Patent Application
20050059035
The present invention relates to nucleotide sequences useful as primers for amplifying portions of the cystic fibrosis transmembrane regulator (CFTR) gene where cystic fibrosis (CF) mutations are known to arise, and use of the amplified sequence to identify the presence or absence of CF mutant sequences in a biological sample.
The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder in the Caucasian population. It affects approximately 1 in 2,500 live births in North America (Boat et al, The Metabolic Basis of Inherited Disease, 6th ed, pp 2649-2680, McGraw Hill, NY (1989)). Approximately 1 in 25 persons are carriers of the disease. The responsible gene has been localized to a 250,000 base pair genomic sequence present on the long arm of chromosome 7. This sequence encodes a membrane-associated protein called the “cystic fibrosis transmembrane regulator” (or “CFTR”). There are greater than 1000 different mutations in the CFTR gene, having varying frequencies of occurrence in the population, presently reported to the Cystic Fibrosis Genetic Analysis Consortium. These mutations exist in both the coding regions (e.g., ΔF508, a mutation found on about 70% of CF alleles, represents a deletion of a phenylalanine at residue 508) and the non-coding regions (e.g., the 5T, 7T, and 9T mutations correspond to a sequence of 5, 7, or 9 thymidine bases located at the splice branch/acceptor site of intron 8) of the CFTR gene.
The major symptoms of cystic fibrosis include chronic pulmonary disease, pancreatic exocrine insufficiency, and elevated sweat electrolyte levels. The symptoms are consistent with cystic fibrosis being an exocrine disorder. Although recent advances have been made in the analysis of ion transport across the apical membrane of the epithelium of CF patient cells, it is not clear that the abnormal regulation of chloride channels represents the primary defect in the disease.
The present invention provides compositions and methods for amplifying CFTR nucleic acid sequences and for using such amplified sequence to identify the presence of absence of CF mutations in the CFTR gene. In particular, nucleic acid primers are provided herein for amplifying segments of the CFTR gene that are known to contain mutant cystic fibrosis (CF) nucleic acid sequence. These primers therefore enable the construction of assays that utilize amplification methods, preferably the polymerase chain reaction (PCR), to amplify the nucleic acid sequences in a biological sample for detection of mutant gene sequence. The present invention therefore further discloses methods for detecting individual mutant CF sequence in the amplified product(s).
In a first aspect, the present invention provides one or more substantially pure nucleic acid sequences, and/or complementary sequences thereof, that can be used as primers to amplify segments of the CFTR gene where CF mutant nucleic acid sequences are known to arise.
The primers of the present invention hybridize to a CFTR coding sequence or a CFTR non-coding sequence, or to a complement thereof. Suitable primers are capable of hybridizing to coding or non-coding CFTR sequence under stringent conditions. The primers may be complementary to CF predetermined nucleic acid sequences that are associated with cystic fibrosis or may flank one or more such sequences. Preferred primers are those that flank mutant CF sequences. Primers may be labeled with any of a variety of detectable agents such as radioisotopes, dyes, fluorescent molecules, haptens or ligands (e.g., biotin), and the like. In a preferred approach, the primer are labeled with biotin. The biotin label is preferably attached to the 5′ end of the primer.
By “predetermined sequence” is meant a nucleic acid sequence that is known to be associated with cystic fibrosis. Predetermined sequence that is known to be associated with cystic fibrosis includes mutant CF nucleotide sequence.
By “mutant CF nucleic acid sequence,” “CF mutant sequences,” or “genotype for cystic fibrosis” is meant one or more CFTR nucleic acid sequences that are associated or correlated with cystic fibrosis. These mutant CF sequences may be correlated with a carrier state, or with a person afflicted with CF. The nucleic acid sequences are preferably DNA sequences, and are preferably genomic DNA sequences; however, RNA sequences such as mRNA or hnRNA may also contain nucleic acid sequences that are associated with cystic fibrosis. Mutations in the cystic fibrosis gene are described, for example, in U.S. Pat. No. 5,981,178 to Tsui et al., including mutations in the cystic fibrosis gene at amino acid positions 85, 148, 178, 455, 493, 507, 542, 549, 551, 560, 563, 574, 1077, and 1092, among others. Also disclosed are mutant DNA at nucleotide sequence positions, 621+1, 711+1, 1717−1 and 3659, which encode mutant CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) polypeptide. Preferred sequences known to be associated with CF are described hereinafter, e.g., in Table 1.
