WO2001021833A2 - Assay for detection of human cftr allele variants using specific diagnostic primers - Google Patents
Assay for detection of human cftr allele variants using specific diagnostic primers Download PDFInfo
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- WO2001021833A2 WO2001021833A2 PCT/GB2000/003597 GB0003597W WO0121833A2 WO 2001021833 A2 WO2001021833 A2 WO 2001021833A2 GB 0003597 W GB0003597 W GB 0003597W WO 0121833 A2 WO0121833 A2 WO 0121833A2
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
Definitions
- This invention relates to a diagnostic method for the simultaneous detection of the variable length polythymidine (polyT) tract alleles in the CFTR gene that are associated with the phenotypic modulation of selected CFTR mutations (Friedman KJ. et al. Hum Mutat. 10: 108-115 (1997)).
- polyT variable length polythymidine
- the invention also relates to a diagnostic method further comprising the simultaneous detection of one or more of the DF508 (Kerem B. et al. Science 245: 1073-1080 (1989)), 621+1G>T (Rozen R. et al. Am. J. Med. Genet. 42: 360-364 (1992)), G542X, 3659delC, A455E, DI507 (Kerem B. et al. Proc. Nat.Acad. Sci. 87: 8447-8451 (1990)), 3849+10kbOT (Highsmith WE. et al. New Eng. J. Med. 331 : 974-980 (1994)), N1303K (Osbome L. et al. Am. J.
- the invention also relates to a diagnostic method further comprising the simultaneous detection of one or more of the G85E (Chalkley G. et al. J. Med. Genet. 28: 875-877 (1991)), 405+lG>A (Dork T. et al. Hum Mol Genet. 2: 1965-1966 1993)), S549R (Kerem B. et al. Proc. Nat.Acad. Sci. 87: 8447-8451 (1990)), (or, depending on which of the two mutations produce this phenotype is tested for, Sangiuolo. et al. Genomics 9: 788-789 (1991)), W1089X (Shoshani T. et al. Hum. Molec. Genet.
- Cystic fibrosis transmembrane conductance regulator functions as a chloride channel and controls the regulation of other transport pathways.
- the CFTR gene maps to chromosome 7q (Riordan J R. et al. Science 245: 1066-1073 (1989)).
- Cystic fibrosis is an autosomal recessive disorder mainly among Caucasians with a frequency of approximately 1/2 500 (Welsh MJ. et al. in Scriver et al. (eds): The Metabolic Basis of Inherited Disease, Vol 3: 7th ed. McGraw-Hill; New York, pp3799-3876 (1995)).
- CF CF
- Treatment is directed at improving nutrition through the use of replacement pancreatic enzymes and vitamins, as well as a high-energy, high-protein and liberal-fat diet.
- enteral supplementation by nightly nasogastric, gastrostomy or jejunostomy infusion of high-energy diets has been used.
- Pulmonary treatment includes antibiotic therapy, either maintained continuously or reserved for exacerbations, and chest physiotherapy consisting of postural drainage, percussion, vibration and assisted coughing.
- Mutations of the CFTR gene are believed to produce an abnormal protein as a component of the chloride channel gate at the cell surface and are classified according to their phenotypic manifestations.
- Class I are nonsense mutations, resulting in the introduction of a stop codon, and in no synthesis of CFTR e.g. G542X, R553X, W1282X.
- Class II mutations cause a block in CFTR assembly in the endoplasmic reticulum or affect transport to the cell membrane, these include ⁇ F508, A455E, and P574H.
- Class HI and IV mutations give rise to defective chloride channel activity or regulation e.g. Rl 17H, which alters amino acid residues in the first CFTR transmembrane domain.
- Class V mutations result in modulations in CFTR synthesis.
- CF CF involving class I and II CFTR mutations (most commonly ⁇ F508 homozygotes)
- chronic pulmonary disease is observed at variable levels with chronic pancreatitis.
- PS-CF sinopulmonary problems with pancreas sufficiency
- CBAVD congenital bilateral absence of vas deferens
- Mutations associated with PS-CF include R334W and R117H; the latter mutation accounts for approximately 0.8% of mutant alleles in Caucasian CF patients (Tsui LC. Trends Genet. 57: 392-398 (1992)).
- the T5 allele results in the most inefficient use of this splice acceptor site (Teng, H. et al. Hum. Mol. Genet. 6: 85-90 (1997)) and so CFTR transcripts from a T5 allele will lack exon 9 sequence.
- Individuals homozygous for the 5T allele were found to generate 90% CFTR mRNA with exon9 skipped (exon9 ⁇ ). While 7T homozygotes generated ⁇ 25% exon9 " transcripts, and 9T ⁇ 15% (Lissens W. et al. Hum. Reprod. 11 : Supp4 55-77 (1996)).
