NO: 33 to SEQ ID NO: 118.

13. The kit of claim 9 further comprising a set of PCR amplification primers for amplifying regions of DNA containing the at least two mutation sites, said set comprising at least two pairs of PCR primers selected from the group of pairs consisting of:

SEQ ID NO.: 11 and SEQ ID NO.: 12, SEQ ID NO.: 13 and SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16, SEQ TD NO.: 1 and SEQ LD NO.: 2, SEQ ID NO.: 17 and SEQ ID NO.: 18, SEQ ID NO.: 3 and SEQ ID NO.: 4, SEQ ID NO.: 19 and SEQ J-D NO.: 20, SEQ ID NO.: 21 and SEQ ID NO.: 22, SEQ LD NO.: 25 and SEQ ID NO.: 26, SEQ J-D NO.: 5 and SEQ ID NO.: 6, SEQ ID NO.: 7 and SEQ ID NO.: 8, SEQ ID NO.: 27 and SEQ ID NO.: 28, SEQ ID NO.: 29 and SEQ ID NO.: 30, SEQ ID NO.: 31 and SEQ ID NO.: 32, SEQ ID NO.: 9 and SEQ ID NO.:10, and SEQ ID NO.: 23 and SEQ ID NO.: 24.

14. The kit of claim 9 further comprising a set of probes.

15. The kit of claim 14 wherein the set of probes are coupled to a support.

16. A kit for use in detecting the presence or absence of a variant nucleotide in at least two mutation sites associated, with cystic fibrosis, said mutation sites selected from the group the of mutations listed in Table 2, said kit comprising a set of PCR amplification primers for amplifying regions of DNA containing the at least two mutation sites, said set comprising at least two pairs of PCR primers selected from the group of pairs consisting of:

SEQ ID NO.: 11 and SEQ ID NO.: 12, SEQ ID NO.: 13 and SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16, SEQ ID NO.: 1 and SEQ LD NO.: 2, SEQ ID NO.: 17 and SEQ ID NO.: 18, SEQ J-D NO.: 3 and SEQ ID NO.: 4, SEQ ID NO.: 19 and SEQ ID NO.: 20, SEQ ID NO.: 21 and SEQ TD NO.: 22, SEQ ID NO.: 25 and SEQ ID NO.: 26, SEQ TD NO.: 5 and SEQ ID NO.: 6, SEQ ID NO.: 7 and SEQ LD NO.: 8, SEQ ID NO.: 27 and SEQ ID NO.: 28, SEQ JO NO.: 29 and SEQ ID NO.: 30, SEQ TD NO.: 31 and SEQ TD NO.: 32, SEQ ID NO.: 9 and SEQ ID NO.: 10, and SEQ ID NO.: 23 and SEQ ID NO.: 24.

17. The kit of claim 16 further comprising a set of at least two tagged allele specific extension primers wherein each tagged allele specific extension primer has a 3 '-end hybridizing portion substantially complementary to a first allele of one of the mutation sites associated with cystic fibrosis and a 5 '-end tag portion complementary to one of a set probes.