By “carrier state” is meant a person who contains one CFTR allele that is a mutant CF nucleic acid sequence, but a second allele that is not a mutant CF nucleic acid sequence. CF is an “autosomal recessive” disease, meaning that a mutation produces little or no phenotypic effect when present in a heterozygous condition with a non-disease related allele, but produces a “disease state” when a person is homozygous, i.e., both CFTR alleles are mutant CF nucleic acid sequences.
By “primer” is meant a sequence of nucleic acid, preferably DNA, that hybridizes to a substantially complementary target sequence and is recognized by DNA polymerase to begin DNA replication.
By “substantially complementary” is meant that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length. In particular, substantially complementary sequences comprise a contiguous sequence of bases that do not hybridize to a target sequence, positioned 3′ or 5′ to a contiguous sequence of bases that hybridize under stringent hybridization conditions to a target sequence.
By “flanking” is meant that a primer hybridizes to a target nucleic acid adjoining a region of interest sought to be amplified on the target. The skilled artisan will understand that preferred primers are pairs of primers that hybridize 3′ from a region of interest, one on each strand of a target double stranded DNA molecule, such that nucleotides may be add to the 3′ end of the primer by a suitable DNA polymerase. Primers that flank mutant CF sequences do not actually anneal to the mutant sequence but rather anneal to sequence that adjoins the mutant sequence.
By “isolated” a nucleic acid (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components which naturally accompany such nucleic acid. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, oligonucleotides, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
By “substantially pure” a nucleic acid, represents more than 50% of the nucleic acid in a sample. The nucleic acid sample may exist in solution or as a dry preparation.
By “complement” is meant the complementary sequence to a nucleic acid according to standard Watson/Crick pairing rules. For example, a sequence (SEQ ID NO: 1) 5′-GCGGTCCCAAAAG-3′ has the complement (SEQ ID NO: 2) 5′-CTTTTGGGACCGC-3′. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA.
By “coding sequence” is meant a sequence of a nucleic acid or its complement, or a part thereof, that can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are joined together by the cell's biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
By “non-coding sequence” is meant a sequence of a nucleic acid or its complement, or a part thereof, that is not transcribed into amino acid in vivo, or where tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, silencers, etc.
In preferred embodiments the substantially pure nucleic acid sequence(s) is(are) a DNA (or RNA equivalent) that is any of the following:
or a complement of one or more of these sequences.
In another aspect, the present invention provides methods of amplifying CF nucleic acids to determine the presence of one or more mutant CF sequences. In accordance with this method, nucleic acid suspected of containing mutant CF sequences are amplified using one or more primers that flank one or more predetermined nucleic acid sequences that are associated with cystic fibrosis under conditions such that the primers will amplify the predetermined nucleic acid sequences, if present. In preferred embodiments, the amplification primers used are one or more of the sequences designated as SEQ ID NO: 3 through SEQ ID NO: 32, or a complement of one or more of these sequences. In preferred embodiments, pairs of primers are used for amplification, the pairs being SEQ ID NOs: 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, and 31 and 32. In further preferred embodiments, the number of pairs of primers is 5 pairs of primers, even more preferably 10 pairs of primers and most preferably 15 pairs of primers.
In the case where the 15 pairs of primers are used in combinations, primer sets are added in the following ratios determined as the moles (mole is defined as mass/molecular weight of a compound) of primers for exon 12 and 21 (SEQ ID NO: 9, 10, 13 and 14) relative to the moles of each other primer sets, the ratio being about 2 for exons 4 and i19 (SEQ ID NOs; 3-6), about 3.2 for exons 19, 7, 11 and i5 (SEQ ID NOs; 7, 8, 15, 16, and 29-32), about 4 for exons 3 and 14 (SEQ ID NOs; 11, 12, 19, 20), about 4.8 for exons 16, 20, 13 and 10 (SEQ ID NOs; 17, 18, 23 and 28), and about 8 for exon 9 (SEQ ID NOs; 22 and 21). Thus, the amount of exon 12 and 21 primers added is about (SEQ ID NO: 9, 10, 13 and 14) 2 fold that of exons 4 and i19 (SEQ ID NOs; 3-6), about 3.2 fold that of exons 19, 7 and i5 (SEQ ID NOs; 7, 8, 15, 16, and 29-32), about 4 fold that of exons 3 and 14 (SEQ ID NOs; 11, 12, 19, 20), about 4.8 fold that of exons 16, 20, 13 and 10 (SEQ ID NOs; 17, 18, 23 and 28), and about 8 fold that of exon 9 (SEQ ID NOs; 22 and 21).