- CFTR gene with the Rl 17H mutation harbors a T5 allele
- the mutant gene will be responsible for CF.
- An Rl 17H mutant CFTR gene that harbors a T7 allele can either result in CF or CBAVD (Kiesewetter S. et al. Nature Genet. 5: 274-278, (1993)).
- polyT splice site variants may therefore explain the clinical heterogeneity observed in some mild cases of CF. It is also possible that the CF clinical phenotypes associated with mutations other than ⁇ F508 result from both the mutation in the CFTR gene and the sequence at exon9 splice site acceptor site. Most CFTR mutations occur on a particular polyT background, most commonly the
- ⁇ F508 is in linkage disequilibrium with the 9T allele and the mild CF mutation Rl 17H is always found on a 5T or 7T background.
- the mutation Rl 17H has been shown to owe its phenotypic heterogeneity (PS CF or CBAVD) to the presence of the 5T or 7T polyT alleles (Friedman KJ. et al. Hum Mutat. 10: 108-115 (1997)).
- Compound heterozygotes having the genotype ⁇ F508/R117H may present as PS-CF, CBAVD or be asymptomatic.
- Diagnostic techniques for phenotypic manifestations of CF include measuring sweat chloride. Elevated sweat chloride concentrations (greater than 60 mmol/1) are almost exclusively observed in patients with CF. However, it is difficult to obtain sufficient sweat from newborns, even after stimulating localised sweating by administering pilocarpine into the skin it is often impossible to collect sufficient sweat for accurate analysis. Sweat chloride concentrations can be measured, for example, by using the Lazar ISM- 146 Micro Chloride electrode. Measurement of elevated immunoreactive trypsinogen (IRT) in a dried blood spot is another screening method for CF. False positives and false negatives are known to occur, with false negatives occurring more frequently in neonates with meconium ileus. The positive predictive value of the test is only 1-7% (Ryley HC. J. Clin. Pathol. 41 : 726-729 (1988),
- the "BM meconium test” identifies infants with high albumin content in the stool resulting from pancreatic insufficiency but it has very low sensitivity (Naylor EW. Semin. Perinatol. 9: 232-249 (1985)).
- Friedman requires the use of each allele-specific primer in a separate reaction.
- ARMS Mutation System
- a diagnostic method for the detection of the 5T, 7T and 9T alleles in intron 8 of the human CFTR gene comprises contacting a test sample of nucleic acid from an individual with a multiplex of diagnostic primers comprising (i) 5T variant primer 5'(N)nAAAGAC3', (ii) 7T variant primer 5'(N*)n*(N)nAAAAGC3' and (iii) 9T variant primer
- N represents additional nucleotides which base pair with the corresponding genomic sequence in the respective allele and n is an integer between 10 and 30 and N* represents additional non-homologous nucleotides which do not base pair with the corresponding genomic sequence in the respective allele and n* is an integer between 5 and 60, in the presence of appropriate nucleotide triphosphates and an agent for polymerisation, such that a diagnostic primer is extended only when the corresponding allelic variant is present in the sample; and detecting the presence or absence of the allelic variant by reference to the presence or absence of a diagnostic primer extension product.
- N* may be any other molecule that serves to reduce the electrophoretic mobility of an extension product such as 3'hexaethylene glycol (HEG) or a combination of such molecules and additional non-homologous nucleotides.
- HOG 3'hexaethylene glycol
- the diagnostic primers for use in the methods of the invention conveniently comprise one or more of
- nucleotide sequence as defined by (N)n in the diagnostic primer is normally selected to be 100%) complementary to the corresponding genomic sequence.
- one or more mismatched bases may be included, for example at the 5' terminus of the primer.
- up to two, three, four or five mismatched base pairs may be included in the nucleotide sequence defined by (N)n. It will also be understood that any mismatched bases must not significantly impair the discriminatory properties of the diagnostic primer.
- n is for example 10, up to 15, up to 20, up to 25, or up to 30.
- Preferred diagnostic primers include
- 5T variant primer 5'TAATTCCCCAAATCCCTGTTAAAGAC3'
- 7T variant primer 5'(N*)n*TAATTCCCCAAATCCCTGTTAAAAAAGC3'
- 9T variant primer 5'(N*)n*TAATTCCCCAAATCCCTGTTAAAAAAAATC3' wherein N* and n* are as defined above.
- Preferred diagnostic 7T and 9T primers are: 7T variant primer
- a diagnostic primer of the invention with a further amplification primer in one or more cycles of PCR amplification.
