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  • 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

Definitions

• the present invention relates to nucleic acid mutations and, in particular, relates to a method of detecting such mutations.
• Point mutations in a nucleic acid strand can generally be characterized as a single base pair distinction in the sequence of a nucleic acid strand when compared to the normal or wild type nucleic acid strand.
• the "wild type" nucleic acid strand is a sequence of base pairs that is most prevalently found or the most common sequence of bases for the particular nucleic acid strand.
• a point mutation in a particular gene may not yield any observable manifestations in the gene product. On the other hand, however, if the point mutation occurs in a critical portion of the gene, the point mutation can give rise to observable manifestations in the gene product thereby resulting in genetic disorders or diseases.
• Cystic fibrosis sickle cell anemia
• Tay Sachs disease thalassemias are examples of genetic disorders that may arise from mutations in a particular gene.
• a competitive PCR format is described in U.S. Patent No. 5,582,989 in which two distinct sets of primer pairs are used to amplify a nucleic acid sequence of interest.
• one set of primers is complementary to the wild type sequence and the other set of primers is complementary to a sequence having a point mutation.
• the wild type set of amplification primers preferentially hybridize with the wild type sequence and therefore are depleted from the reaction mixture at a higher rate than the point mutation set of primers.
• the point mutation primers are depleted from the reaction mixture at a higher rate than the wild type primers.
• the differential rate with which one primer set is used over the other serves as the basis for distinguishing the wild type sequence 5 from the sequence having a point mutation.
• PCR is known to amplify sequences to which the primers are not perfectly complementary. Accordingly, either set of the competitive primers could prime elongation of either the wild type or mutant sequence.
• LCR nucleic acid amplification reaction
• LCR has also been employed to detect point mutations. LCR is sometimes the preferred amplification reaction for detecting point mutations because LCR probes can be designed such that the point mutation occurs at the point of ligation. Accordingly, if an LCR probe set is designed to
• 2o LCR probes are subject to a phenomenon known as blunt end ligation where probes are spuriously ligated in the absence of a target sequence.
• blunt end ligation probes are spuriously ligated in the absence of a target sequence.
• no distinction could be made between a wild type and mutated sequence. While methods have been devised to avoid blunt end ligation, these methods may require multiple enzymes to
• the present invention provides a method for distinguishing a wild type sequence from a mutated version of the wild type sequence.
• the method is one for characterizing a nucleic acid sequence contained in a test sample or otherwise detecting a mutant nucleic 35 acid sequence in a test sample.
• the method comprises the steps of (a) forming a reaction mixture by contacting a test sample containing one of at least two possible target sequences (e.g.
• a mutant or wild-type sequence with a consensus primer set for amplifying either of the at least two possible nucleic acid sequences; b) subjecting the reaction mixture to amplification conditions such that copies of the target sequence are produced; c) exposing the target sequence copies to a probe sequence specific for either of the at least two target sequences; and d) detecting whether or not a copy sequence/probe hybrid was formed as an indication of the presence of a mutant nucleic acid sequence in the test sample.
• a kit for detecting a mutant nucleic acid comprising a consensus set of primers for amplifying at least two distinct target sequences, and a probe sequence that completely hybridizes with only one of the at least two distinct target sequences.
• the method disclosed herein provides a nucleic acid amplification based method of distinguishing a mutant nucleic acid sequence from a wild type nucleic acid sequence.
• the method provides a means for detecting mutations in a nucleic acid sequence. While the method can be employed to detect large mutations such as multiple base deletions, the method can discriminate point mutations from wild type nucleic acid sequences.
• the amplification reaction can be performed in a single reaction vial.
• the present invention generally comprises the steps of contacting a test sample suspected of containing either a wild type or mutant target nucleic acid sequence with ampiification reaction reagents comprising a set of amplification primers.
• the so-formed reaction mixture is then subjected to amplification conditions such that the amplification primers prime synthesis of copies of either the mutant or wild type target sequence, whichever happens to be in the test sample.
• the amplification primers are consensus sequences capable of amplifying both the mutant and wild type target sequence.
• a hybridization probe is then hybridized to a copy of the target sequence and this hybrid product can be detected. Depending upon whether the probe was specific for the wild type sequence or mutant sequence detection of a hybrid product is an indication of the presence of the wild type or mutant target sequence in the sample.
• the method can distinguish between a wild type or mutant sequence on the basis of whether or not the probe sequence hybridizes with the amplification product and therefore whether or not a 5 probe/amplification product is formed which can then be detected as an indication of the presence of the wild-type or mutant sequence in the test sample.
• test sample means anything suspected of containing a mutant or wild type target sequence.