The method of identifying the presence or absence of mutant CF sequence by amplification can be used to determine whether a subject has a genotype containing one or more nucleotide sequences correlated with cystic fibrosis. The presence of a wildtype or mutant sequence at each predetermined location can be ascertained by the invention methods.
By “amplification” is meant one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. A target nucleic acid may be either DNA or RNA. The sequences amplified in this manner form an “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.
The nucleic acid suspected of containing mutant CF sequence may be obtained from a biological sample. By “biological sample” is meant a sample obtained from a biological source. A biological sample can, by way of non-limiting example, consist of or comprise blood, sera, urine, feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample and/or chorionic villi. Convenient biological samples may be obtained by, for example, scraping cells from the surface of the buccal cavity. The term biological sample includes samples which have been processed to release or otherwise make available a nucleic acid for detection as described herein. For example, a biological sample may include a cDNA that has been obtained by reverse transcription of RNA from cells in a biological sample.
By “subject” is meant a human or any other animal which contains as CFTR gene that can be amplified using the primers and methods described herein. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. A human includes pre and post natal forms. Particularly preferred subjects are humans being tested for the existence of a CF carrier state or disease state.
By “identifying” with respect to an amplified sample is meant that the presence or absence of a particular nucleic acid amplification product is detected. Numerous methods for detecting the results of a nucleic acid amplification method are known to those of skill in the art.
In another aspect the present invention provides kits for one of the methods described herein. In various embodiments, the kits contain one or more of the following components in an amount sufficient to perform a method on at least one sample: one or more primers of the present invention, one or more devices for performing the assay, which may include one or more probes that hybridize to a mutant CF nucleic acid sequence, and optionally contain buffers, enzymes, and reagents for performing a method of detecting a genotype of cystic fibrosis in a nucleic acid sample.
The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.
The present invention provides specific primers that aid in the detection of mutant CF genotype. Such primers enable the amplification of segments of the CFTR gene that are known to contain mutant CF sequence from a nucleic acid containing biological sample. By amplifying specific regions of the CFTR gene, the invention primers facilitate the identification of wildtype or mutant CF sequence at a particular location of the CFTR gene. Accordingly, there is provided a substantially purified nucleic acid sample comprising one or more nucleic acids having sequences selected from the group consisting of:
or a complementary nucleic acid sequence thereof.