- a convenient example of this aspect is set out in our European patent number EP-B1-0332435. Any convenient amplification primer may be useed provided that the resulting amplification products are of a suitable size for separation and analysis.
- the further amplification primer is conveniently the polyT common primer
- GTACATAAAACAAGCATCTATTGAAAATATCTGAC is used in combination with the intron 8 5T, 7T and 9T allele-specific primers.
- a diagnostic method for the detection of the 5T, 7T and 9T alleles in intron 8 of the human CFTR gene comprises contacting a test sample of nucleic acid from an individual with a multiplex of diagnostic primers comprising (i) 5T variant primer 5'(N)nAAAGAC3', (ii) 7T variant primer 5'(N*)n*(N)nAAAAGC3' and (iii) 9T variant primer
- N represents additional nucleotides which base pair with the corresponding genomic sequence in the respective allele and n is an integer between 10 and 30 and N* represents additional non-homologous nucleotides which do not base pair with the corresponding genomic sequence in the respective allele and n* is an integer between 5 and 60, and a common amplification primer in the presence of appropriate nucleotide triphosphates and an agent for polymerisation, and subjecting the mixture to PCR amplification such that a diagnostic primer is extended only when the corresponding allelic variant is present in the sample; and detecting the presence or absence of the allelic variant by reference to the presence or absence of a corresponding PCR amplification product.
- primer set 1 The above aspects of the invention are referred to as primer set 1 and conveniently illustrated by reference to Table 1 and the disclosure of a specific primer mixl. References to primer “mixes” and “sets” are not intended to be limiting and the terms are used throughout the text interchangeably.
- N* is one from a group of labels that can be conjugated to primers whereby an individual label is associated specifically to one allele- specific primer and n* may be 0 or an integer greater than 1.
- An example of labels according to this definition of N* is a group of molecules such as fluorophores for example, (Brown T. and Brown DJS. in Newton CR. (ed): PCR - Essential Data: 1 st ed. Wiley; Chichester, pp57- 71 (1995)).
- the primers may be manufactured using any convenient method of synthesis. Examples of such methods may be found in standard textbooks, for example "Protocols For Oligonucleotides And Analogues: Synthesis And Properties;” Methods In Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1 st Edition.
- any of the above diagnostic methods may, if appropriate, also be configured so that extension of the diagnostic primer indicates the absence of the respective CFTR gene polyT allele.
- the polyT methods of this invention may be used with any known CFTR testing procedure.
- Two or more of the above diagnostic primers are conveniently used as a multiplex and more conveniently with a suitable amplificiation primer. Preferably several or all of the above primer sequences are used in a single multiplex reaction.
- Convenient primers include: W1282X mutant primer 5'(N)nGCAACAGTCA3', 1717-lOA mutant primer 5'(N)nTTGGTAATTA3 ⁇ G542X mutant primer 5'(N)nATAGTTCTCT3', N1303K mutant primer 5'(N)nGGGATCCATC3', DF508 mutant primer 5'(N)nAAACACCATT3' and
- Preferred diagnostic primers include W1282X mutant primer 5'TCTTGGGATTCAATAACTTTGCAACAGTCA3', 1717-1G>A mutant primer 5'TCTCGAATTTTCTATTTTTGGTAATTA3', G542X mutant primer 5 ⁇ GTTTGCAGAGAAAGACAATATAGTTCTCT3', N1303K mutant primer 5 GATCACTCCACTGTTCATAGGGATCCATC3', DF508 mutant primer 5'GTATCTATATTCATCATAGGAAACACCATT3', and 3849+ 1 Okb OT mutant primer 5 ' GAAC ATTTCCTTTC AGGGTGTCTT ACGC A3 ' .
- primer set 2 A The above aspects of the invention are referred to as primer set 2 A and illustrated by reference to Table 3 and the specific primer mix 2A.
- Primer mix 2A is conveniently used in combination with primer mix 1 as set out in Table 1.
- Two or more of the above diagnostic primers are conveniently used as a multiplex and more conveniently with a suitable amplificiation primer.
- Preferably several or all of the above primer sequences are used in a single multiplex reaction.
- Convenient diagnostic primers include: DF508 non-mutant primer 5'(N)nAAACACCACA3', Rl 17H mutant primer 5'(N)nGCGATAGACT3', 621+1G>T mutant primer 5'(N)nGAAGTATTGA3', R334W mutant primer 5 '(N)nATCATCCTGT3 ', Rl 162X mutant primer 5'(N)nTCTGTGAGTT3 ⁇ R553X mutant primer 5'(N)nTTCTTGCTGA3' and
- Preferred diagnostic primers include:
- Rl 17H mutant primer5'AGCCTATGCCTAGATAAATCGCGATAGACT3' 621+lOT mutant primer 5'TGCCATGGGGCCTGTGCAAGGAAGTATTGA3', R334W mutant primer 5'CCTATGCACTAATCAAAGGAATCATCCTGT3', Rl 162X mutant primer 5'TATTTTTATTTCAGATGCGATCTGTGAGTT3', R553X mutant primer 5 TATTCACCTTGCTAAAGAAATTCTTGCTGA3', G551D mutant primer 5'GCTAAAGAAATTCTTGCTCGTTGTT3'.
