WO1993018177A1 - Diagnostic de la mucoviscidose par amplification enzymatique du genome multiplex specifique d'un allele - Google Patents

Diagnostic de la mucoviscidose par amplification enzymatique du genome multiplex specifique d'un allele Download PDF

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Publication number
WO1993018177A1
WO1993018177A1 PCT/US1993/002259 US9302259W WO9318177A1 WO 1993018177 A1 WO1993018177 A1 WO 1993018177A1 US 9302259 W US9302259 W US 9302259W WO 9318177 A1 WO9318177 A1 WO 9318177A1
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seq
primer
polymerase chain
primer sets
chain reaction
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PCT/US1993/002259
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English (en)
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Paolo Fortina
Saul Surrey
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The Children's Hospital Of Philadelphia
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Priority to AU39185/93A priority Critical patent/AU3918593A/en
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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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention is directed to the diagnosis of cystic fibrosis using a novel multiplex allele-specific polymerase chain reaction system.
  • Cystic fibrosis is the most common lethal autosomal recessive disease in Caucasians affecting approximately 1 in 2000 newborns with a carrier frequency of about 1 in 25 individuals. More than 120 different mutations have been reported in the CFTR gene; Kerem, et al.. Science 245: 1073-1080 (1989); Zielenski, et al., Genomics 10: 214-228 (1991); Dean, et al.. Cell j51: 863-870 (1990); Cutting, et al., Nature 364: 366-369 (1990); Kere , et al. Proc . Natl . Acad . Sci . U.S.A.
  • Multiplex PCR has also been used for simultaneous amplification of multiple target sequences, permitting mutant allele scanning using two lanes of an agarose gel.
  • This strategy involves appropriate choice of primer pairs so that PCR fragments (either normal or mutant) are generated of different size which can be easily resolved by comparison of samples run in parallel lanes of a gel.
  • deletional forms of X-linked diseases such as Lesch-Nyhan syndrome and Duchenne muscular dystrophy are immediately obvious with this procedure, since missing exons are readily apparent in the amplification pattern.
  • the methods of the present invention comprise the steps of obtaining genomic DNA from a patient suspected of carrying a genetic mutation characteristic of cystic fibrosis and selecting at least two primer sets for detecting the normal and mutant allele characteristic of cystic fibrosis.
  • Each primer set is comprised of two primer pairs, a first primer pair comprising a specific primer for a normal allele, and a second primer pair comprising a specific primer for a mutant allele.
  • Each pair further comprises a common primer.
  • a polymerase chain reaction is performed in accordance with methods of the present invention using said genomic DNA and said at least two primer sets whereby primer pairs comprising a specific primer for a normal allele are used simultaneously and primer pairs comprising a specific primer for a mutant allele are used simultaneously. Two or more polymerase chain reaction products are detected whereby the detection of a polymerase chain reaction product of a specific primer for a mutant allele indicates the likelihood that said patient carries a mutation characteristic of the phenotype cystic fibrosis.
  • each specific primer is differentially labeled.
  • the genomic DNA and all differentially labeled primer sets are used simultaneously to perform the polymerase chain reaction.
  • kits comprising four dNTPs and at least two primer sets selected from the group consisting of CF-W1468 GGCACCATTAAAGAAAATATCATCTT (SEQ ID NO: 1) ,
  • G551D-N GAAATTCTTGCTCGTTGAC SEQ ID NO: 7
  • G551D-M GAAATTCTTCGTCGTTGAT
  • IVS-11 CAACTGTGGTTAAAGCAATAGTGT
  • G542X-N GTGTGATTCCACCTTCTCC (SEQ ID NO: 9)
  • G542X-M GTGTGATTCCACCTTCTCA (SEQ ID NO: 10)
  • Figure 1 is a schematic representation of a strategy for multiplex amplification of the areas encompassing the most common cystic fibrosis mutations. Approximate location of five common cystic fibrosis mutations is indicated (A) within the CFTR gene. Multiplex amplification is accomplished using allele specific primer sets. Common primers (CF-508RP, 5' IVS- 11 and 5' IVS-21) and a mixture selected from five normal (CF- 1468, R553X-N, G551D-N, G542X-N and N1303K-N) and/or five mutant primers ( ⁇ F508, R553X-M, G551D-M, G542X-M and N1303K-M) are used in each PCR reaction. Size of expected PCR products in base pairs is also shown.