• the test sample ⁇ o is or can be derived from any biological source ⁇ , such as, for example, blood, cells, ocular lens fluid, cerebral spinal fluid, milk, ascots fluid, synovial fluid, peritoneal fluid, amniotic fluid, tissue, fermentation broths, cell cultures and the like.
• the test sample can be used (i) directly as obtained from the source or (ii) following a pre-treatment
• test sample can be pre-treated prior to use by, for example, preparing serum or plasma from blood, disrupting cells or viral particles, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, 0 adding reagents, purifying nucleic acids, and the like.
• Wild-type and mutant sequences as used herein have their ordinary meanings. Thus, as mentioned above, a wild-type sequence is the most common or ordinary sequence, while the mutant sequence is one that diverges from the wild-type sequence. Such divergences from
• 25 or mutations of the wild-type sequence can be in the form of deletions, insertions, or substitutions in the range of from 1 base pair to 5 base pairs.
• mutant sequences having a single base pair mutation or "point mutation" are detected according to the method provided herein.
• target sequence as used herein means a nucleic acid sequence that is detected, amplified, or both amplified and detected using the method herein provided. Additionally, while the term target sequence is sometimes referred to as single stranded, those skilled in the art will recognize that the target sequence may actually be double
• primer sequences employed according to the present invention will amplify both strands of the target sequence.
• Primer sequences will generally comprise deoxyribonucleic acid (DNA), or ribonucleic acid (RNA) and are commonly used in the art for 5 purposes of priming extension of target sequence copies.
• a primer set will typically comprise two nucleic acid sequences one of which hybridizes to one strand of the target sequence and one of which hybridizes to other strand of the target sequence in such a way that the primers flank the target sequence.
• the primer ⁇ o sequences typically are consensus primers and will therefore amplify both the wild type sequence and a mutant version thereof.
• the primers are designed such that any anticipated mutation occurs downstream from the primer so that any mutant portion of the target sequence is copied through primer extension.
• Probe sequences will also generally comprise DNA or RNA and are typically less than 25 base pairs in length and more typically less than 20 base pairs in length. Additionally, probe sequences are typically at least ten base pairs in length.
• Probe sequences can be designed to hybridize with either a wild
• probes are designed to hybridize with the region of the target sequence this is (i) distinct from the primer sequences and (ii) spans the anticipated site of a mutation. Additionally, the anticipated mutation is located close to the center of the probe. Typically, the mutation is located within
• a probe of twenty base pairs can be designed such that the anticipated mutation occurs between base pairs four and seventeen, preferably between base pairs seven and fourteen, using base pairs ten and eleven as the centermost sequence of two base pairs.
• Primer and probe sequences can routinely be synthesized using a variety of techniques currently available. For example, a sequence of 5 DNA can be synthesized using conventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc. (Foster City, CA); DuPont, (Wilmington, DE); or Milligen, (Bedford, MA). Similarly, and when desirable, the sequences can be labeled using methodologies well known in the art such as described in U.S. Patent ⁇ o Applications Numbered 5,464,746; 5,424,414; and 4,948,882 all of which are herein incorporated by reference.
• label means a molecule or moiety having a property or characteristic which is capable of detection.
• a label can be directly detectable, as with, for example, radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles and the like; or a label may be indirectly
• directly detectable labels may require additional components such as, for example, substrates, triggering reagents, light, and the like to enable detection of the label.
• indirectly detectable labels they are typically used in combination with
• a conjugate is typically a specific binding member which has been attached or coupled to a directly detectable label.
• Coupling chemistries for synthesizing a conjugate are well known in the art and can include, for example, any chemical means and/or physical means that does not destroy the specific binding property of
• binding member means a member of a binding pair, i.e., two different molecules where one of the molecules through, for example, chemical or physical means specifically binds to the other molecule.
• binding pair i.e., two different molecules where one of the molecules through, for example, chemical or physical means specifically binds to the other molecule.
• 35 pairs other specific binding pairs include, but are not intended to be limited to, avidin or streptavidin and biotin; haptens and antibodies specific for haptens; complementary nucleotide sequences; enzyme cofactors or substrates and enzymes; and the like.
• primers and probes employed according to the method herein provided are preferably differentially with labels 5 having different purposes.
• probes can be labeled with one type of label and primers can be labeled with another type of label.
• one type of label can be employed to capture any hybrids on a solid phase reagent comprising a solid support material such as, for example, latex, plastic, glass, nyten, nitrocellulose, paper, ⁇ o or magnetic metal in any of the well known configurations such as, for example, a bead, microparticle or strip or coated or attached to a specific binding member.
• the detection label can be employed to indicate the presence of the hybrid on the solid support material.