- primer set 2B The above aspects of the invention are referred to as primer set 2B and are conveniently illustrated by reference to Table 3 and the specific primer mix 2B.
- Primer mix 2B is conveniently used in combination with mixl as set out in Table 1.
- We have also devised novel diagnostic primer sequences for the A455E, 2183AA>G, 3659delC, DI507, 1078delT, R347P, S1251N and E60X mutations of the human CFTR gene using ARMS allele specific amplification. Therefore the polyT multiplex of the present invention is conveniently accompanied by the use in a separate ARMS reaction of one or more of :
- N and n are as previously defined, in the presence of appropriate nucleotide triphosphates and an agent for polymerisation, such that a diagnostic primer is extended only when the corresponding allelic variant is present in the sample; and detecting the presence or absence of the allelic variant by reference to the presence or absence of a diagnostic primer extension product.
- Two or more of the above diagnostic primers are conveniently used as a multiplex and more conveniently with a suitable amplificiation primer.
- Preferably several or all of the above primer sequences are used in a single multiplex reaction.
- the diagnostic primer conveniently comprises one or more of A455E mutant primer 5'(N)nAGTTGTTGTA3', 1078delT mutant primer 5'(N)nAGGGTTCCTG3', R347P mutant primer 5'(N)nTTGTTCTACC3', DI507 mutant primer 5'(N)nGAAAATAACT3', 3659delC mutant primer 5'(N)nTAAACCTAGA3', 2183AA>G mutant primer 5'(N)nAAAAGATAGC3', S 125 IN mutant primer 5 ' (N)nCAGGGAAGC A3 ' and
- Preferred diagnostic primers include A455E mutant primer 5 TCAAGATAGAAAGAGGACAGTTGTTGTA3', 1078delT mutant primer 5'CCTTCTTCTTCTCAGGGTTCCTG3',
- primer set 2C R347P mutant primer 5'CACCATCTCATTCTGCATTGTTCTACC3', DI507 mutant primer 5'GCCTGGCACCATTAAAGAAAATAACT3', 3659delC mutant primer 5 ⁇ TGCCAACAGAAGGTAAACCTAGA3', 2183AA>G mutant primer 5'CAAACTCTCCAGTCTGTTTAAAAGATAGC3', S1251N mutant primer 5'GGAAGAACTGGATCAGGGAAGCA3' and E60X mutant primer 5'TTAGGATTTTTCTTTGAAGCCAGTTA3'.
- primer set 2C primer set 2C and conveniently illustrated by reference to Table 4 and the specific primer mix 2C.
- Primer mix 2C is conveniently used in combination with mixl as set out in Table 1.
- diagnostic primers are conveniently used as a multiplex and more conveniently with a suitable amplificiation primer. Preferably several or all of the above primer sequences are used in a single multiplex reaction.
- the diagnostic primer conveniently comprises one or more of
- N, n, are as defined above and n is an integer between 6 and 26.
- Preferred diagnostic primers include: G85E mutant primer
- primer set 3 GATTTATGTTCT ATGGAATCTTTTT AT ATTTAGTGA3 '
- DI 152H mutant primer 5 ⁇ AAGATGATAAGACTTACCAAGCTATCCACTTG3' The above aspects of the present invention are referred to as primer set 3 and conveniently illustrated by reference to Table 5 and the specific mix 3.
- Mix 3 may for example be used in combination with mixl as set out in Table 1.
- the primers may be manufactured using any convenient method of synthesis. Examples of such methods may be found in standard textbooks, for example "Protocols For Oligonucleotides And Analogues: Synthesis And Properties " Methods In Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1993; 1 st Edition. It will be appreciated that any of the above diagnostic methods may, if appropriate, also be configured so that extension of the diagnostic primer indicates the absence of the respective CFTR gene mutation or polyT allele.
- the test sample of nucleic acid is preferably a blood sample but may also conveniently be a sample of any body fluid, or tissue obtained from an individual. The individual is any convenient mammal, preferably a human being.
- test sample may equally be a nucleic acid sequence corresponding to the sequence in the test sample. That is to say that all or a part of the region in the sample nucleic acid may firstly be amplified using any convenient technique such as PCR before use in the method of the invention.