  • Common primers CF-508RP, 5' IVS- 11 and 5' IVS-21
  • Figure 2 shows PCR amplification of normal DNA in regions encompassing the most common cystic fibrosis mutations. All amplifications were done with either normal (N) or mutant (M) primers corresponding to the ⁇ F508, G551D, G542X and N1303K regions of the human CFTR gene. Amplifications were done with DNA from normal controls using normal primers (lanes 2,4,6 and 8) or mutant primers for the same regions (lanes 3,5,7 and 9). PCR products are sized relative to markers generated from a Hae III digest of ⁇ X174 Rf DNA (lane 1) . Arrows show location of each PCR product using indicated primers.
  • Figure 3 shows the detection of common cystic fibrosis mutations by multiplex allele specific polymerase chain reaction (MASPCR) .
  • MASPCR multiplex allele specific polymerase chain reaction
  • PCR products are sized relative to markers generated from a Hae III digest of 0X174 Rf DNA (lanes 1 and 12) .
  • Figure 4 is a graphical representation of the detection of the ⁇ F508 allele in the CFTR gene by fluorescence- based allele-specific PCR.
  • Panels A-D are graphic representations of fluorescence emission from the reactions with three (Panel A) , two (Panel B) or single colors (Panels C and D) , displayed by analysis software for comparison of overlapping signals. The third color (red) is not shown.
  • Panels E through G are electrophoretograms showing signal traces through lanes 20, 21 and 22, respectively, of panel A with the different colors displayed separately.
  • genomic DNA is obtained from a patient suspected of carrying a genetic mutation characteristic of a disease such as CF.
  • Genomic DNA may be extracted by methods described by Poncz, et al. , Hemoglobin 6_: 27-33 (1982) or with an automated extractor (Applied Biosystems, Inc., Foster City, CA) .
  • Other methods for extraction of genomic DNA known to those skilled in the art are also encompassed by the present invention.
  • the gene responsible for cystic fibrosis is the cystic fibrosis transmembrane conductance regulator (CFTR) gene. More than 120 different mutations have been reported in the CFTR gene. Kerem, et al. , Science 245: 1073-1080 (1989); Zielenski, et al. , Genomics 3 ): 214-228 (1991); Dean, et al. , Cell 61: . 863-870 (1990); Cutting, et al. , Nature 364: 366-369 (1990); Kerem, et al. Proc . Natl . Acad. Sci . U.S .A. : 8447-8451 (1990) and Cystic Fibrosis Genetic Analysis Consortium, Am . J. - Hum. Genet. 47: 354-359 (1990) . Some common mutations are set forth in Table I.
  • MASPCR can be used to detect mutations such as the mutations of Table I by the appropriate choice of primers.
  • a primer strategy such as the strategy set forth in Figure 1 can be used to detect at least two of the mutations of Table I simultaneously.
  • Primers are designed so that the size of the resulting PCR products differ, thereby facilitating detection.
  • Oligonucleotide primers of the present invention can be synthesized by procedures known to those skilled in the art such as by solid state phosphoramidite synthesis.
  • At least two primer sets for detecting at least two mutations characteristic of a disease such as cystic fibrosis are selected.
  • four primer sets are selected useful for diagnosing four mutations characteristic of cystic fibrosis.
  • five primer sets are selected which are useful for detecting five mutations characteristic of cystic fibrosis.
  • Each primer set is comprised of two primer pairs.
  • a first primer pair is comprised of a primer specific for a normal allele.
  • a second primer pair is comprised of a primer specific for' a mutant allele such as an allele specific for cystic fibrosis.
  • the specific primers differ from each other only at their terminal 3' nucleotide.
  • the 3' nucleotide of the specific primer of the first primer pair is specific for the nucleic acid sequence of a normal allele.
  • the 3' nucleotide of the specific primer of the second primer pair may be specific for the nucleic acid sequence of a cystic fibrosis mutation.
  • Each primer pair further comprises a common primer.
  • the nucleic acid sequence of the common primer is the same for both primer pairs comprising a primer set. Under proper annealing temperatures and polymerase chain reaction conditions, these primers pairs only direct amplification of their complementary allele.
  • genomic DNA from a patient homozygous for the ⁇ F508 mutation will be amplified by the second primer pair (mutant specific) .
  • the first primer pair, specific for the normal allele will not amplify genomic DNA from a patient homozygous for a mutant allele.
  • each specific primer is differentially labeled. resulting in differentially labeled PCR products.
  • Any label known to those skilled in the art which can be easily differentiated clinically are encompassed by the present invention.
  • dyes known to those skilled in the art may be useful to distinguish PCR products based upon color differentiation.
  • fluorescent dyes such as FAMTM (blue) , JOEfTM (green) , TAMRATM (yellow) and ROXTM (red) (Applied Biosystems, Inc., Foster City, CA) may be used.