The invention nucleic acids are useful for primer-directed amplification of CFTR gene segments known to contain CF mutations. The primers may be used individually or, more preferably in pairs that flank a particular CF gene sequence. Thus, SEQ ID NO: 3, 5′-GCGGTCCCAAAAGGGTCAGTTGTAGGAAGTCACCAAAG-3′ (g4e1F), and SEQ ID NO: 4, 5′-GCGGTCCCAAAAGGGTCAGTCGATACAGAATATATGTGCC-3′ (g4e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 5, 5′-GCGGTCCCAAAAGGGTCAGTGAATCATTCAGTGGGTATAAGCAG-3′ (g19i2F), and SEQ ID NO: 6, 5′-GCGGTCCCAAAAGGGTCAGTCTTCAATGCACCTCCTCCC-3′ (q19i3R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 7, 5′-GCGGTCCCAAAAGGGTCAGTAGATACTTCAATAGCTCAGCC-3′ (g7e1F), and SEQ ID NO: 8, 5′-GCGGTCCCAAAAGGGTCAGTGGTACATTACCTGTATTTTGTTT-3′ (g7e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 9, 5′-GCGGTCCCAAAAGGGTCAGTGTGAATCGATGTGGTGACCA-3′ (s12e1F), and SEQ ID NO: 10, 5′-GCGGTCCCAAAAGGGTCAGTCTGGTTTAGCATGAGGCGGT-3′ (s12e1R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 11, 5′-GCGGTCCCAAAAGGGTCAGTTTGGTTGTGCTGTGGCTCCT-3′ (g14be1F), and SEQ ID NO: 12, 5′-GCGGTCCCAAAAGGGTCAGTACAATACATACAAACATAGTGG-3′ (g14be2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 13, 5′-GCGGTCCCAAAAGGGTCAGTGAAAGTATTTATTTTTTCTGGAAC-3′ (q21e1F), and SEQ ID NO: 14 5′-GCGGTCCCAAAAGGGTCAGTGTGTGTAGAATGATGTCAGCTAT-3′ (q21e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 15, 5′-GCGGTCCCAAAAGGGTCAGTCAGATTGAGCATACTAAAAGTG-3′ (g11e1F), and SEQ ID NO: 16, 5′-GCGGTCCCAAAAGGGTCAGTTACATGAATGACATTTACAGCA-3′ (g11e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 17, 5′-GCGGTCCCAAAAGGGTCAGTAAGAACTGGATCAGGGAAGA-3′ (g20e1F), and SEQ ID NO: 18, 5′-GCGGTCCCAAAAGGGTCAGTTCCTTTTGCTCACCTGTGGT-3′ (g20e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 19, 5′-GCGGTCCCAAAAGGGTCAGTGGTCCCACTTTTTATTCTTTTGC-3′ (q3e2F), and SEQ ID NO: 20 5′-GCGGTCCCAAAAGGGTCAGTTGGTTTCTTAGTGTTTGGAGTTG-3′ (q3e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 21, 5′-GCGGTCCCAAAAGGGTCAGTTGGATCATGGGCCATGTGC-3′ (g9e9F), and SEQ ID NO: 22, 5′-GCGGTCCCAAAAGGGTCAGTACTACCTTGCCTGCTCCAGTGG-3′ (g9e9R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 23, 5′-GCGGTCCCAAAAGGGTCAGTAGGTAGCAGCTATTTTTATGG-3′ (g13e2F), and SEQ ID NO: 24, 5′-GCGGTCCCAAAAGGGTCAGTTAAGGGAGTCTTTTGCACAA-3′ (g13e2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 25 5′-GCGGTCCCAAAAGGGTCAGTGCAATTTTGGATGACCTTC-3′ (q16i1F), and SEQ ID NO: 26 5′-GCGGTCCCAAAAGGGTCAGTTAGACAGGACTTCAACCCTC-3′ (q16i2R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 27, 5′-GCGGTCCCAAAAGGGTCAGTGGTGATTATGGGAGAACTGG-3′ (q10e10F), and SEQ ID NO: 28, 5′-GCGGTCCCAAAAGGGTCAGTATGCTTTGATGACGCTTC-3′ (q10e11R), are preferably used together as forward (F) and reverse (R) primers; SEQ ID NO: 29, 5′-GCGGTCCCAAAAGGGTCAGTTTCATTGAAAAGCCCGAC-3′ (q19e12F), and SEQ ID NO: 30, 5′-GCGGTCCCAAAAGGGTCAGTCACCTTCTGTGTATTTTGCTG-3′ (q19e13R) are preferably used together as forward (F) and reverse (R) primers; and SEQ ID NO: 31, 5′-GCGGTCCCAAAAGGGTCAGTAAGTATTGGACAACTTGTTAGTCTC-3′ (q5e12F), and SEQ ID NO: 32, 5′-GCGGTCCCAAAAGGGTCAGTCGCCTTTCCAGTTGTATAATTT-3′ (q5e13R), are preferably used together as forward (F) and reverse (R) primers.
