WO1995011990A1

WO1995011990A1 - Assay for the detection of genetic abnormalities

Info

Publication number: WO1995011990A1
Application number: PCT/EP1994/003538
Authority: WO
Inventors: Giorgio Martinazzo; Roberta Bichi; Stanislavo Marcolini; Elisabetta Turchetti; Angela Pelliccia
Original assignee: Raggio-Italgene S.P.A.
Priority date: 1993-10-29
Filing date: 1994-10-27
Publication date: 1995-05-04
Also published as: EP0725834A1; AU7992494A

Abstract

A process for detecting in an analyte target sequences respectively excluding (wild-type) or including a genetic mutation, comprises the steps of: reacting the analyte under hybridising conditions with (i) first and second oligonucleotide probes and (ii) first and third oligonucleotide probes, the probes being respectively complementary to first, second and third regions of the target sequences, wherein the first region is on one side of the mutation, the second and third probes each span the mutation site and are respectively complementary to the wild-type and mutated regions, and wherein the first probe is separable or detectable and the second and third probes are respectively separable or detectable; and, if necessary, amplifying either or each target sequence; and separating and detecting the resultant hybrids that carry two probes.

Description

ASSAY FOR THE DETECTION OF GENETIC ABNORMALITIES Field of the Invention

This invention relates to a process for detecting the nucleic acid sequences that occur in chromosomal or genetic abnormalities.

Background of the Invention

Genetic abnormalities are the cause of various undesirable conditions in humans, both inherited and non- inherited, including neoplastic conditions. It is clearly of vital importance to detect such abnormalities, whether caused by chromosomal translocation, transposition, intergene or intragene recombination, insertion, deletion or point mutation, at an early stage, by a simple and reliable test. Genetic aberrations include those that have now been characterised in chromosome 7. In particular, this chromosome is associated with cystic fibrosis (CF) , an autosomal recessive disease. Only people carrying the mutation(s) on both the alleles (homozygous) are affected with CF, whereas people carrying the mutation on a single allele (heterozygous) are not affected. To date, about 67% of CF chromosomes in cystic fibrosis patients carry the ΔF 508 mutation, which is a 3 base-pair deletion in exon 10 of the CFTR gene located on chromosome 7, resulting in the loss of a single phenylalanine residue in the expressed protein. About 46% of all the CF patients are homozygous for the ΔF 508 deletion, while another 43% are heterozygous for this deletion and other mutations. Unfortunately, there are many other mutations which cover the remaining 33%; of these mutations, the most common comprise no more than 2 to 3% of CF mutations and are mostly single-point mutations.

In recent years, many methods for identifying nucleic acid sequences have been developed. They are generally solid-phase methods that are time-consuming, difficult to quantitate and not easily adaptable for clinical and diagnostic laboratories. If radioactive labelling is avoided, for ease of operation, it is at the expense of the sensitivity of the method. This drawback can be overcome by amplifying the sequence to be detected, e.g. using the polymerase chain reaction (PCR) as disclosed in EP-A- 0200362 and EP-A-0258017.

In order to detect whether a mutation is present or not, Allele Specific Oligonucleotides (ASO) have been used.

Its suitability to detect even a single point mutation in membrane hybridisation is widely described in the literature.

GB-A-2169403 describes a method for the identification of nucleic acids, in which two independently-labelled oligonucleotide probes are reacted in a single solution, under hybridising conditions, with a target analyte. If the analyte contains a sequence that hybridises to both probes, this may readily be detected by virtue of the fact that one label allows separation of the hybrid and the other its detection. The same or similar techniques are described in, for example, EP-A-0128332, EP-A-0145356, EP- A-0159719, EP-A-0177191, EP-A-0192168 and EP-A-0198662. Oligonucleotide probes and their use in detecting chromosomal abnormalities are described in, for example, US-A-4701409, US-A-5015568, US-A-5024934, EP-A-0181635 and EP-A-0252685. O-A-9219775 (Applicant: Raggio-Italgene SpA) describes a process of the general type described in GB-A- 2169403, i.e. using homogeneous hybridisation, but is applied to the detection of chromosomal abnormalities, e.g. translocations, using capture and reporter probes that are respectively complementary to different regions of the target sequence, e.g. on opposite sides of the translocation. That invention is based on the principle that two oligonucleotide probes, appropriately designed, complementary respectively either to the normal or to the mutated sequence, will anneal only onto the homologous target and not onto the other one. Summary of the Invention

The present invention is a process for detecting in an analyte target sequences respectively excluding (wild-type) or including a genetic mutation, comprising the steps of: reacting the analyte under hybridising conditions with (i) first and second oligonucleotide probes and (ii) first and third oligonucleotide probes, the probes being respectively complementary to first, second and third regions of the target sequences, wherein the first region is on one side of the mutation, the second and third probes each span the mutation site and are respectively complementary to the wild-type and mutated regions, and wherein the first probe is separable or detectable and the second and third probes are respectively detectable or separable; and, if necessary, amplifying either or each target sequence; and separating and detecting the resultant hybrids that carry two probes.