• test sample As previously mentioned, the test sample, primer set, and test sample
• the probe (collectively the reaction mixture) are subjected to amplification conditions and "amplification conditions" are those conditions well known to those skilled in the art which promote the synthesis of at least one copy of the target sequence. Hence, thermal cycling of the reaction mixture is included in the term amplification
• PCR polymerase chain reaction
• the probe sequence can be hybridized to the target sequence copies and surprisingly according to the method provided herein, a point mutation in a wild type target sequence can be distinguished from the wild type sequence. For example, after
• wild-type probe a probe which would hybridize completely with the wild type sequence (wild-type probe) is exposed to the amplification product. Whether or not this probe binds to the amplification product is contingent upon whether the wild type sequence or mutant sequence was initially present in the test sample.
• the wild type sequence were present in the test sample it would have been amplified and the probe will bind to the amplified wild type sequence. Hence, detection of the probe/amplified wild type target sequence would indicate the presence of the wild type sequence and absence of a mutant sequence.
• the wild type probe 5 would not hybridize, no hybrid would form and therefore no hybrid could be detected. Hence, based upon a lack of detection, the sequence present in the test sample could be characterized as the mutant sequence.
• probe sequence could be-configured to hybridize ⁇ o perfectly with a mutant sequence ("mutant probe").
• mutant probe easily can be configured based upon known mutations and positions of such mutations in various genes of various organisms. In any event, detection of a hybrid between a mutant probe and the amplification product would indicate the presence of the mutant sequence, whereas,
• the amplification product can be contacted with two probes, one mutant probe and one wild-type probe.
• the two probes can be differentially labeled such so that upon 0 formation of a hybrid, a wild-type probe hybrid can be distinguished from a mutant probe hybrid.
• one probe can be labeled with a first binding member and the other probe can be labeled with a second binding member.
• the probes, and any hybrids formed with the probes can be separated on different solid phase 5 reagents having distinct specific binding members separately specific for the first and second binding members attached to the probes.
• solid phase reagent yields a signal
• probes can be differentially labeled with detection type labels and distinguished on this basis.
• U.S. Patent Application Serial No. 08/362,036 filed December 22, 1994 describes methods for
• the method can be applied to haploid organisms to distinguish a wild-type from a mutant sequence, the method can also be applied to polyploid organisms to distinguish between homozygous wild-type, heterozygous, and homozygous mutant sequences.
• an organism having a heterozygous allele can be distinguished from a homozygous allele by applying the method and detecting a signal, but a signal that is diminished as compared to a signal which would result from the homozygous allele. Whether or not a signal is diminished easily can be established by performing the ⁇ o method on a standard comprising sequences corresponding to the homozygous allele using probe sequences perfectly complementary with the standards. The signal derived from the standard then can be used as a standard baseline homozygous signal to which a potentially heterozygous signal can be compared. Similarly, either homozygous
• 15 variation can be established simply by performing the assay and detecting a signal in the range of the standard or no signal.
• the amplification reaction and probe hybridization reaction are performed in a single reaction vessel. Additionally, the primers and probes are selected such
• the probe sequence has a lower melt temperature than the primer sequences.
• the amplification primers, hybridization probe and test sample are placed under amplification conditions whereby copies of the target sequence (an amplicon) are produced at temperatures above the Tm of the probe(s).
• the amplicon is double
• primers are provided to amplify a target sequence and its complementary strand.
• the double stranded amplicon is then thermally denatured to produce single stranded amplicon members. After denaturation, the mixture is cooled, i.e., re-natured, to enable the formation of complexes between the probes and single stranded
• the rate of temperature reduction from the denaturation temperature down to a temperature at which the probes will bind to single stranded amplicons is preferably quite rapid (for example five seconds to 15
• SEQUENCE ID NO. 3 A portion of a representative sequence ⁇ o from exon 10 of wild type Leiden Factor V is designated herein as SEQUENCE ID NO. 1 , and a portion of the similar representative sequence from exon 10 of mutant Leiden Factor V is designated herein as SEQUENCE ID NO. 2.
• SEQUENCE ID NO. 2 differs from SEQUENCE ID NO. 1 at only one base, which is the point mutation which distinguishes
• SEQUENCE ID NO. 7 are specific for a region in exon 10 of both wild , type and mutant Leiden Factor V.
• SEQUENCE ID NO. 4 is specific for a region from base -66 to -87 in intron 10 of both wild type and mutant Leiden Factor V.
• SEQUENCE ID NO. 5 is specific for a region in exon 10 o of wild type Leiden Factor V only.
• SEQUENCE ID NO. 6 is specific for a region in mutant Leiden Factor V only.
• SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 7 are used as amplification primers specific for both wild type and mutant Leiden Factor V.
• SEQ ID NO. 5 is used as an internal 5 hybridization probe for the wild type Leiden Factor V amplification product.
• SEQ ID NO. 6 is used as an internal hybridization probe for the mutant Leiden Factor V amplification product.
