EP0975804A2

EP0975804A2 - Amplification based mutation detection

Info

Publication number: EP0975804A2
Application number: EP98915634A
Authority: EP
Inventors: Cynthia Jou; Ronald L. Marshall; Christi P. Scheffel
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1997-04-18
Filing date: 1998-04-17
Publication date: 2000-02-02
Also published as: JP2001522243A; WO1998048052A2; CA2287506A1; WO1998048052A3

Abstract

Provided herein is an amplification based method for detecting mutant nucleic acid sequences.

Description

AMPLIFICATION BASED MUTATION DETECTION

This application is a continuation-in-part application of U.S. Patent Application Serial No. 08/514,704, filed August 14, 1995, the full text of which is herein incorporated by reference.

Field of the Invention

The present invention relates to nucleic acid mutations and, in particular, relates to a method of detecting such mutations.

Background of the Invention

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, for example, 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. Similarly, larger mutations, on the order of several base pair distinctions from the wild type sequence, can also give rise to genetic disorders. Cystic fibrosis, sickle cell anemia, Tay Sachs disease and thalassemias are examples of genetic disorders that may arise from mutations in a particular gene.

A variety of methods exist which can be employed to detect a mutation in a nucleic acid sequence. In particular, 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. According to this method, 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. Hence, if the wild type sequence is present, 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. Conversely, in the event the sequence contains the point mutation, 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. Unfortunately, however, 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. ι o Another well known 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

15 hybridize perfectly with the wild type sequence but contains a mismatch at the point of ligation when hybridized with a sequence containing a point mutation, ligation will not occur. Thus, the distinction between ligated versus unligated probes can be exploited to determine if a wild type or mutant sequence is present. However,

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. As a result, 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

25 achieve amplification.

Thus, there is a need for an amplification based method for effectively discriminating between a wild type and mutated sequence which employs a minimum number of reagents.

so Summary of the Invention

The present invention provides a method for distinguishing a wild type sequence from a mutated version of the wild type sequence.

Hence, 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. Also provided is 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.

Detailed Description of the I nvention

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. Hence, 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. Advantageously, 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. Hence, 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. Accordingly, 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.

The term "test sample" as used herein, means anything suspected of containing a mutant or wild type target sequence. The test sample ι 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

15 to modify the character of the sample. Thus, the 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. Preferably, mutant sequences having a single base pair mutation or "point mutation" are detected according to the method provided herein.

30 A "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

35 stranded. Thus, in cases where the target is double stranded, 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. Additionally, the primer ι o sequences typically are consensus primers and will therefore amplify both the wild type sequence and a mutant version thereof. Preferably, 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.

15 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

20 type sequence or a mutant sequence. In either case, 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

25 five base pairs on either side of the centermost base pair or centermost sequence of two base pairs of the probe, and more preferably within two base pairs from the centermost base pair or centermost sequence of two base pairs of the probe, depending upon whether the probe contains an even or odd number of bases. Thus, in

30 the case of a point mutation for example, 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. In the case of a probe having an odd number of base

35 pairs, for example nineteen base pairs, the single base pair in the center would be located at position ten, and a mutation typically would be located between base pairs four and sixteen, and is preferably located between base pairs seven and thirteen.

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 ι o Applications Numbered 5,464,746; 5,424,414; and 4,948,882 all of which are herein incorporated by reference.

The primer and probe sequences employed according to the present method can be labeled to assist in detecting any hybrid sequences formed between copies of the target sequence and the probe

15 sequence. The term "label" as used herein 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

20 detectable, as with, for example, specific binding members. It will be understood that directly detectable labels may require additional components such as, for example, substrates, triggering reagents, light, and the like to enable detection of the label. When indirectly detectable labels are used, they are typically used in combination with

25 a "conjugate". 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

30 the specific binding member or the detectable property of the label.

As used herein, "specific 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. In addition to antigen and antibody specific binding

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.

When labeled, primers and probes employed according to the method herein provided are preferably differentially with labels 5 having different purposes. For example, probes can be labeled with one type of label and primers can be labeled with another type of label. Thus, for example, 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, ι 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.

As previously mentioned, the test sample, primer set, and

15 optionally 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

20 conditions and is a well known procedure for generating multiple copies of a target sequence. Additionally, methods for amplifying a nucleic acid sequence that employ thermal cycling are well known and include the polymerase chain reaction (PCR) which has been described in U.S. Patents 4,683, 195 and 4,683,202 herein incorporated by

25 reference.

After amplification, 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

30 amplification of the target sequence, 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.

35 In particular, if 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. On the other hand, if a mutant sequence was present in the test sample, 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.