Given the importance of assaying for genetic abnormalities, the present invention provides a number of valuable characteristics. Firstly, for example, it is simple to use, e.g. by relatively unskilled personnel in hospitals and less specialised laboratories; it is quick, non-radioactive and requires only simple equipment. Secondly, the absorbance readings allow a quantitative measurement of the final signal. With other methods, such as gel electrophoresis/Southern blotting, or dot-blotting, this quantitative determination of signal is only possible with the use of sophisticated instrumentation. These traditional methods are much more prone to subjective interpretation. The quantitation of the signal allows much easier comparison of results between experiments, and between laboratories, and allows better QC of reagents and procedures. Thirdly, the system will only generate a signal if both reporter and capture probes (complementary to or at least including sequences on either side of the mutation or breakpoint) bind. This provides a very high degree of specificity and helps minimise the risk of false positives. The use of two probes, internally "nested" with respect to primers used for amplification by PCR, also reduces the risk of obtaining false positives due to the detection of PCR artefacts such as truncated elongations, primer concatenamers and other problems related to the specificity of the PCR reaction, as well as to the known imprecision of the Taq I Polymerase enzyme. Description of the Invention

The present invention is particularly useful for the detection of nucleic acid sequences comprising point mutations.

The nucleic acids in the analyte preferably comprise double-stranded DNA. They may be amplified by the action of DNA polymerase which is capable of synthesising in the

5'-3' direction a complementary strand from a template, in the presence of a primer which is complementary to an extreme portion of the single-stranded analyte sequence. Preferably, amplification occurs for both strands of the analyte sequence, and the DNA polymerase is heat-stable.

It is important that the primers be of such length and composition as not to allow hybridisation to occur with themselves or with portions of the analyte DNA segment which is complementary to the other primer. Accordingly, the extension products are synthesised employing a DNA polymerase, which is preferably heat-stable, and extends the terminal portion to the 3' position of each primer.

The extension products are then separated from their templates by means of high temperature denaturation (92-

94°C) . The passage is repeated through a number of cycles sufficient to increase the amount of the target sequence up to the concentration at which it can be detected.

Due to high number of mutations involved in cystic fibrosis at least, it would be extremely useful to amplify different fragments of the CFTR gene which are known to include the majority of the most frequent mutations. This can be accomplished by the use of as many couples of specific PCR primers as the number of DNA fragments to be amplified. The primers composition must fulfill the above specifications. This procedure is known to those skilled in the art as "multiplex PCR".

When the amplification cycles are completed, a suitable amount of the analyte sequence is caused to react with a suitable concentration of NaOH, e.g. 0.08N NaOH, so as to cause denaturation of the double-stranded segment. Alternatively, denaturation can be carried out through exposure of the DNA to a temperature of 90-97°C, e.g. 94-97°C for 5-10 minutes, and then cooling suddenly down to 0°c

Once denaturation is completed, a second pair of oligonucleotides is employed. These are probes which are different from the primers employed in the amplification procedure and which are both complementary to the same strand of the analyte DNA, in zones which are the same as or different to those employed for amplification. The probes are then contacted with the reaction mixture at an excess concentration with respect to the analyte sequence. The second pair of probes comprises a capture oligonucleotide and a reporter (or detectable) oligonucleotide. Each probe can be conjugated through its 5' end with a reactive group, to provide an appropriate label or capture means.