- Any convenient enzyme for polymerisation may be used provided that it does not affect the ability of the DNA polymerase to discriminate between normal and mutant template sequences to any significant extent.
- convenient enzymes include thermostable enzymes which have no significant 3 '-5' exonuclease activity, for example Taq DNA polymerase, particularly "Ampli Taq Gold”TM DNA polymerase (PE Applied Biosystems), Stoffel fragment, or other appropriately N-terminal deleted modifications of Taq or Tth (Thermus thermophilus) DNA polymerases.
- the diagnostic methods of the invention can be used in combination to enable the presence or absence of mutations or polyT alleles of the CFTR gene to be detected simultaneously (i.e. the method comprises several multiplex tests).
- Primer Mix 1 can be used in isolation or in combination with any other cystic fibrosis diagnostic tests. Because the multiplex polyT test using the mix 1 primer mix adds value to the information derived from other cystic fibrosis diagnostic tests it is convenient to use the mix 1 primer mix in combination with the mix 2A primer mix. Similarly, it is convenient to use the mix 1 primer mix in combination with the mix 2B primer mix. Similarly, it is convenient to use the mix 1 primer mix in combination with the mix 2C primer mix.
- the tests may provide genotype information for the 1717-1G>A, G542X, W1282X, N1303K, 3849+1 OkbOT, 621+lOT, R553X, G551D, R117H, R1162X, R334W, A455E, 2183AA>G, 3659delC, 1078delT, DI507, R347P, S 125 IN, E60X, G85E, 405+lOA, S549R, W1089X, and DI 152H CFTR gene mutations by the inclusion of primers specific for the normal CFTR gene sequences that correspond to the respective mutated CFTR gene sequences associated with these mutations.
- each multiplex test may have any of the primers described above in combination the prefe ⁇ ed diagnostic primer multiplexes are the mix 1 comprising primers for the 5T, 7T and 9T alleles.
- Mix 2A comprising primers for the 1717-1G>A, G542X, W1282X, N1303K, DF508 and 3849+lOkbOT mutations;
- mix 2B comprising primers for the 621+lOT, R553X, G551D, Rl 17H, Rl 162X and R334W mutations and the normal CFTR gene sequence corresponding to the DF508 mutation;
- mix 2C comprising primers for the A455E, 2183AA>G, 3659delC, 1078delT, DI507, R347P, S1251N and E60X mutations;
- mix 3 comprising primers for the G85E, 405+lOA, S549R, W1089X, and DI 152H mutations.
- the further amplification primer is either a 1717-1G>A common, G542X common, W1282X common, N1303K common, DF508 common or 3849+lOkbOT common primer.
- the 1717-lOA common primer TAATCTCTACCAAATCTGGATACTATACC is conveniently used in combination with the 1717-1G>A mutant primer
- the G542X common primer TAATCTCTACCAAATCTGGATACTATACC is conveniently used in combination with the G542X mutant primer
- the W1282X common primer GAATTCCCAAACTTTTAGAGACATC is conveniently used in combination with the W1282X mutant primer
- CTTGATGGTAAGTACATGGGTTTTTCTTAT is conveniently used in combination with the N1303K mutant primer
- the DF508 common primer CCAGACTTCACTTCTAATTATGATTATGGG is conveniently used in combination with the DF508 mutant primer
- TTGTGGATCAAATTTCAGTTGACTTGTCATC is conveniently used in combination with the 3849+1 OkbOT mutant primer.
- any convenient control primer may be used.
- the amplification products from the Mix 1 primer mix serve as internal controls and so an additional amplification control is not required.
- the further amplification primer is either a 1717-1G>A common, G542X common, W1282X common, N1303K common, DF508 common or 3849+lOkbOT common primer.
- the DF508 common primer GACTTCACTTCTAATGATGATTATGGGAGA is conveniently used in combination with the DF508 normal primer
- the 621+1G>T common primer GTTTCACATAGTGTATGACCCTCTATATACACTCATT is conveniently used in combination with the 621+1G>T mutant primer
- the R553X common primer ATCTAAAATTGGAGCAATGTTGTTTTTGACC is conveniently used in combination with the R553X mutant primer
- the G551D common primer ATCTAAAATTGGAGCAATGTTGTTTTTGACC is conveniently used in combination with the G551D mutant primer
- GTTTCACATAGTGTATGACCCTCTATATACACTCATT is conveniently used in combination with the Rl 17H mutant primer
- the Rl 162X common primer TTTTGCTGTGAGATCTTTGACAGTCATTT is conveniently used in combination with the Rl 162X mutant primer
- TTTGTTTATTGCTCCAAGAGAGTCATACCA is conveniently used in combination with the R334W mutant primer. Any convenient control primer may be used.