Conversely, the probe sequence could be-configured to hybridize ι o perfectly with a mutant sequence ("mutant probe"). Such probes 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,

15 the absence of a hybrid and therefore no detectable signal, would indicate the presence of the wild type sequence in the test sample._.

According to another embodiment, 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. For example, one probe can be labeled with a first binding member and the other probe can be labeled with a second binding member. In this manner, 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. Hence, depending upon which solid phase reagent yields a signal, by virtue of a label associated with the primer sequences, it can be determined which probe formed a hybrid with the amplification

30 product and thereby indicate whether the wild-type or mutant sequence was present in the test sample. Alternatively, the probes can be differentially labeled with detection type labels and distinguished on this basis. For example, U.S. Patent Application Serial No. 08/362,036 filed December 22, 1994, describes methods for

35 distinguishing two different types of signal generating groups. While 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. In 5 particular, 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 ι 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.

In accordance with another embodiment, the amplification reaction and probe hybridization reaction are performed in a single reaction vessel. Additionally, the primers and probes are selected such

20 that 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). In the usual case, the amplicon is double

25 stranded because 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

30 amplicon members, in the event the probes are perfectly or completely hybridizable with the single stranded members of the amplicon. 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

35 minutes, preferably 1 minute to three minutes) and particularly through the temperature range in which the polymerase enzyme is active for primer extension.

Examples

5 The following examples demonstrate detection of wild type and mutant Leiden Factor V using the DNA oligomer primers and probes herein provided. These DNA primers and probes are identified as SEQUENCE ID NO. 3, SEQUENCE ID NO. 4, SEQUENCE ID NO. 5, SEQUENCE ID NO. 6 and SEQUENCE ID NO. 7. A portion of a representative sequence ι 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

1 5 mutant Leiden Factor V from wild type. SEQUENCE ID NO. 3 and

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.

In the following examples, 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.

Example 1 0 Preparation of Leiden Factor V Primers and Probes

A. wild type and mutant Leiden Factor V Primers 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.

B. wild type and mutant Leiden Factor V Probes 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).

Example 2 Sensitivity of Leiden Factor V Detection

Purified wild type and mutant Leiden Factor V genomic DNA was used to assess the sensitivity of the wild type and mutant Leiden Factor V primer/probe sets. 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. 5 Recombinant 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 μM 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 ι o used in a total reaction volume of 0.2 ml, with sample volume of 25 μl. 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.

15 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

20 15°C within 2 minutes. Samples were held at 15°C for a minimum of 5 minutes, and thereafter until reaction products were analyzed and detected.

Reaction products were detected on the Abbott LCx® system (available from Abbott Laboratories, Abbott Park, IL). A suspension of

25 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),

30 with the rate of conversion of MUP to MU measured and reported as counts/second/second (c/s/s).

Data from this experiment is presented in TABLE 1 and shows detection of at least 50 pg of wild type Leiden Factor V purified DNA using primers with the wild type probe, and detection of at least 50 pg

35 of mutant Leiden Factor V purified DNA using the same primers but with the mutant probe. Fifty pg of DNA is equivalent to 6.25 molecules DNA and thus both the wild type and mutant assays show detection of at least 6 molecules of DNA. Additionally, both probes show excellent specificity, in that each probe only detects the type of DNA it is specific for and neither probe type detects the opposite type of DNA (present as the non-specific DNA negative control).

TABLE 1

A. Specificity using biotin labeled probes Specificity of wild type and mutant probes was assessed further using samples with a wild type or mutant homozygous genotype and mutant heterozygous samples. The purified wild type and mutant homozygous DNA used was that DNA prepared in Example 1. Mutant heterozygous 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), and quantitated by taking the absorbance reading at 260 nm using a spectrophotometer. The genotype of the samples was confirmed using the standard methodology of PCR amplification followed by restriction enzyme digestion and gel electrophoresis or Southern blot analysis. Mutant heterozygous and wild type and mutant homozygous purified DNA were then diluted to various levels and PCR amplified, hybridized and detected using the SEQ ID NO. 3 and 4 primers, SEQ ID NO. 5 and 6 biotin-labeled probes and methodology described in Example 1. All samples were tested in duplicate. The average of the results from this experiment are given in Table 2.

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. As expected, 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.

TABLE 2

(ND = Not Determined)

The above experiment was repeated except the SEQ ID NO. 7 primer was used instead of the SEQ ID NO. 3 primer. The results, summarized in Table 3, support the same conclusions as above, showing the excellent specificity of the wild type and mutant probes. Additionally, both primer sets (SEQ ID NO. 3 and 4 or SEQ ID NO. 7 and 4) allow amplification of both wild type and mutant Leiden Factor V for hybridization by the specific probe.