Preferably, the capture probe is conjugated to, a hapten such as fluorescein isothiocyanate (FITC) . Then, separation is by means of anti-hapten antibodies, e.g. anti-FITC, which are immobilised on a solid phase such as plastics beads, microplates, coated tubes, latex or, preferably, agnetisable microparticles which are attracted onto magnetic plates. After capture, the liquid phase containing free detection probes may be removed by washing. The reporter molecule may be conjugated to a reporter molecule. Reporter molecules include haptens, enzymes and radioactive labels, or include any substrate that provides a chromogenic, fluorescent or chemiluminescent signal. By way of example, e.g. the reporter probe is conjugated to biotin which may be detected by means of avidin conjugated to an enzyme. The reporter probe may be conjugated to an enzyme or other material whose presence can then be detected by reaction therewith. For an enzyme, detection is conducted by means of: incubation with a substrate which is specific for the enzyme, e.g. fluorescent, chemiluminescent or, preferably, chromogenic; termination of the reaction, e.g. by adding a stop solution; and colorimetric or other appropriate reading of the solution itself. By way of illustration, the enzyme is an alkaline phosphatase, the specific chromogenic substrate is phenolphthalein monophosphate, and the colorimetric reading is carried out at a wavelength of 550-554 nm.

Next, or at the same time as the addition of the probes, a neutralising solution, e.g. 0.5 M Tris, pH 7.5, is added to the reaction mixture, in such an amount as to buffer the NaOH and allow the hybridisation of the probes to the analyte DNA to occur.

After a suitable incubation period at a constant temperature, e.g. 30 minutes at +37°C, an excess amount of a solid phase consisting of magnetisable microparticles coated with an anti-FITC antibody which is capable of binding the whole amount of the FITC-labelled separator probe, both the free and that reacted with the DNA sequence, is added to the reaction mixture, so forming the analyte sequence-probes complex. After a suitable incubation period at a constant temperature, e.g. 10 minutes at +37°C, the reaction tubes are put on a magnetic plate which, in a short time, e.g. 3 minutes, causes the magnetisable particles to settle onto the bottom of the tube itself. The supernatant is then removed by decantation, by turning the magnetic plate upside down, the magnetised particles adhering to the bottom of the tube. The tubes are then removed from the magnetic plate and the solid phase is resuspended in a suitable washing solution, e.g. 0.5 ml of 0.075M Tris-buffered saline, pH = 7.5, allowed to settle and decanted again. The washing cycle is repeated as often as is necessary to remove any non-specific binding of the reagents, and in particular of the reporter probe which is conjugated to the enzyme, with the solid phase. During decantation and washing, all those reactants which are not specifically linked to the magnetic particles are removed from the reaction tube.

Next, a suitable amount of a chromogenic substrate which is enzyme-specific, e.g. 200 βl of phenolphthalein monophosphate, is added to the magnetic particles and allowed to react for the time required at a constant temperature, e.g. 1 hour at 37°C. After this period, the reaction is stopped by adding a stop solution, e.g. 750 μl of a Na₂C0₃ solution, pH 12.

The addition of the stop solution causes the formation and the stabilisation of colour, the absorbance value of which is measured at a suitable wavelength, for instance

554 nm, on a colorimeter. A colour development which is significantly higher than that of blank samples indicates that, during amplification, some extension products were formed starting from the specific primers and from the analyte DNA sequence that has acted as a template. In the absence of the analyte sequence, no formation of specific extension products would have occurred, which products are the only compounds capable of acting as bridges between the magnetic particles and the reporter probe that bears the enzyme capable of generating the signal.

The method for conjugating a reactive group to the oligonucleotide probes obviously depends on the group type that is to be employed; generally the preferred bond occurs through the OH group in the 5' and/or 3' position of the oligonucleotide. During automated synthesis of the oligonucleotide, employing phosphoroa idite chemistry, it is possible to introduce an aliphatic amine at the 5' end employing the Aminolink 2 (ABI) reactant or the Aminomodifier II (Clontech) reactant or to the 3' end using appropriately-derivatised 3'-CPG columns; these one or two amino groups can be reacted successively with a specific hapten, for instance FITC or a biotin-hydroxy-succinimide ester, or any other group containing an ester which is activated and capable of reacting with a primary amine.

For conjugation with the enzyme, it is generally preferred to use heterobifunctional reactants such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) and 2-iminothiolane (2-IT) , available from Pierce. For instance, SMCC is capable of reacting with the primary amine in the 5' position of the reporter probe give a derivative having a maleimido group free; the 2-IT is capable of reacting with the NH₂ groups of lysines of the alkaline phosphatase so as to give a derivative having a free -SH group. The maleimido groups and the -SH group, if caused to react under suitable conditions, react spontaneously so as to form a very stable carbon-sulphur covalent bond. In this way, it is possible to obtain conjugates in which the reporter probe is linked through its 5' end to the alkaline phosphatase through a long and flexible carbon atom chain, keeping the oligonucleotide capability of specifically hybridising with a complementary sequence unaltered, and keeping also unaltered the capability of the enzyme to interact with its specific substrate, to generate a coloured solution.