Accordingly, there is provided a method of amplifying a nucleic acid sequence, comprising, contacting a nucleic acid containing sample with reagents suitable for nucleic acid amplification including one or more pairs of primers flanking one or more predetermined nucleic acid sequences that are correlated with cystic fibrosis, and amplifying said one or more predetermined nucleic acid sequences, if present, wherein said primers are one or more pairs of nucleic acids selected from the group consisting of:
The above pairs of primers have been designed for multiplex use. Thus, one may simultaneously in a single sample amplify one or more CFTR gene segments. In preferred embodiment, five pairs of primers are used to amplify at least five CFTR gene segments. In a more preferred embodiment, ten pairs may be used and in most preferred embodiment, all 15 pairs of primers may be used.
The identify of mutations characteristics of each amplified segment for each primer pair are shown in the following table.
The table below identifies preferred primer pairs and characteristics of the amplified product.
The nucleic acid to be amplified may be from a biological sample such as an organism, cell culture, tissue sample, and the like. The biological sample can be from a subject which includes any eukaryotic organism or animal, preferably flugi, invertebrates, insects, arachnids, fish, amphibians, reptiles, birds, marsupials and mammals. A preferred subject is a human, which may be a patient presenting to a medical provider for diagnosis or treatment of a disease. The biological sample may be obtained from a stage of life such as a fetus, young adult, adult, and the like. Particularly preferred subjects are humans being tested for the existence of a CF carrier state or disease state.
The sample to be analyzed may consist of or comprise blood, sera, urine, feces, epidermal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample and/or chorionic villi, and the like. A biological sample may be processed to release or otherwise make available a nucleic acid for detection as described herein. Such processing may include steps of nucleic acid manipulation, e.g., preparing a cDNA by reverse transcription of RNA from the biological sample. Thus, the nucleic acid to be amplified by the methods of the invention may be DNA or RNA.
Nucleic acid may be amplified by one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. The sequences amplified in this manner form an “amplicon.” In a preferred embodiment, the amplification by the is by the polymerase chain reaction (“PCR”) (e.g., Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., European Patent Appln. 50,424; European Patent Appln. 84,796, European Patent Application 258,017, European Patent Appln. 237,362; Mullis, K., European Patent Appln. 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194). Other known nucleic acid amplification procedures that can be used include, for example, transcription-based amplification systems or isothermal amplification methods (Malek, L. T. et al., U.S. Pat. No. 5,130,238; Davey, C. et al., European Patent Application 329,822; Schuster et al., U.S. Pat. No. 5,169,766; Miller, H. I. et al., PCT appln. WO 89/06700; Kwoh, D. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173 (1989); Gingeras, T. R. et al., PCT application WO 88/10315; Walker, G. T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)). Amplification may be performed to with relatively similar levels of each primer of a primer pair to generate an double stranded amplicon. However, asymmetric PCR may be used to amplify predominantly or exclusively a single stranded product as is well known in the art (e.g., Poddar et al. Molec. And Cell. Probes 14:25-32 (2000)). This can be achieved for each pair of primers by reducing the concentration of one primer significantly relative to the other primer of the pair (e.g. 100 fold difference). Amplification by asymmetric PCR is generally linear. One of ordinary skill in the art would know that there are many other useful methods that can be employed to amplify nucleic acid with the invention primers (e.g., isothermal methods, rolling circle methods, etc.), and that such methods may be used either in place of, or together with, PCR methods. Persons of ordinary skill in the art also will readily acknowledge that enzymes and reagents necessary for amplifying nucleic acid sequences through the polymerase chain reaction, and techniques and procedures for performing PCR, are well known. The examples below illustrate a standard protocol for performing PCR and the amplification of nucleic acid sequences that correlate with or are indicative of cystic fibrosis.
In another aspect, the present invention provides methods of detecting a cystic fibrosis genotype in a biological sample. The methods comprise amplifying nucleic acids in a biological sample of the subject and identifying the presence or absence of one or more mutant cystic fibrosis nucleic acid sequences in the amplified nucleic acid. Accordingly, the present invention provides a method of determining the presence or absence of one or more mutant cystic fibrosis nucleic acid sequences in a nucleic acid containing sample, comprising: contacting said sample with reagents suitable for nucleic acid amplification including one or more pairs of nucleic acid primers flanking one or more predetermined nucleic acid sequences that are correlated with cystic fibrosis, amplifying said predetermined nucleic acid sequence(s), if present, to provide an amplified sample; and identifying the presence or absence of said one or more predetermined sequences in said amplified sample, whereby the presence or absence of said one or more mutant cystic fibrosis nucleic acid sequences is determined; wherein said pairs of nucleic acid primers are selected from the group consisting of:
One may analyze the amplified product for the presence of absence of any of a number of mutant CF sequences that may be present in the sample nucleic acid. As already discussed, numerous mutations in the CFTR gene have been associated with CF carrier and disease states. For example, a three base pair deletion leading to the omission of a phenylalanine residue in the gene product has been determined to correspond to the mutations of the CF gene in approximately 70% of the patients affected by CF. The table below identifies preferred CF sequences and identifies which of the primer pairs of the invention may be used to amplify the sequence.