  • Differential labels may be linked to oligonucleotide primers of the present invention by methods known to those skilled in the art, such as by linker molecules.
  • Linker molecules useful in the present invention may be selected from any of a variety of linker molecules available to those skilled in the art, such as a reactive aminohexyl linker (Aminolink) .
  • differential label may be incorporated during synthesis of the oligonucleotide primers.
  • PCR products are labeled by differential recognition by a labeled probe or chemical moiety such as a rhodamine coupled antibody.
  • the specific primer for the N1303K mutant allele may be labeled with yellow dye and the N1303K normal allele labeled with blue dye. By detecting a yellow signal, one skilled in the art would be apprised that the patient has a N1303K mutant allele. A blue signal would indicate a normal allele.
  • N and M indicate normal and mutant primers, respectively.
  • Common primer indicates primers having a sequence common to both primer pairs.
  • Normal and mutant indicate primers having a sequence specific for a normal or mutant allele, respectively.
  • Bold letters indicate single-base mutations. Amount (pMoles) of each primer used in multiplex reactions per primer pair is also indicated. Sequences are provided in 5' to 3' direction.
  • primer sets useful to detect a particular disease such as cystic fibrosis can be identified using methods known to those skilled in the art.
  • the first member in each set is the specific primer for a normal allele
  • the second member of each set is the specific primer for the corresponding mutant allele
  • the third member of each set is the common primer included in each primer pair.
  • the names of the primers generally correspond to the mutation they are designed to detect.
  • N1303K-N and N1303K-M are the primers useful for amplification of the N1303K normal and N1303K mutant alleles, respectively.
  • At least two mutant alleles can be detected simultaneously by methods of the present invention. It is encompassed by some embodiments of the present invention to perform two polymerase chain reactions per diagnosis. In one PCR reaction mixture, primer pairs for normal alleles from each primer set are used. In a second reaction mixture, all primer pairs for mutant alleles from each primer set are used. Thus each polymerase chain reaction in such a diagnostic test causes amplification of genomic DNA using either primers specific for mutant or normal alleles, i.e. half of each primer set per reaction, an entire primer set per diagnosis. Resulting PCR products are run in parallel on gels to detect the presence or absence of bands.
  • diagnosis of cystic fibrosis is accomplished in some embodiments of the present invention, by comparison of normal and mutant polymerase chain reaction products.
  • Figure 3 shows five, two PCR diagnoses using four primer sets each. In each diagnosis, one PCR reaction was performed with primers specific for normal alleles (lanes 2,4,6,8 and 10) and one PCR reaction was performed with primers specific for mutant alleles (lanes 3,5,7,9 and 11).
  • Diagnosis may be used in the context of the present invention to encompass a procedure whereby two or more primer sets are amplified and interpreted in order to determine the presence or absence of selected normal and mutant alleles in a particular genomic DNA sample. As has been exemplified above, a diagnosis may encompass one or more polymerase chain reactions.
  • both members of each primer pair of at least two primer sets are used simultaneously in a single polymerase chain reaction which is run on a single lane of a gel.
  • Differential labels as described above, are useful herein for distinguishing polymerase chain reaction products, especially those having similar mobilities. Thus PCR products can be distinguished by mobility and label.
  • labels such as fluorescent labels may be particularly amenable to automated methods.
  • At least two of the primer sets provided in Table II are selected. It is still more preferred in some embodiments of the present invention to select all of the primer sets of Table II.
  • the primer sets 1 ( ⁇ F508/CF-W1468) , 3 (G551D-N/G551D-M) , 4 (G542X-N/G542X- M) and 5 (N1303K-N/N1303K-M) are selected.
  • the primer sets 1 ( ⁇ F508/CF- 1468) , 3 (G551D-N/G551D-M) and 4 (G542X-N/G542X-M) may be selected in accordance with some embodiments of the present invention.
  • the primer sets 1 ( ⁇ F508/CF-W1468) and 3 (G551D- N/G551D-M) are selected.
  • Kits are also provided by the present invention comprising four dNTPs and at least two primer sets selected from the primer sets provided in Table II.
  • Genomic DNA samples were obtained from normal controls and patients either homozygous or heterozygous for the common CF mutations, ⁇ F508, G551D, R553X, G542X and N1303K. Genomic DNA was extracted using protocols previously described in Poncz, M.D., et al. , Hemoglobin 6.: 27-33 (1982) or with an automated extractor (ABI, Foster City, CA) . Genotypes were confirmed either by manual DNA sequence analysis or by allele-specific oligonucleotide hybridization.