Magnetisable particles coated with anti-FITC antibodies are commercially available (from Bangs Laboratories Inc., Advanced Magnetics) or they can be prepared by well-known procedures. Specific substrates for the phosphatase and stop solutions are also commercially available (from Sigma) .

The extension products can be generated by the exposure of the primers, hybridised to their templates, to a DNA polymerase which is preferably heat-stable, e.g. the Taq polymerase disclosed in EP-A-0258017. The DNA polymerase will replicate the sequence of the template, so synthesising some fresh DNA from the primers in the 5'-3' direction. A heat-stable polymerase is preferred, but it is not indispensable because the simplest way of denaturing the double-stranded extension product is by exposure to high temperatures (about 95°C) during the cycles of the PCR, as disclosed in US-A-4683202. By employing different procedures for denaturating the extension products, other polymerases can be used, including the Klenow fragment. As indicated in the "Background of the Invention", above, cystic fibrosis (CF) is associated with point mutations and/or deletions; a technique according to the invention for its ready detection will now be described, as an illustrative embodiment of the invention. DNA is first extracted from a suitable source, such as peripheral blood, dried blood spots, mouth-brush washes, etc. The DNA sequence carrying the (eventual) mutation is amplified by means of PCR using appropriate primers: CF homozygous patients will yield only mutated DNA fragments; CF carriers, heterozygous, will yield both mutated and normal fragments; unaffected patients will yield only normal DNA fragments. In a multiplex system, the DNA sequence(s) carrying the (eventual) mutation(s) are amplified by means of multiplex PCR using four appropriate pairs of primers which can amplify, by reaction in a single tube, four CTFR gene exons, namely exon 10, where at least 5 important mutations can be detected, exon 11 where at least 6 mutations can be detected, exon 20 where at least 2 mutations can be detected, and exon 21 where at least 1 mutation can be detected.

Oligonucleotide probes, nested with respect to the primers used for the amplification and capable of annealing onto the same single-stranded DNA fragment are prepared as follows: (1) a reporter probe complementary to a conserved region of the amplified fragment, e.g. conjugated to the enzyme alkaline phosphatase, acts as a tracer; it will hybridise to a sequence common to both the normal and the mutated DNA fragment;

(2) a capture probe spanning the mutation site and complementary to the wild-type sequence is conjugated with, say, FITC; under appropriate hybridisation conditions, this capture probe will only anneal to the normal amplified DNA fragment;

(3) another capture probe spanning the mutation site and complementary to the mutated sequence is conjugated with, say, FITC; under appropriate hybridisation conditions, it will only anneal to the mutated amplified fragment. After PCR amplification, a patient sample is diluted (if necessary) , denatured and reacted in turn, under hybridising conditions, with a set of detection probes that contain either reagents 1 and 2 (Normal probes) or reagents 1 and 3 (Mutated probes) , for each of the reactions to be assayed.

Following the usual procedure of magnetic separation, washing steps and substrate reaction, a colour signal will be generated; the interpretation of the results is as follows:

(A) sample positive with the normal probe negative with the mutated probe result: X-mutation unaffected patient (B) sample positive with the normal probe positive with the mutated probe result: X-mutation heterozygous patient

(C) sample negative with the normal probe positive with the mutated probe result: X-mutation homozygous patient, CF- affected Merging the results for all the mutations, a further result can be as follows:

(D) sample heterozygous for two different mutations result: compound homozygous patient, CF-affected.

A schematic representation of the principle of the assay procedure is shown in the accompanying drawing. The abbreviations that are used there are: Chr. 7, chromosome 7; ΔF 508, site of the deletion; probe N, probe complementary to the normal sequence; probe D, probe complementary to the deleted sequence; FITC, fluorescein isothiocyanate; Ab, antibody; ALP, alkaline phosphatase; PMP, phenolphtalein monophosphate; P, phenolphtalein; A_₅₅₀, absorbance at 550 nm.

The assay protocol can be summarised as follows:

1. DNA extraction 2. PCR amplification

3. Sample denaturation

4. Sample hybridisation with either normal or mutated probes

5. Solid phase separation 6. Signal generation

More specifically, the following deletion method may be used: to perform the test, a magnetic separator is required, which is constituted by a tube rack that can be slid into a magnetic plate to allow magnetic sedimentation of the paramagnetic beads.