CF mutations including those known under symbols: 2789+5G>A; 711+1G>T; W1282X; 3120+1G>A; d1507; dF508; (F508C, 1507V, 1506V); N1303K; G542X, G551D, R553X, R560T, 1717-1G>A: R334W, R347P, 1078delT; R117H, I148T, 621+1G>T; G85E; R1162X, 3659delC; 2184delA; A455E, (5T, 7T, 9T); 3849+10kbC>T; and 1898+1G>A, are described in U.S. patent application Ser. No. 396,894, filed Apr. 22, 1989, application Ser. No. 399,945, filed Aug. 29, 1989, application Ser. No. 401,609 filed Aug. 31, 1989. and U.S. Pat. Nos. 6,011,588 and 5,981,178, which are hereby incorporated by reference in their entirety. Any and all of these mutations can be detected using nucleic acid amplified with the invention primers as described herein.
CF mutations in the amplified nucleic acid may be identified in any of a variety of ways well known to those of ordinary skill in the art. For example, if an amplification product is of a characteristic size, the product may be detected by examination of an electrophoretic gel for a band at a precise location. In another embodiment, probe molecules that hybridize to the mutant or wildtype CF sequences can be used for detecting such sequences in the amplified product by solution phase or, more preferably, solid phase hybridization. Solid phase hybridization can be achieved, for example, by attaching the CF probes to a microchip. Probes for detecting CF mutant sequences are well known in the art. See Wall et al. “A 31-mutation assay for cystic fibrosis testing in the clinical molecular diagnostics laboratory,” Human Mutation, 1995;5(4):333-8, which specifies probes for CF mutations ΔF508 (exon 1), G542X (exon 11), G551D (exon 11), R117H (exon 4), W1282X (exon 20), N1303K (exon 21), 3905insT (exon 20), 3849+10Kb (intron 19), G85E (exon 3), R334W (exon 7), A455E (exon 9), 1898+1 (exon 12), 2184delA (exon 13), 711+1 (exon 5), 2789+5 (exon 14b), Y1092x exon 17b), ΔI507 (exon 10), S549R(T-G) (exon 11), 621+1 (exon 4), R1162X (exon 19), 1717-1 (exon 11), 3659delC (exon 19), R560T (exon 11), 3849+4(A-G) (exon 19), Y122X (exon 4), R553X (exon11), R347P (exon 7), R347H (exon 7), Q493X (exon 10), V520F (exon 10), and S549N (exon 11). Probes for additional CF mutations include those shown in Table 17.
CF probes for detecting mutations as described herein may be attached to a solid phase in the form of an array as is well known in the art (see, U.S. Pat. Nos. 6,403,320 and 6,406,844). For example, the full complement of 24 probes for CF mutations with additional control probes (30 in total) can be conjugated to a silicon chip essentially as described by Jenison et al., Biosens Bioelectron. 16(9-12):757-63 (2001) (see also U.S. Pat. Nos. 6,355,429 and 5,955,377). Amplicons that hybridized to particular probes on the chip can be identified by transformation into molecular thin films. This can be achieved by contacting the chip with an anti-biotin antibody or streptavidin conjugated to an enzyme such as horseradish peroxidase. Following binding of the antibody(or streptavidin)-enzyme conjugate to the chip, and washing away excess unbound conjugate, a substrate can be added such as tetramethylbenzidine (TMB) {3,3′,5,5′Tetramethylbenzidine} to achieve localized deposition (at the site of bound antibody) of a chemical precipitate as a thin film on the surface of the chip. Other enzyme/substrate systems that can be used are well known in the art and include, for example, the enzyme alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate as the substrate. The presence of deposited substrate on the chip at the locations in the array where probes are attached can be read by an optical scanner. U.S. Pat. Nos. 6,355,429 and 5,955,377, which are hereby incorporated by reference in their entirety including all charts and drawings, describe preferred devices for performing the methods of the present invention and their preparation, and describes methods for using them.