Table 3

(ND = Not Determined)

B. Specificity using carbazole-labeled probes Specificity of wild type and mutant probes was tested using carbazole as the label instead of biotin used in previous experiments. The samples tested were prepared as in Example 2. Heterozygous and wild type homozygous purified DNA were diluted to various levels and PCR amplified, hybridized and detected using the SEQ ID NO. 3 and 4 primers and SEQ ID NO. 5 and 6 carbazole-labeled probes described in Example 1 , and methodology as in Example 2 except that reaction products were detected on the Abbott LCx® system using a suspension of anti- carbazole coated microparticles (available from Abbott Laboratories, Abbott Park, IL). All samples were tested in duplicate. The average of the results from this experiment are given in Table 4. TABLE 4

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. Again, 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.

While the invention has been described in detail and with reference to specific embodiments, it will be apparent to one skilled in the art that various changes and modifications may be made to such embodiments without departing from the spirit and scope of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: C. Jou

C. Scheffel

R. Marshall

(ii) TITLE OF INVENTION: AMPLIFICATION BASED MUTATION DETECTION (iii) NUMBER OF SEQUENCES: 12 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Abbott Laboratories

(B) STREET: 100 Abbott Park Road

(C) CITY: Abbott Park

(D) STATE: Illinois (E) COUNTRY: USA

(F) ZIP: 60064-3500

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Macintosh

(C) OPERATING SYSTEM: System 7.0.1

(D) SOFTWARE: Microsoft Word 5.1a

(Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Paul D. Yasger

(B) REGISTRATION NUMBER: 37,477

(C) DOCKET NUMBER:

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 847/937-2341

(B) TELEFAX: 847/938-2623

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: geno ic DNA (wild type)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

CTTAGAGTTT GATGAACCCA CAGAAAATGA TGCCCAGTGC TTAACAAGAC 50 CATACTACAG TGACGTGGAC ATCATGAGAG ACATCGCCTC TGGGCTAATA 100

GGACTACTTC TAATCTGTAA GAGCAGATCC CTGGACAGGC GAGGAATACA 150

G 151

(2) INFORMATION FOR SEQ ID NO : 2 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 151 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA (mutant)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTTAGAGTTT GATGAACCCA CAGAAAATGA TGCCCAGTGC TTAACAAGAC 50

CATACTACAG TGACGTGGAC ATCATGAGAG ACATCGCCTC TGGGCTAATA 100

GGACTACTTC TAATCTGTAA GAGCAGATCC CTGGACAGGC AAGGAATACA 150

G 151 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ACCCACAGAA AATGATGCCC AG 22

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

TGCCCCATTA TTTAGCCAGG AG 22

(2) INFORMATION FOR SEQ ID NO : 5 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

GACAGGCGAG GAA 13

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : GACAGGCAAG GAA 13

(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

GGACTACTTC TAATCTGTAA GAGC 24

Claims

C l a i m sWhat is claimed is:

1. A method for detecting a mutant nucleic acid sequence in a test sample comprising the steps of: a) forming a reaction mixture by contacting a test

5 sample suspected of containing one of at least two possible target sequences with a consensus primer set for amplifying said at least two possible nucleic acid sequences; b) subjecting said reaction mixture to amplification conditions such that copies of said target sequence are produced; ╬╣ o c) exposing said target sequence copies to a probe specific for one of said 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 said test sample.

2. The method of claim 1 wherein steps a, b and c take place in a single reaction vessel.

3. The method of claim 2 wherein the melt temperature of said probe is less than the melt temperature of the members of the primer set.

4. The method of claim 1 wherein said mutant nucleic acid sequence is a nucleic acid sequence containing a point mutation.

5. The method of claim 1 wherein said method further comprises exposing said target sequence copies to a second probe sequence wherein i ) said probe sequences are differentially 5 labeled, i i ) one probe completely hybridizes with said mutant sequence, and i i i ) the other probe completely hybridizes with a wild-type sequence.

6. The method of claim 1 wherein the test sample is taken from a polyploid organism and the method distinguishes a homozygous allele from a heterozygous allele.

7. A kit for detecting a mutant nucleic acid comprising a) a consensus set of primers for amplifying at least two distinct target sequences; and b) a probe sequence that completely hybridizes with one of said at least two distinct target sequences.

8. The kit of claim 7 wherein said at least two distinct target sequences comprise a wild-type sequence and a mutant sequence and said kit further comprises a second probe sequence wherein a) one probe completely hybridizes with said mutant sequence and b) the other probe completely hybridizes with said wild-type sequence.