- the further amplification primer is either a 621+1G>T common, R553X common, G551D common, Rl 17H common, Rl 162X common, R334W common or A455E common primer.
- the A455E common primer is either a 621+1G>T common, R553X common, G551D common, Rl 17H common, Rl 162X common, R334W common or A455E common primer.
- GACTGACTGACTGACTGACTGAAATGGAGACTTTTTGTTTATGTGGTTACTAA is conveniently used in combination with the A455E mutant primer
- the 2183AA>G common primer GTATGATAGAGATTATATGCAATAAAACATTAACA is conveniently used in combination with the 2183 AA>G mutant primer
- TGTGTCTAATATTGATTCTACTGTACAATAATAA is conveniently used in combination with the 3659delC mutant primer
- the 1078delT common primer ATTTTTCCAAACTTCATTAGAACTGATCTATTGAC is conveniently used in combination with the 1078delT mutant primer
- the DI507 common primer CACAGTAGCTTACCCATAGAGGAAACA is conveniently used in combination with the DI507 mutant primer
- ATTTTTCCAAACTTCATTAGAACTGATCTATTGAC is conveniently used in combination with the R347P mutant primer
- the S 125 IN common primer GCTCACCTGTGGTATCACTCCAA is conveniently used in combination with the S 125 IN mutant primer
- AATCAAACTATGTTAAGGGAAATAGGACAACTAA is conveniently used in combination with the E60X mutant primer. Any convenient control primer may be used.
- the further amplification primer is either a G85E common, 405+1 G>A common, S549R common, W1089X common, DI 152H common primer.
- the G85E and the 405+lOA common primer CGATTCGATTCAGTTTTCTGTGGTTTCTTAGTGTTTGGA is conveniently used in combination with the G85E mutant primer and/or the 405+lG>A mutant primer
- the S549R common primer GTAATTTTTTTACATGAATGACATTTACAGCAA is conveniently used in combination with the S549R mutant primer
- GGAAATTATTTGTTTAACAATAAAACAATGGAA is conveniently used in combination with the W1089X mutant primer and the DI 152H common primer CCAACAACACCTCCAATACCAGTAAC is conveniently used in combination with the D 1152H mutant primer.
- control primers from unrelated regions of the genome, namely, part of the human apolipoprotein B gene and part of the ornithine decarboxylase gene.
- a variety of methods may be used to detect the presence or absence of diagnostic primer extension products and/or amplification products. These will be apparent to the person skilled in the art of nucleic acid detection procedures. Preferred methods avoid the need for radiolabelled reagents. Particular detection methods include size separation of amplification products, for example as described in our patents numbers EP 0 332 435 & US 5595890, "ALEX" product detection (Haque K. et al. Diag. Mol. Pathol. &: 248-252 (1998)), the detection of amplification incorporated "Sunrise” probes (Nazarenko LA. et al. Nucl. Acids Res.
- patent number EP 0 731 177 "taqman” product detection for example as described in patent numbers US-A- 5487972 & US-A-5210015; and "Molecular Beacons” product detection outlined in patent number WO-95/13399.
- primer mixes of the invention may be conveniently packaged with instructions for use in the method of the invention and appropriate packaging and sold as a kit.
- Convenient primer mixes include intron 8 polyT allele-specific primers and CFTR mutation- specific primers, equivalent normal-specific primers; all as hereinbefore disclosed.
- the kits will conveniently include one or more of the following: appropriate nucleotide triphosphates, for example one or more of dATP, dCTP, dGTP, and dTTP, a suitable polymerase as previously described, and a convenient buffer solution.
- Table 1 shows a specific primer mix 1
- Table 2 shows a specific primer mix 2A
- Table 3 shows a specific primer mix 2B
- Table 4 shows a specific primer mix 2C
- Table 5 shows a specific primer mix 3
- Figure 1 shows the relative location of the PCR product bands on an agarose electrophoresis gel corresponding to (PolyT)
- Fisure 2 shows the relative location of the PCR product bands on an agarose electrophoresis gel corresponding to a CFTR DF508 heterozygote, DF508 homozygote, DF508/ DI507 compound heterozygote, G542X heterozygote, G551D heterozygote, 1078delT heterozygote.
- Figure 3 shows the relative location of the PCR product bands on an agarose electrophoresis gel corresponding to a (Middle Eastern Panel)
- Figure 4 shows diagramatically the relative sizes in base pairs and the relative location of the PCR product bands on an agarose electrophoresis gel corresponding to the CFTR intron 8 5T, 7T and 9T alleles.