Each crude PCR mixture is diluted 1:4 with the sample diluent to perform the detection procedure. Then for each sample and the relevant controls the following reagents are pipetted into polystyrene reaction tubes in duplicate: 20 μl of diluted sample

20 μl of denaturing solution

Shake the tubes i Incubate at 37°C for 5 min

100 μl of relevant detection probes sol. (either normal or mutated) Shake the tubes I Incubate at 37°C for 15 min

100 μl of separation reagent

Shake the tubes I Incubate at 37°C for 10 min

Slide the rack into the separator Allowmagnetic sedimentation for 4 min

Decant the supernatant by inverting the separator 500 μl of wash solution Shake the tubes 1 Slide the rack into the separator

Allowmagnetic sedimentation for 3 min

Decant the supernatant by inverting the separator Repeat the washing step other two times (three washing steps in total)

200 μl of substrate solution

Shake the tubes 1 Incubate at 37°C for 30 min 750 μl of stop solution i Slide the rack into the separator

Allow magnetic sedimentation for 5 min

Read the absorbances at 550 n with a spectrophotometer.

The following Examples illustrate the present invention. Oligonucleotides (SEQ. ID. Nos. 1-24) were synthesised with an ABI PCT Mate 391 synthesiser and purified by HPLC with a Waters 625 Baseline instrument. FITC and alkaline phosphatase conjugates were prepared and purified as described above. All sequences except primers are NH₂-terminated. Example

Three mutations have been selected to demonstrate the effectiveness of the process of the invention: (a) ΔF 508: it is the most frequent; (b) G 542X: the second most frequent; it features a G to T mutation at nt 1756 in exon 11 of the CFTR gene; (c) N1303K: this mutation features a G to C mutation at nt 4041 in exon 21 of the CFTR gene. Sequences 1 and 2, 6 and 7, and 11 and 12 are respective PCR primers for (a), (b) and (c) . Sequences 3, 8 and 13 are the respective "normal" capture probes. Sequences 4, 9 and 14 are the respective deleted (15) or mutated (20 and 25) capture probes. Sequences 5, 10 and 15 are the respective ALP reporters.

The results are shown in the following Tables 1 to 3. The given values are each the mean of two replicates (A^) . The "normal probe" is specific for the normal sequence; the "mutated probe" is specific for the mutated sequence.

Table 1 (ΔF 508 Test)

Sample Normal probe Mutated probe

Blank 0.030 0.034

Normal 1.562 0.118

Heterozygous 0.732 1.177

Homozygous 0.033 2.327

Table 2 (G 542X Test)

Sample Normal probe Mutated probe

Blank 0.016 0.018

Normal 1.572 0.071

Heterozygous 0.735 0.628

Homozygous 0.076 1.429

Table 3 (N 1303K Test)

Sample Normal probe Mutated probe

Blank 0.073 0.095

Normal 1.234 0.054

Heterozygous 1.616 2.170

Homozygous 0.016 2.310

These results show the practicality of the invention in the detection of single point mutations in specific segments of DNA. Specificity is good; the positive:negative (P/N) ratio is generally higher than 20/1; the positive/positive (P/P) ratio ranges between 1-1.5. Sensitivity is not a critical point; in genetic diseases involving single point mutations, the target to be amplified will always be present either as two copies per cell or as a single copy; as a consequence, the detection limit of the system is not a critical issue. The total assay time, after amplification, can be reduced to less than one hour; the test is easy to use, fast, practicable and cost-effective, whereas molecular biology techniques used to detect single mutations, such as RFLP, SSCP and TGGE, are complicated and time-consuming. The possibility of automation opens the route to a wide range of applications of this technique to the detection at the molecular level of genetic diseases, metabolic disorders, mutated oncogenes/oncosuppressors.

With relation to the following Sequences, those numbered 3, 4, 5, 8, 9, 10, 13, 14, 15 adn 18 to 24 are each NH₂-terminated at both ends. Example 2

A multiplex system was used to conduct single-tube amplification of the following four CFTR exons:

Primer sequences for exons 10, 11 and 21 are as in Example 1; the primers for exon 20 are Sequences 16 and 17. Sequences 18 and 19 are the ALP reporter probe and normal capture probe for R 553X and G 551D, Sequences 20 and 21 are the respective mutated capture probes, and Sequences 22 to 24 are the ALP reporter probe, normal capture probe and mutated capture probe for W 1282X.