The binding of amplified nucleic acid to the probes on the solid phase following hybridization may be measured by methods well known in the art including, for example, optical detection methods described in U.S. Pat. No. 6,355,429. In preferred embodiments, an array platform (see, e.g., U.S. Pat. No. 6,288,220) can be used to perform the methods of the present invention, so that multiple mutant DNA sequences can be screened simultaneously. The array is preferably made of silicon, but can be other substances such as glass, metals, or other suitable material, to which one or more capture probes are attached. In preferred embodiments, at least one capture probe for each possible amplified product is attached to an array. Preferably an array contains 10, more preferably 20, even more preferably 30, and most preferably at least 60 different capture probes covalently attached to the array, each capture probe hybridizing to a different CF mutant sequence. Nucleic acid probes useful as positive and negative controls also may be included on the solid phase or used as controls for solution phase hybridization.
In still another approach, wildtype or mutant CF sequence in amplified DNA may be detected by direct sequence analysis of the amplified products. A variety of methods can be used for direct sequence analysis as is well known in the art. See, e.g., The PCR Technique: DNA Sequencing (eds. James Ellingboe and Ulf Gyllensten) Biotechniques Press, 1992; see also “SCAIP” (single condition amplification/internal primer) sequencing, by Flanigan et al. Am J Hum Genet. 2003 April;72(4):931-9. Epub Mar. 11, 2003.
In yet another approach for detecting wildtype or mutant CF sequences in amplified DNA is single nucleotide primer extension or “SNuPE.” SNUPE can be performed as described in U.S. Pat. No. 5,888,819 to Goelet et al., U.S. Pat. No. 5,846,710 to Bajaj, Piggee, C. et al. Journal of Chromatography A 781 (1997), p. 367-375 (“Capillary Electrophoresis for the Detection of Known Point Mutations by Single-Nucleotide Primer Extension and Laser-Induced Fluorescence Detection”); Hoogendoom, B. et al., Human Genetics (1999) 104:89-93, (“Genotyping Single Nucleotide Polymorphism by Primer Extension and High Performance Liquid Chromatography”); and U.S. Pat. No. 5,885,775 to Haff et al. (analysis of single nucleotide polymorphism analysis by mass spectrometry). In SNuPE, one may use as primers such as those specified in Table 17.
Still another approach for detecting wildtype or mutant CF sequences in amplified DNA is oligonucleotide ligation assay or “OLA”. The OLA uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. See e.g., Nickerson et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927, Landegren, U. et al. (1988) Science 241:1077-1080 and U.S. Pat. No. 4,998,617.
These above approaches for detecting wildtype or mutant CF sequence in the amplified nucleic acid is not meant to be limiting, and those of skill in the art will understand that numerous methods are known for determining the presence or absence of a particular nucleic acid amplification product.
In another aspect the present invention provides kits for one of the methods described herein. In various embodiments, the kits contain one or more of the invention primers in an amount suitable for amplifying a specified CFTR sequence from at least one nucleic acid containing sample. The kit optionally contain buffers, enzymes, and reagents for amplifying the CFTR nucleic acid via primer-directed amplification. The kit also may include one or more devices for detecting the presence or absence of particular mutant CF sequences in the amplified nucleic acid. Such devices may include one or more probes that hybridize to a mutant CF nucleic acid sequence, which preferably is attached to a bio-chip device, such as any of those described in U.S. Pat. No. 6,355,429. The bio-chip device optionally has at least one capture probe attached to a surface on the bio-chip that hybridizes to a mutant CF sequence. In preferred embodiments the bio-chip contains multiple probes, and most preferably contains at least one probe for a mutant CF sequence which, if present, would be amplified by a set of flanking primers. For example, if five pairs of flanking primers are used for amplification, the device would contain at least one CF mutant probe for each amplified product, or at least five probes. The kit also preferably contains instructions for using the components of the kit.