- Figure 5 shows diagramatically the size in base pairs and the relative location of the
- Example 1.1 Materials Provided in a diagnostic kit containing 50tests (polyT)
- Example 2.1 Materials Provided in a diagnostic kit containing 50 tests (CF20)
- Example 3.1 Materials Provided in a diagnostic kit containing 50tests (Middle Eastern) 1. 50 Vial 3 (colour coded) containing primers (DI 152H, W1089X, G85E,
- 1 vial normal DNA control contains human DNA unaffected by the mutations detected by the kit, in buffer. 6. Instructions for use.
- the samples may be stored at room temperature overnight or at 2-8 °C for up to 7 days before analysis by gel electrophoresis.
- the samples may be stored at room temperature overnight or at 2-8 °C for up to 7 days before analysis by gel electrophoresis.
- a 50 Base-Pair Ladder (Amersham Pharmacia Biotech) at 1.5 ⁇ g/15 ⁇ L was prepared in the loading dye (80 ⁇ L distilled water / lO ⁇ L loading dye / lO ⁇ L 50 Base-Pair Ladder). 15 ⁇ L of this dilution was run adjacent to samples as a molecular weight marker.
- Electrophoresis was carried out at 5 to 6 V/cm between electrodes until the dye front had migrated 5 cm from the loading wells towards the anode (1.5 to 2 hours).
- the negative control should show no bands in the tracks within the area co ⁇ esponding to the upper and lower control bands.
- a diagnostic band should not be interpreted if a similar band is also seen in the negative control for that PCR run as this is indicative of contamination with genomic DNA.
- An individual has two copies of the CFTR gene. Where these copies have the same sequence for any given site, an individual is described as being homozygous for this site. Where the copies differ in sequence at a given site, an individual is described as being heterozygous for this site.
- the bands should be of similar intensity to the 250bp band in the 50bp ladder, loaded at 1.5mg/15ml (Amersham Pharmacia Biotech).
- heteroduplexes may occasionally be formed from the PCR products and may therefore be visible in some test sample tracks. The position of these heteroduplexes is indicated in figure
- a 7T/7T individual may have a heteroduplex band larger than the 7T allele but not as large as the 9T allele.
- a 5T/7T individual may have a heteroduplex that runs in a similar position to the 9T allele. Therefore if all three diagnostic bands are visible the individual's genotype is 5T/7T.
- the heteroduplex if present, is generally weaker than the corresponding 5T and 7T diagnostic products.
- a 5T/9T individual occasionally has a heteroduplex which run larger than the 9T allele.
- a diagnostic 2183AA>G band may indicate that the
- 2184insG mutation is present. 4.
- the mix formulation for the 2A mix is presented below (Table 2).
- the 25 ⁇ l total reaction volume also includes lx ARMS, lOOmM dNTP, 1.5U AmpliTaq Gold, lx CRS
- the mix formulation for the 2B mix is presented below (Table 3).
- the 25 ⁇ l total reaction volume also includes lx ARMS, lOOmM dNTP, 1.5U AmpliTaq Gold, lx CRS.
- the mix formulation for the 2C mix is presented below (Table 4).
- the 25 ⁇ l total reaction volume also includes lx ARMS, lOOmM dNTP, 1.5U AmpliTaq Gold, lx CRS.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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EP00960854A EP1242619A2 (en) | 1999-09-24 | 2000-09-19 | Assay for detection of human cftr allele variants using specific diagnostic primers |
CA002385920A CA2385920A1 (en) | 1999-09-24 | 2000-09-19 | Assay |
AU73022/00A AU7302200A (en) | 1999-09-24 | 2000-09-19 | Assay |
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GB9922527.8 | 1999-09-24 | ||
GBGB9922527.8A GB9922527D0 (en) | 1999-09-24 | 1999-09-24 | Assay |
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WO2001021833A2 true WO2001021833A2 (en) | 2001-03-29 |
WO2001021833A3 WO2001021833A3 (en) | 2002-07-25 |
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PCT/GB2000/003597 WO2001021833A2 (en) | 1999-09-24 | 2000-09-19 | Assay for detection of human cftr allele variants using specific diagnostic primers |
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EP (1) | EP1242619A2 (en) |
AU (1) | AU7302200A (en) |
CA (1) | CA2385920A1 (en) |
GB (1) | GB9922527D0 (en) |
WO (1) | WO2001021833A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008016334A1 (en) * | 2006-08-03 | 2008-02-07 | National University Of Singapore | Multiplex analysis of nucleic acids |