The results are shown in Tables 4 to 6. a indicates that each result is the average of two replicates, b and c indicate the probes respectively specific for the normal and mutated sequences, and d indicates the synthetic sequence.

Table 4 (R 553X)

Sample CFN Probe^b CFM Probe^c

Blank 0.021 0.014

Normal 2.492 0.068

Heterozygous 1.021 1.086

Homozygous 0.038 1.954

Table 5 (G 551D)

Sample CFN Probe^b CFM Probe^c

Blank 0.046 0.035

Normal 1.981 0.068

Heterozygous 2.166 1.600

Homozygous 0.079 1.847

Table 6 (W 1282X)

Sample CFN Probe^b CFM Probe⁰

Blank 0.016 0.018

Normal 1.535 0.085

Heterozygous 1.605 1.156

Homozygous 0.048 1.466

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Raggio Italgene SpA

(B) STREET: Via delle Antille, 29

(C) CITY: POMEZIA

(D) STATE:

(E) COUNTRY: Italy

(F) POSTAL CODE (ZIP) : 1-00040

(ii) TITLE OF INVENTION: ASSAY FOR THE DETECTION OF GENETIC ABNORMALITIES

(iii) NUMBER OF SEQUENCES: 15

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GACTTCACTT CTAATGATGA 20

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CTCTTCTAGT TGGCATGCT 19 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GAAACACCAA AGATGATATT 20

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AACACCAATG ATATTTTCTT T 21

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCTTCTGTAT CTATATTCA 19

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CAAATGCTTG CTAGACCAAT AAT 23 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCAAGTTTGC AGAGAAAGAC AA 22

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: AGTTCTTGGA GAAGGTGG 18

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AGTTCTTTGA GAAGGTGG 18

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 10 : GGAGGTCAAC GAGCAAGAAT 20 (2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATTCATACTT TCTTCTTCTT TTCTTT 26

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AGTTAGCAGC CTTACCTCAT CT 22

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AGAAAAAACT TGGATCCC 18

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AGAAAAAAGT TGGATCCC 18 (2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GATCAAGAAA TATGGAAAGT TGC 23

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: ATCCCATCAC TTTTACCTT 19

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: AGTCCTTTTG CTCACCTGT 19

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: TTGGAGAAGG TGGAATCACA 20 (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GGAGGTCAAC GAGC 14

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GGAGGTCAAT GAGC 14

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GAGATCAACG AGCAA 15

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: CGATGGTGTG TCTTGGGA 18 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CAACAGTGGA GGAAA 15

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: CAACAGTGAA GGAAAG 16

Claims

CLAIMS:

1. A process for detecting in an analyte target sequences respectively excluding (wild-type) or including a genetic mutation, comprising the steps of: reacting the analyte under hybridising conditions with (i) first and second oligonucleotide probes and (ii) first and third oligonucleotide probes, the probes being respectively complementary to first, second and third regions of the target sequences, wherein the first region is on one side of the mutation, the second and third probes each span the mutation site and are respectively complementary to the wild-type and mutated regions, and wherein the first probe is separable or detectable and the second and third probes are respectively detectable or separable; and, if necessary, amplifying either or each target sequence; and separating and detecting the resultant hybrids that carry two probes.

2. A process according to claim 1, which comprises amplifying the target sequence prior to the hybridisation reaction.

3. A process according to claim 2, wherein the amplification occurs through the action of a DNA polymerase which synthesises a complementary chain in the 5'-3' direction from a single-strand template, in the presence of primers which are complementary to regions of the target sequence, and the oligonucleotide probes are internally nested with respect to those regions.

4. A process according to claim 3, wherein both strands of double-stranded DNA are amplified and the DNA polymerase is heat-stable.

5. A process according to any preceding claim, wherein the or each separable probe is conjugated to a hapten, and separation is conducted by means of anti-hapten antibodies immobilised on a solid phase.

6. A process according to claim 5, wherein the solid phase comprises magnetisable microparticles.

7. A process according to claim 5, wherein the hapten is fluorescein isothiocyanate and the antibody is an anti- FITC.

8. A process according to any preceding claim, wherein the or each detectable probe is conjugated to an enzyme, and the detection comprises incubation with a substrate, e.g. chromogenic, specific for the enzyme.

9. A process according to any of claims 1 to 7, wherein the or each detectable probe is conjugated to biotin and the detection comprises reaction with a streptavidin-enzyme conjugate.

10. A process according to any preceding claim, wherein the abnormality is a mutation or deletion associated with cystic fibrosis.