  • Unlabelled oligonucleotide primers were prepared by standard phosphoramidite chemistry. Caruthers, M.H. , et al. , Methods in Enzymol . 154: 287-313 (1987). Oligonucleotide primers were prepared for fluorescent labeling following standard phosphoramidite chemistry preparation by attachment of a reactive aminohexyl linker group (Aminolink) to the 5' end of the primer. Draper, D. and L.E. Gold, Biochemistry 19.: 1774- 1781 (1980) .
  • oligonucleotide primers were dissolved in 0.5M NaHC0 3 /Na 2 C0 3 , pH 9.0 buffer. Fluorescent dye-N-hydroxyl succinimide esters were dissolved in DMSO and added to oligonucleotide primer aliquots. The reaction was allowed to proceed at room temperature for 2- 24 hours. ABI 370 user bulletin (1989) . The dye labeled primer was then removed from excess reactants via high performance liquid chromatography purification. Sequences of the oligonucleotide primers are as provided in Table II.
  • PCR was performed according to methods previously described by Saiki, R.K. , et al. , Science 239: 487-489 (1988).
  • Reaction mixtures (25 ⁇ l) contained lOOng of genomic DNA, 1.5 ⁇ M of each dNTP, the common primers (CF-508RP, 5' IVS-11 and 5' IVS-21) and normal and/or mutant primers in a buffer containing 6.7mM MgCl 2 , 16.6 mM (NF 4 ) 2 S0 4 , 5.0 ⁇ M jSME, 6.8 mM EDTA, 67.0 mM Tris HC1 pH 8.8, 10% (v/v) DMSO.
  • the mixture was heated at 95°C for 5 minutes to denature the DNA, .and then quickly chilled on ice.
  • Taq DNA polymerase 1.5 U, Perkin Elmer, Norwalk, CT
  • the samples were then subjected to 30 cycles on a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) with denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute and extension at 72°C for 1 minute. The last cycle had a 5 minute extension at 72°C.
  • Approximately 15 ⁇ l of the PCR product were then analyzed following electrophoresis on a 3% (w/v) agarose (NuSieve GTG) gel.
  • the normal specific primer (CF-W1468) is labeled with a blue tag and the mutant specific primer (CF- ⁇ F508) with a green tag.
  • the common primer (CF-508RP) was unlabeled, and internal size standards were labeled with a red tag (not shown) .
  • PCR was performed and the PCR product was then analyzed following electrophoresis on a 2% (w/v) agarose (NuSieve GTG) gel using a multi-line argon ion laser such as a GENE SCANNERTM (Applied Biosystems, Inc. Foster City, CA) to detect the fluorescently labeled PCR products. Results are provided in Figure 4.
  • panel A a graphic recreation of the real-time fluorescence emission from the reactions during electrophoresis is shown, displaying two colors (a third not shown) .
  • Panel B excludes the red fluorescence signal (not shown) from the marker fragments
  • panel C displays only the blue signal
  • panel D shows only the green signal.
  • the lower band in each lane represents excess primer not incorporated into the PCR reaction.
  • both blue and green signals indicates that both normal and ⁇ F508 specific primers were included in each reaction.
  • the reaction in lane 20 is from a normal control consistent with the incorporation of only blue-labeled wild-type primers into a PCR product 79 bp in size (panel C) .
  • Lane 21 contains a reaction using genomic DNA from an individual homozygous for the ⁇ F508 mutation, showing appropriate incorporation of only green-labeled mutant primers into a PCR product 76 bp in length (panel D) .
  • the sample in lane 22 contains DNA from a heterozygote for the ⁇ F508 deletion, yielding amplification with both primers, and resulting in both blue 79 bp (panel C) and green 76 bp (panel D) bands.
  • Panels E, F and G show electrophoretograms of lanes 20, 21, and 22, respectively, of panel A, which represents signal traces within each lane and indicate the relative positions and areas of the bands.
  • Diagnosis is carried out as provided in Example 5, except that both normal and mutant allele primer sets are combined in a single PCR reaction mixture.
  • Each reaction contains three common primers; CF-508RP, 5' IVS-11 and 5' IVS- 21; four mutant and four normal primers for the ⁇ F508, G551D, G542X and N1303K sequences.
  • Primers are labeled as follows: CF- 1468 (blue) , ⁇ F508 (green) , G542X-N (yellow) , G542X-M (red) , G551D-N (blue) , G551D-M (green) N1303K-N (yellow) and N1303K-M (red) .
  • a fluorescently labeled marker lane is run in a separate lane to facilitate sizing PCR products.