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EP0497527A1 (en) * | 1991-01-31 | 1992-08-05 | Zeneca Limited | Detection method for nucleotide sequences |
WO1993018177A1 (en) * | 1992-03-13 | 1993-09-16 | The Children's Hospital Of Philadelphia | Diagnosis of cystic fibrosis using allele specific multiplex polymerase chain reactions |
WO1997042345A1 (en) * | 1996-05-04 | 1997-11-13 | Zeneca Limited | Method for detecting a nucleic acid base sequence |
US5853989A (en) * | 1991-08-27 | 1998-12-29 | Zeneca Limited | Method of characterisation of genomic DNA |
EP0928832A2 (en) * | 1998-01-13 | 1999-07-14 | Zeneca Limited | Cystic fibrosis test based on the detection of mutations in the CFRE gene by ARMS |
-
1999
- 1999-09-24 GB GBGB9922527.8A patent/GB9922527D0/en not_active Ceased
-
2000
- 2000-09-19 EP EP00960854A patent/EP1242619A2/en not_active Withdrawn
- 2000-09-19 AU AU73022/00A patent/AU7302200A/en not_active Abandoned
- 2000-09-19 CA CA002385920A patent/CA2385920A1/en not_active Abandoned
- 2000-09-19 WO PCT/GB2000/003597 patent/WO2001021833A2/en not_active Application Discontinuation
Patent Citations (5)
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EP0497527A1 (en) * | 1991-01-31 | 1992-08-05 | Zeneca Limited | Detection method for nucleotide sequences |
US5853989A (en) * | 1991-08-27 | 1998-12-29 | Zeneca Limited | Method of characterisation of genomic DNA |
WO1993018177A1 (en) * | 1992-03-13 | 1993-09-16 | The Children's Hospital Of Philadelphia | Diagnosis of cystic fibrosis using allele specific multiplex polymerase chain reactions |
WO1997042345A1 (en) * | 1996-05-04 | 1997-11-13 | Zeneca Limited | Method for detecting a nucleic acid base sequence |
EP0928832A2 (en) * | 1998-01-13 | 1999-07-14 | Zeneca Limited | Cystic fibrosis test based on the detection of mutations in the CFRE gene by ARMS |
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Title |
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BRAUN A ET AL: "DETECTING CFTR GENE MUTATIONS BY USING PRIMER OLIGO BASE EXTENSION AND MASS SPECTROMETRY" CLINICAL CHEMISTRY, AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY. WINSTON, US, vol. 43, no. 7, 1997, pages 1151-1158, XP001019094 ISSN: 0009-9147 * |
COHEN J ET AL: "Relation between mutations of the Cystic Fibrosis Gene and idiopathic pancreatitis" THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 339, no. 10, September 1998 (1998-09), pages 653-58, XP001053192 * |
FRIEDMAM K ET AL: "Cystic fibrosis syndrome: A new paradigm for inherited disorders and implications for molecular diagnosis" CLINICAL CHEMISTRY, vol. 45, no. 7, 1999, page 92931 XP002188789 * |
FRIEDMAN K ET AL: "Rapid characterization of the variable length Polythymidine Tract in the Cystic Fibrosis gene (CFTR)" HUMAN MUTATION, vol. 10, no. 2, 1997, pages 108-15, XP001053200 * |
GELFI C ET AL: "Rapid capillary zone electrophoresis in isoelectric histidine buffer: high resolution of the poly-T tract allelic variants in intron 8 of the CFTR gene" CLINICAL CHEMISTRY, vol. 44, no. 5, 1998, pages 906-13, XP002188788 * |
HABER P ET AL: "Alcoholic Pancreatitis and polymorphisms of the variable length tract in the cystic fibrosis gene" ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH, vol. 23, no. 3, March 1999 (1999-03), pages 509-12, XP001053196 * |
MAK V ET AL: "Proportion of cystic fibrosis gene mutations not detected by routine testing in men with Obstructive Azoospermia" JAMA, vol. 281, no. 23, June 1999 (1999-06), pages 2217-24, XP001053208 * |
SHARER N ET AL: "Mutations of the cystic fibrosis gene in patients with chronic pancreatitis" THE NEW JOURNAL OF ENGLAND MEDICINE, vol. 339, no. 10, 3 September 1998 (1998-09-03), pages 645-52, XP001053194 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008016334A1 (en) * | 2006-08-03 | 2008-02-07 | National University Of Singapore | Multiplex analysis of nucleic acids |
JP2009545314A (en) * | 2006-08-03 | 2009-12-24 | ナショナル ユニヴァーシティー オブ シンガポール | Nucleic acid multiplex analysis method |
Also Published As
Publication number | Publication date |
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CA2385920A1 (en) | 2001-03-29 |
WO2001021833A3 (en) | 2002-07-25 |
AU7302200A (en) | 2001-04-24 |
EP1242619A2 (en) | 2002-09-25 |
GB9922527D0 (en) | 1999-11-24 |
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