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WO1993018178A1 - DIAGNOSTIC DE LA THALASSEMIE β PAR MUTATION REFRACTAIRE A AMPLIFICATION MULTIPLEX - Google Patents

DIAGNOSTIC DE LA THALASSEMIE β PAR MUTATION REFRACTAIRE A AMPLIFICATION MULTIPLEX Download PDF

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WO1993018178A1
WO1993018178A1 PCT/US1993/002260 US9302260W WO9318178A1 WO 1993018178 A1 WO1993018178 A1 WO 1993018178A1 US 9302260 W US9302260 W US 9302260W WO 9318178 A1 WO9318178 A1 WO 9318178A1
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seq
ivs
primer
crpl
primer sets
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PCT/US1993/002260
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Paolo Fortina
Saul Surrey
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The Children's Hospital Of Philadelphia
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Priority to AU38049/93A priority Critical patent/AU3804993A/en
Publication of WO1993018178A1 publication Critical patent/WO1993018178A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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 ⁇ - thalassemia using a novel multiplex amplification refractory mutation system.
  • Beta thalassemia is a heterogenous genetic disease associated with defective expression of ⁇ chain human hemoglobin (Hb) .
  • Hb human hemoglobin
  • IVS-1 nt 1, IVS-1 nt 6, codon 39, IVS-1 nt 110, IVS-2 nt 1, IVS-2 nt 745 changes are the most common mutations among Mediterraneans, and account for approximately 92% of the ⁇ -thalassemia defects in this area.
  • a wide variety of strategies and techniques are currently available to detect mutations. Orkin, et al.. Nature 296; 627 (1982); Saiki, et al. , N. Engl . J. Med 319: 537 (1988); Losekoot, M.
  • ⁇ -thalassemia has been diagnosed by using denaturing gradient gel electrophoresis and direct sequencing of PCR amplified genomic DNA. This procedure is limited by the labor intensive nature of the procedure.
  • the amplification refractory mutation system is another method which has been used to diagnose ⁇ --thalassemia. Varawalla, et al. , Brit . J. of Haematology 7_8.: 242-247
  • Multiplex PCR is another procedure which has been used for the diagnosis of diseases characterized by mutant alleles. Multiplex is useful for the simultaneous amplification of multiple target sequences permitting multiple mutant alleles to be scanned in a single lane 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.
  • the methods of the present invention comprise the steps of obtaining genomic DNA from a patient suspected of carrying a genetic mutation characteristic of /3-thalassemia and selecting at least two primer sets for detecting at least two mutations characteristic of ⁇ -thalassemia.
  • 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 ⁇ - thalassemia.
  • each specific primer is differentially labeled.
  • the genomic DNA and all differentially labeled primer sets are used simultaneously to perform the polymerase chain reaction.
  • IVS-1 ntl-N TAAACCTGTCTTGTAACCTTGATACGAAC SEQ ID NO: 1
  • IVS-1 ntl-M TTAAACCTGTCTTGTAACCTTGATACGAAT SEQ ID NO: 2
  • jS-CRPl ACCTCACCCTGTGGAGCCAC SEQ ID NO: 3
  • IVS-1 nt6-N TCTCCTTAAACCTGTCTTGTAACCTTCATA (SEQ ID NO: 4), IVS-1 nt6-M TCTCCTTAAACCTGTCTTGTAACCTTCATG (SEQ ID NO: 5) , and 3-CRPl ACCTCACCCTGTGGAGCCAC (SEQ ID NO: 3) ; IVS-1 ntllO-N ACCAGCAGCCTAAGGGTGGGAAAATAGTCC (SEQ ID NO: 6), IVS-1 ntllO-M ACCAGCAGCCTAAGGGTGGGAAAATAGTCT (SEQ ID NO: 1 ) , and ⁇ -CRPl ACCTCACCCTGTGGAGCCAC (SEQ ID NO: 3);
  • IVS-2 nt 1-N AAGAAAACATCAAGGGTCCCATAGACTGAC SEQ ID NO: 10
  • IVS-2 nt 1-M AAGAAAACATCAAGGGTCCCATAGACTGAT
  • ⁇ -CRPl ACCTCACCCTGTGGAGCCAC SEQ ID NO: 3
  • Figure 1 is a schematic representation of the strategy for multiplex amplification of the areas encompassing the most common ⁇ -thalassemia mutations in Mediterraneans. Approximate locations of the five most common ⁇ -thalassemia mutations are indicated (A) within the ⁇ -globin gene. Multiplex amplification is accomplished using a common upstream primer ( ⁇ -CRPl) and a mixture selected from five normal and/or five mutant primers in each polymerase chain reaction. The size of expected PCR products in base pairs is also shown.