The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.
A. Extraction of DNA
Suitable samples may include fresh tissue, e.g., obtained from clinical swabs from a region where cells are collected by soft abrasion (e.g., buccal, cervical, vaginal, etc. surfaces) or biopsy specimens; cells obtained by amniocentesis or chorionic villus sampling; cultured cells, or blood cells; or may include fixed or frozen tissues. The following example describes preparation of nucleic acids from blood.
50 μL of whole blood was mixed with 0.5 ml of TE (10 mM Tris HCl, 1 mM EDTA, pH 7.5) in a 1.5 mL microfuge tube. The sample was spun for 10 seconds at 13,000×g. The pellet was resuspended in 0.1 mL of TE buffer with vortexing, and pelleted again. This procedure was repeated twice more, and then the final cell pellet was resuspended in 100 μl of K buffer 50 mM KCl, 10 mM Tris HCl, 2.5 mM MgCl2, 0.5% Tween 20, 100 μg/mL proteinase K, pH 8.3) and incubated 45 minutes at 56° C., then 10 minutes at 95° C. to inactivate the protease.
B. Amplification from DNA
Individual amplifications were prepared in a volume of 13.5 μl, which was added to 96 well microtiter plates. Each amplification volume contained 2 μl of the DNA sample (generally 10-100 ng of DNA), 11.5 μl of PCR-Enzyme Mix (PCR-Enzyme mix stock was prepared with 11.3 μl master mix, 0.25 μl MgCl2 (from 25 mM stock), and 0.2 μl of FasStar Taq (source for last two reagents was Roche Applied science, Cat. No. 2 032 937). Master mix contained 5′ biotinylated primers, Roche PCR buffer with MgCl2, Roche GC rich solution (cat. No. 2 032 937), bovine serum albumin (BSA) (New England BioLabs, Cat no. B9001B), and NTPs (Amersham Biosciences, Cat no. 27-2032-01).
The final concentration in the PCR for MgCl2 was 2.859 mM, for BSA was 0.725 μg/μl, and for each dNTP was 0.362 mM. Primer final concentrations of biotinylated primers were 0.29 μM for each of SEQ ID NOs: 9, 10, 13 and 14 (exon 12 and 21), 0.145 μM for each of SEQ ID NOs: 3-6 (exons 4 and i19), 0.091 μM for each of SEQ ID NOs: 7, 8, 15, 16, and 29-32 (exons 19, 7, i5 and 11), 0.072 μM for each of SEQ ID NOs: 11, 12, 19 and 20 (exon 3 and 14), 0.060 μM for each of SEQ ID NOs: 17, 18 and 23-28 (exons 16, 20, 13 and 10), and 0.036 μM for each of SEQ ID NOs: 21 and 22, (exon 9).
PCR was conducted using the following temperature profile: step 1: 96° C. for 15 minutes; step 2: 94° C. for 15 seconds; step 3: decrease at 0.5° C./second to 56° C.; step 4: 56° C. for 20 seconds; step 5: increase at 0.3° C./second to 72° C., step 6: 72° C. for 30 seconds; step 7: increase 0.5° C. up to 94° C.; step 8: repeat steps 2 to 7 thirty three times; step 9: 72° C. for 5 minutes; step 10: 4° C. hold (to stop the reaction).
C. Detection of CF Amplicons
The presence of CF sequences in the amplicons was determined by hybridizing the amplified product to a solid phase strip containing an array of 50 probes specific for CF mutations and CF wildtype sequence (LINEAR ARRAY CF GOLD 1.0™, Roche Diagnostics) in accordance with the manufacturer's instructions. Detection of hybridized amplicons was by streptavidin-HRP conjugate and development using the TMB as substrate.
The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.
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. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
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.
Other embodiments are within the following claims. 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.
This application is a continuation-in-part of U.S. application Ser. No. 10/659,582, filed Sep. 9, 2003, the entire contents of which are incorporated by reference herein for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10659582 | Sep 2003 | US |
| Child | 10775650 | Feb 2004 | US |