• A. wild type and mutant Leiden Factor V Primers were designed to detect the exon 10/intron 10 target sequence of both wild type and mutant Leiden Factor V by oligonucleotide hybridization PCR. 5 These primers were SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 7. SEQ ID NO. 3 and SEQ ID NO. 7 are specific for a region in exon 10 of both wild type and mutant Leiden Factor V. SEQ ID NO. 4 is specific for a region in intron 10 of both wild type and mutant Leiden Factor V.
• Primer sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with adamantane at their 5' ends using standard cyanoethyl phosphoramidite coupling chemistry as described in U.S. Patent No. 5,424,414 incorporated herein by reference.
• Probes were designed to hybridize with the amplified exon 10/intron -40 target sequence of wild type or mutant Leiden Factor V by oligonucleotide hybridization. These probes were SEQ ID NO. 5 for wild type Leiden Factor V and SEQ ID NO. 6 for mutant Leiden Factor V. Probe sequences were synthesized using standard oligonucleotide synthesis methodology and haptenated with either 2 carbazoles at the 3' end, or a biotin, followed by 6 thymines and another biotin at the 3' end using standard cyanoethyl phosphoramidite coupling chemistry as described in U.S. Patent No. . 5,464,746 (herein incorporated by reference).
• Wild type Leiden Factor V DNA was purified from whole blood or lymphocytes separated from whole blood, using the QIAgen nucleic acid extraction procedure and column methodology as described by the manufacturer (QIAgen, Inc., Chatsworth, CA). Mutant Leiden Factor V purified DNA was obtained from Beth Israel Deaconess Medical Center, Boston, MA, where it was purified from whole blood by phenol/chloroform extraction. Purified DNA was quantitated by taking the absorbance reading at 260 nm using a spectrophotometer and diluted to the range of 1000 to 0.05 ng/test.
• Dilutions of the purified wild type or mutant Leiden Factor V DNA were PCR amplified and detected using SEQ ID NO. 3 and SEQ ID NO. 4 primers with the corresponding probe, either the SEQ ID NO. 5 (wild type) biotin-labeled probe or the SEQ ID NO. 6 (mutant) biotin-labeled probe, in separate reactions.
• PCR was performed using final concentrations of 50 mM Bicine (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.25, 150 mM potassium acetate, 8% w/v glycerol, 0.001% bovine serum albumin (BSA), 0.1 mM EDTA and 0.02% sodium azide.
• Thermus thermophilus polymerase was used at a concentration of 5 units/reaction, with dNTPs (dATP, dGTP, dTTP and dCTP) present at a final concentration of 150 ⁇ M each. Primers were used at a concentration of 250 nM each, and probes at a concentration of 5 nM each. A final concentration of 2.5 mM -Manganese acetate was ⁇ o used in a total reaction volume of 0.2 ml, with sample volume of 25 ⁇ l. Two negative controls were tested and consisted of either water or 1000 ng of the opposite purified DNA, i.e. purified mutant DNA was a negative control when tested using the wild type probe and vice versa. Reaction mixtures were amplified in an LCx® Thermal Cycler.
• reaction mixtures were first incubated at 97°C for 4 minutes, followed by 40 cycles of PCR amplification at 94°C for 60 seconds then 55°C for 90 seconds. After the reaction mixtures were thermal cycled, the mixtures were maintained at 97°C for 5 minutes and probe oligo hybridization was accomplished by lowering the temperature to
• strepavidin coated microparticles (Bangs Laboratories, Inc., Fisher, IN) and an anti-adamantane antibody/alkaline phosphatase conjugate (available from Abbott Laboratories, Abbott Park, IL) were used in conjunction with the LCx® to capture and detect the reaction products.
• the enzyme substrate used was methyl-umbelliferyl phosphate (MUP),
• the wild type probe detected both homozygous wild type and heterozygous mutant Leiden Factor V samples but did not detect homozygous mutant Leiden Factor V samples as- positive.
• the mutant probe detected both homozygous mutant and heterozygous mutant Leiden Factor V samples but did not detect homozygous wild type Leiden Factor V samples as positive.
• both probes detected the mutant heterozygous samples since they contain one wild type and one mutant allele, and these probes did so in a dose dependent manner. Thus, both probes showed excellent specificity.
• the wild type probe detected both homozygous wild type and heterozygous mutant Leiden Factor V samples as positive.
• the mutant probe detected the heterozygous mutant Leiden Factor V samples but did not detect homozygous wild type Leiden Factor V samples as positive.
• both probes detected the heterozygous mutant samples since they contain one wild type and one mutant allele, and did so in a dose dependent manner. Thus, both probes were shown to be efficacious when labeled with carbazole or biotin.
• MOLECULE TYPE genomic DNA (mutant)

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