  • ⁇ -CRPl common upstream primer
  • Figure 2 shows PCR amplification of normal DNA using normal primers (lanes 2, 4, 6, 8 and 10) or mutant primers for the same regions (lanes 3, 5, 7, 9 and 11). Multiplex amplification of four regions in normal DNA encompassing IVS-2 nt l, codon 39, IVS-1 nt 110 and IVS-l nt 6 with normal primers (lane 12) or mutant primers (lane 13) is also shown. Lanes 14 and 15 show the same pattern of multiplex allele-specific PCR using the IVS-l nt 6 normal and mutant primers, respectively. PCR products are sized relative to markers generated from a Hae III digest of 0X174 Rf DNA (lane 1) . Arrows show location of each PCR product using indicated primers. Figure 3 shows the detection of common
  • Multiplex amplification was done using separate reactions containing a mixture of either normal or corresponding mutant primer pairs with normal genomic DNA (lanes 10, 11, and 12), DNA from a heterozygote for the IVS-2 nt 1 mutation (lanes 2 and 3) , DNA from a heterozygote for the IVS-l nt 1 mutations (lanes 4 and 5) , DNA from a compound heterozygote for the codon 39 and IVS-l nt 1 mutations (lanes 6 and 7) , DNA from a compound heterozygote for -29 and codon 24 mutations (lanes 8 and 9) , respectively.
  • PCR products are sized relative to markers generated from a Hae III digest of 0X174 Rf DNA (lanes 10, 11, and 12), DNA from a heterozygote for the IVS-2 nt 1 mutation (lanes 2 and 3) , DNA from a heterozygote for the IVS-l nt 1 mutations (lanes 4 and 5) , DNA from a compound heterozy
  • genomic DNA is obtained from a patient suspected of carrying a genetic mutation characteristic of a disease such as ⁇ -thalassemia.
  • Genomic DNA may be extracted by methods described by Poncz, et al., Hemoglobin j5: 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.
  • a multiplex amplification refractory mutation system can be used to detect mutations such as the mutations described above 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 ⁇ -thalassemia mutations.
  • 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 ⁇ -thalassemia are selected.
  • four primer sets are selected useful for diagnosing four mutations characteristic of ⁇ -thalassemia.
  • five primer sets are selected which are useful for detecting five mutations characteristic of ⁇ -thalassemia.
  • Each primer set is comprised of two primer pairs.
  • a first primer pair is comprised of a 3' primer specific for a normal allele.
  • a second primer pair is comprised of a 3' primer specific for a mutant allele such as an allele specific for ⁇ -thalassemia.
  • a mismatched residue is incorporated at the third nucleotide in from the 3 ' nucleotide, Newton, et al., Nucleic Acid Research 1/7: 2503 (1989), in both normal and mutant 3' primers in order to ensure selective amplification.
  • the 3 ' primers differ from each other only at their terminal 3' nucleotide.
  • the 3 ' nucleotide of the 3 ' primer of the first primer pair is substantially complementary to the nucleic acid sequence of a normal allele.
  • the 3' nucleotide of the 3 ' primer of the second primer pair may be substantially complementary to the nucleic acid sequence of a ⁇ -thalassemia mutation.
  • Each 3 ' primer has a mismatched incorporated at the third nucleotide in from its 3' nucleotide.
  • 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.