  • the PCR product is then analyzed following electrophoresis on a 3% (w/v) agarose (NuSieve GTG) gel using a multi-line argon ion laser such as a GENE SCANNERTM (Applied Biosystems, Inc. Foster City, CA) to detect the fluorescently labeled PCR products.
  • Multicolored bands indicate heterozygosity for an allele, while single colored bands indicate homozygosity for an allele.
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Abstract

L'invention concerne des procédés de diagnostic de maladies telles que la mucoviscidose par un système d'amplification enzymatique du génome multiplex spécifique d'un allèle tel qu'illustré par les gels d'électrophorèse et les tracés de densitométrie de la figure 4. L'invention concerne aussi des nécessaires utiles pour diagnostiquer des maladies telles que la mucoviscidose.
PCT/US1993/002259 1992-03-13 1993-03-11 Diagnostic de la mucoviscidose par amplification enzymatique du genome multiplex specifique d'un allele WO1993018177A1 (fr)

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WO1996001908A1 (fr) * 1994-07-08 1996-01-25 Visible Genetics Inc. Procede, reactifs et kit de detection ciblee et de diagnostic du retinoblastome
WO1996041012A1 (fr) * 1995-06-07 1996-12-19 Genzyme Corporation Sequence d'amorce universelle pour une amplification multiplex de l'adn
US5843660A (en) * 1994-09-30 1998-12-01 Promega Corporation Multiplex amplification of short tandem repeat loci
EP0928832A2 (fr) * 1998-01-13 1999-07-14 Zeneca Limited Test pour la mucoviscidose basé sur la révélation de mutations dans le gène CFTR par ARMS
EP1061136A2 (fr) * 1999-06-09 2000-12-20 Hitachi, Ltd. Procédé de préparation d'un échantillon et un appareil de préparation d'un échantillon pour l'analyse de l'ADN
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WO1995006137A1 (fr) * 1993-08-27 1995-03-02 Australian Red Cross Society Detection de genes
WO1996001908A1 (fr) * 1994-07-08 1996-01-25 Visible Genetics Inc. Procede, reactifs et kit de detection ciblee et de diagnostic du retinoblastome
US5550020A (en) * 1994-07-08 1996-08-27 Visible Genetics Inc. Method, reagents and kit for diagnosis and targeted screening for retinoblastoma
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US6063567A (en) * 1994-07-08 2000-05-16 Visible Genetics Inc. Method, reagents and kit for diagnosis and targeted screening for retinoblastoma
GB2304411B (en) * 1994-07-08 1999-02-10 Visible Genetics Inc Method, reagents and kit for diagnosis and targeted screening for retinoblastoma
US5843660A (en) * 1994-09-30 1998-12-01 Promega Corporation Multiplex amplification of short tandem repeat loci
US6479235B1 (en) 1994-09-30 2002-11-12 Promega Corporation Multiplex amplification of short tandem repeat loci
US7008771B1 (en) 1994-09-30 2006-03-07 Promega Corporation Multiplex amplification of short tandem repeat loci
US6221598B1 (en) 1994-09-30 2001-04-24 Promega Corporation Multiplex amplification of short tandem repeat loci
US5882856A (en) * 1995-06-07 1999-03-16 Genzyme Corporation Universal primer sequence for multiplex DNA amplification
US6207372B1 (en) 1995-06-07 2001-03-27 Genzyme Corporation Universal primer sequence for multiplex DNA amplification
WO1996041012A1 (fr) * 1995-06-07 1996-12-19 Genzyme Corporation Sequence d'amorce universelle pour une amplification multiplex de l'adn
EP0928832A2 (fr) * 1998-01-13 1999-07-14 Zeneca Limited Test pour la mucoviscidose basé sur la révélation de mutations dans le gène CFTR par ARMS
EP0928832A3 (fr) * 1998-01-13 2004-02-11 AstraZeneca AB Test pour la mucoviscidose basé sur la révélation de mutations dans le gène CFTR par ARMS
EP1061136A2 (fr) * 1999-06-09 2000-12-20 Hitachi, Ltd. Procédé de préparation d'un échantillon et un appareil de préparation d'un échantillon pour l'analyse de l'ADN
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EP1061136A3 (fr) * 1999-06-09 2002-05-08 Hitachi, Ltd. Procédé de préparation d'un échantillon et un appareil de préparation d'un échantillon pour l'analyse de l'ADN
US6518027B2 (en) 1999-06-09 2003-02-11 Hitachi, Ltd. Sample preparation method and a sample preparation apparatus for DNA analysis
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