  • these primers pairs only direct amplification of their complementary allele. For example, normal genomic DNA will be amplified by the first primer pair which is specific for a normal allele.
  • the second primer pair specific for a mutant allele will not amplify genomic DNA from a normal patient.
  • each 3' 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.
  • luorescent dyes such as FAMTM (blue) , JOETM (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 IVS-l nt 1 mutant allele may be labeled with yellow dye and the IVS-l nt 1 normal allele labeled with blue dye. By detecting a yellow signal, one skilled in the art would be apprised that the patient has a IVS-l nt 1 mutant allele. A blue signal would indicate a normal allele.
  • Coordinates are given relative to the cap site (+1) of the ⁇ - globin gene, with "N” indicating normal and "M” mutant primers, respectively.
  • ⁇ -CRPl refers to the common primer having a sequence common to both primer pairs.
  • Bold letters identify 3 ' mutation and second base change at 3 nucleotides in from the 3' nucleotide.
  • Picomoles (pM) of each primer used per primer pair is also indicated. Sequences are provided in the 5' to 3' direction.
  • primer sets useful to detect a particular disease 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.
  • 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 ⁇ -thalassemia is accomplished in some embodiments of the present invention, by comparison of normal and mutant polymerase chain reaction products.
  • Figure 3 shows four, two PCR diagnoses using five primer sets each. In each diagnosis, one PCR reaction was performed with 3' primers specific for normal alleles (lanes 2,4,6 and 8) and one PCR reaction was performed with primers specific for mutant alleles (lanes 3,5,7 and 9).
  • 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 I are selected. It is still more preferred in some embodiments of the present invention to select all of the primer sets of Table I.
  • the primer sets 1 (IVS-l nt 1 N and M) , 3 (IVS-l nt 110 N and M) , 4 (Codon 39 N and M) and 5 (IVS-2 nt 1 N and M) are selected.
  • the primer sets 3 (IVS-l nt 110 N and M) and 4 (Codon 39 N and 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 I. The following examples are illustrative, but should not be construed as limiting the present invention. EXAMPLES EXAMPLE 1 DNA Samples
  • Genomic DNA samples were obtained from normal controls and patients either homozygous or heterozygous for the common ⁇ - thalassemia Mediterranean mutations.
  • 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 DNA sequence analysis using PCR based, cycle- sequencing approach employing laser-activated fluorescence- emission DNA sequencer; Tamary, et al., Araer. J " . Hemat. in press (1992); Trifillis, et al.. Blood 78.: 3298 (1991);
  • Unlabelled oligonucleotide primers were prepared on a 38OB DNA Synthesizer (ABI, Foster City, CA) by the phosphoramidite method at 0.2 mmol scale with (2-O-cyano- ethyl)-phosphoramidites; Caruthers, M.H., et al.. Methods in Enzymol . 154: 287-313 (1987); and were then purified. 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) .
  • each modified primer was then reacted with an N-hydroxyl succinimide ester derivative of a distinct dye.
  • 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 I.
  • Reaction mixtures (25 ⁇ l) contained lOOng of genomic DNA, 1.5 /iM of each dNTP, the common primer 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 ⁇ ME, 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 was added before overlaying the samples with 25 ⁇ l of mineral oil. 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.
  • Diagnosis is carried out as provided in Example 5, except that both normal and mutant allele primers are combined in a single PCR reaction mixture.
  • Each reaction contains the common primer and five mutant and five normal primers for the IVS-l nt l, IVS-2 nt 1, IVS-l nt 6, codon 39 and IVS-l nt 110 regions of the human ⁇ -globin gene.
  • Primers are labeled as follows: IVS-l nt 1-N (biue) , IVS-l nt 1-M (green) , IVS-l nt 6-N (yellow) , IVS-l nt 6-M (red) , IVS-l nt 110-N (blue) , IVS-l nt 110-M (green) Codon 39-N (yellow) , Codon 39-M (red) , IVS-2 nt 1-N (blue) , IVS-2 nt 1-M (green) .
  • a fluorescently labeled marker lane in 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.
  • MOLECULE TYPE DNA (genomic)
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Abstract

L'invention concerne des procédés permettant de diagnostiquer des maladies telles que la thalassémie β par l'utilisation d'un système de mutation réfractaire à amplification multiplex. L'invention concerne également des nécessaires utiles pour diagnostiquer des maladies telles que la thalassémie β.
PCT/US1993/002260 1992-03-13 1993-03-11 DIAGNOSTIC DE LA THALASSEMIE β PAR MUTATION REFRACTAIRE A AMPLIFICATION MULTIPLEX WO1993018178A1 (fr)

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WO1995006137A1 (fr) * 1993-08-27 1995-03-02 Australian Red Cross Society Detection de genes
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
EP0688873A3 (fr) * 1994-06-23 1999-02-24 Bayer Ag Test DNA rapide pour la détection de staphylococcus aureus résistant à la chinolone dans du matériel clinique
WO1999010529A1 (fr) * 1997-08-22 1999-03-04 Zeneca Limited Procedes d'analyse des polymorphismes de synthese du ltc4 et utilisation diagnostique
US6221598B1 (en) 1994-09-30 2001-04-24 Promega Corporation Multiplex amplification of short tandem repeat loci
US6432639B1 (en) * 1997-09-10 2002-08-13 Dna Sciences Laboratories, Inc. Isolated CYP3A4 nucleic acid molecules and detection methods
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WO2012137110A1 (fr) * 2011-04-06 2012-10-11 Koninklijke Philips Electronics N.V. Marqueurs d'association pour le caractère de bêta-thalassémie
US8809518B2 (en) 2000-10-05 2014-08-19 Riken Oligonucleotide linkers comprising a variable cohesive portion and method for the preparation of polynucleotide libraries by using said linkers
EP4034680A4 (fr) * 2019-09-25 2023-10-25 Council of Scientific & Industrial Research Trousse pour la détection de mutations à l'origine de troubles génétiques

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GENOMICS, Volume 2, issued 1988, M.H. SKOLNICK et al., "Simultaneous Analysis of Multiple Polymorphic Loci Using Amplified Sequence Polymorphisms (ASPs)", pages 273-279. *
GENOMICS, Volume 7, issued 1990, R.A. GIBBS et al., "Multiplex DNA Deletion Detection and Exon Sequencing of the Hypoxanthine Phosphoribosyltransferase Gene in Lesch-Nyhan Families", pages 235-244. *
PROC. NATL. ACAD. SCI., Volume 86, issued April 1989, (U.S.A.), D.Y. WU et al., "Allele-Specific Enzymatic Amplification of Beta-Globin Genomic DNA for Diagnosis of Sickle Cell Anemia", pages 2757-2760. *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006137A1 (fr) * 1993-08-27 1995-03-02 Australian Red Cross Society Detection de genes
US5972602A (en) * 1993-08-27 1999-10-26 Australian Red Cross Society Quantitative PCR-based method of gene detection
EP0688873A3 (fr) * 1994-06-23 1999-02-24 Bayer Ag Test DNA rapide pour la détection de staphylococcus aureus résistant à la chinolone dans du matériel clinique
US6015666A (en) * 1994-06-23 2000-01-18 Bayer Aktiengesellschaft Rapid DNA test for detecting quinolone-resistant Staphylococcus aureus pathogens in clinical material
US6221598B1 (en) 1994-09-30 2001-04-24 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
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
US5882856A (en) * 1995-06-07 1999-03-16 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
US6207372B1 (en) 1995-06-07 2001-03-27 Genzyme Corporation Universal primer sequence for multiplex DNA amplification
GB2342717A (en) * 1997-08-22 2000-04-19 Zeneca Ltd Methods for analyzing ltc4 synthase polymorphisms and diagnostic use
US6316196B1 (en) 1997-08-22 2001-11-13 Zeneca Limited Methods for analyzing LTC4 synthase polymorphisms and diagnostic use
GB2342717B (en) * 1997-08-22 2001-11-28 Zeneca Ltd Methods for analyzing ltc4 synthase polymorphisms and diagnostic use
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