IL102382A - Method for detecting nucleotide sequences and test kit therefor - Google Patents

Description

Method for detecting nucleotide sequences and test kit therefor Nurit EYAL t»»»N n>m C:- 85772 METHOD FOR DETECTING NUCLEOTIDE SEQUENCES AND TEST KIT THEREFOR FIELD OF THE INVENTION The present invention relates to the identification of specific nucleotide sequences and to the detection of mutations at particular sites within nucleotide sequences.

In particular, the present invention concerns a method and kit for the detection of the presence of a certain RNA or DNA sequence in a sample of genetic material. The method and kit of the present invention are very sensitive to small alterations in the sequence and thus are very useful for the detection of point mutations, i.e. a single base-pair alteration in a DNA sequence coding for a specific protein.

The present method and kit of the invention are also useful for identifying the presence of foreign genetic material in a sample of genetic material, for example for detecting the presence of specific bacterial or viral nucleotide sequences in plants and animals, in particular, in humans, thereby providing means for determining the cause of a disease associated with such bacteria or viruses.

- - BACKGROUND OF THE INVENTION AND PRIOR ART The prior art which is considered to be relevant to the following disclosure is listed at the end of the description before the claims. In the following description these references will be identified by indicating, in brackets, their number from the aforementioned list.

In recent years with the development of methods such as the polymerase chain reaction (PCR) and various automated DNA sequencing techniques, an extremely large number of human genes have been isolated, identified and fully sequenced. One of the consequences of such developments has been the elucidation of the genetic basis of many diseases such as, for example, Cystic Fibrosis, Hemophilia, Duchenne (DMD) and Becker (BMD) muscular dystrophy, Lesch-Nyhan syndrome, β-thalassemia, Sickle Cell Anemia, Phenylketonuria, Tay-Sachs, Gaucher and many others. A large number of genetic diseases have been shown to be caused by a point mutation in the gene or an alteration in a small number of nucleotides, i.e. a single base pair change at one or more specific sites within the gene, or a deletion of a small number of nucleotides, e.g. a deletion of a single codon (3 nucleotide base pairs). As a consequence of such point mutations, or small deletions, the normal protein encoded by such genes is either not produced or produced in an inactive form. Furthermore, many cancers have been shown to be associated with point mutations in certain genes.

In view of the aforementioned development it is now possible to obtain genetic material from an individual, to amplify a certain gene region, using PCR technology, and then sequence this region using automated sequencing techniques. The sequenced gene region may then be compared with the known normal sequence to determine whether the individual has a mutation at any particular site in this region. In this way, it is possible to determine specifically whether an individual has a certain disease, whether an individual is a "carrier", i.e. is heterozygous for the - - mutation causing such a disease. If the sequencing is performed on foetal cells, to determine the chances of the foetus to bear a certain inherited disease and if possible to treat the disease using genetic therapy, or if not, to terminate the pregnancy.

Such techniques have also become important for a number of applications including forensic medicine where usually only minute samples are available; in parenteral testing, in analyzing a sample for the presence of DNA of a certain pathogen, e.g. DNA of viral origin such as HIV, etc.

Generally, two sequencing techniques are available, the most widely used today being based on the dideoxynucleotide chain termination procedure(1), which involves the use of dideoxynucleotides which can be incorporated by a DNA polymerase at a 3' end of an elongating DNA chain but once incorporated blocks the further elongation of the chain. Other methods which have been used to determine the presence of point mutations in known DNA sequences are, for example, so-called ligase chain reaction (LCR), which is a modification of the PCR method; hybridization procedures under highly stringent hybridization conditions, etc. The aforementioned procedures have various drawbacks including complexity which allows these methods to be used only by highly skilled personnel, the use of radioactive reagents in many procedures; inadequacy of initial results in most procedures requiring subsequent DNA sequencing; inaccuracy of results; etc.

In an attempt to overcome the above drawbacks in the DNA sequencing methods, new methods have been developed which provide for more rapid and safer DNA sequencing techniques. One such approach involves the use of a set of four chain-terminating fluorescently labelled dideoxynucleotides(2_4). In this method succinyl fluorescein dyes are used wherein each dideoxynucleotide will receive a different dye having different absorption and emission spectra. Thus, DNA molecules labelled with each of the different dideoxynucleotides may be distinguished from one another.

- - Using these dideoxynucleotides it is possible to sequence a DNA segment by carrying out a single reaction in which all four of the differently labelled dideoxynucleotides are added together into a single reaction mixture and the resulting labelled oligonucleotide fragments may then be resolved by polyacrylamide gel electrophoresis in only one sequencing lane on the gel. The gel is scanned by a fluorimeter capable of distinguishing between the different fluorescent labels. The sequence of the different labels along the lane is then translated to the sequence of tested DNA segment.

Recently a novel method for the detection of point mutations has been disclosed based on a single nucleotide primer extension(5,6). By this method the DNA containing the putative mutation site is amplified by the use of PCR and for each amplified fragment several reaction mixtures are then prepared. Each reaction mixture contains a primer whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5' end of the mutation site; a radioactively labelled nucleotide corresponding to the normal coding sequence at the tested site or to a suspected mutant sequence at the site; and a DNA polymerase for incorporating the radio-labelled nucleotide into the primer. The primers are then separated from the template and the occurrence of radioactive labelling on the primers is determined and on this basis the subject may be identified as being healthy (normal), a homozygous or a heterozygous carrier of the mutated gene.

The aforementioned method is believed to be the closest one to the present invention, the present invention having considerable advantages, one of which being the fact that the method of the invention does not require the carrying out of several different reactions and the entire procedure may be carried out in a single reaction vessel. The invention entails several other advantages as will become apparent from the following description.

It is an object of the present invention to provide a simple, rapid and highly accurate method for detecting specific nucleotide sequences in a sample.

It is an object, in accordance with a preferred embodiment of the present invention, to provide a method allowing the identification of mutations at specific sites within a certain nucleotide sequence.

It is another object of the present invention to provide a diagnostic kit to be used for carrying out the above method of the invention.

SUMMARY OF THE INVENTION The present invention provides a method for the detection of the presence of a specific nucleotide sequence in a sample containing DNA or RNA comprising: a) providing an oligonucleotide primer having a sequence complementary to said specific nucleotide sequence short of one. nucleotide at its 3' end complementary to the nucleotide at the 5' end of said specific nucleotide sequence; b) contacting said primer with the sample under conditions in which said primer will anneal to said nucleotide sequence if present in the sample; c) contacting the annealed product obtained in step b) with reverse transcriptase or DNA polymerase, as the case may be, and with one or more chain elongation terminator nucleotides carrying a detectable label; d) incubating for a time sufficient for incorporation of a terminator nucleotide complementary to the nucleotide at the 5' end of said specific nucleotide sequence into the 3' end of the primer to obtain an extended primer; e) removing all non-incorporated terminator nucleotides; and - - f) determining the presence of a label on the extended primer, labelling being an indication of the presence of said nucleotide sequence in said sample.

A specific embodiment of the method of the present invention is identification of point mutations at specific sites in a gene. In this case, the primer will have a complementary sequence to that of the normal gene immediately flanking the 3' end of the putative mutation site. The chain elongation terminator nucleotide, for example, a dideoxynucleotide (ddNTP) will be complementary to either the normal nucleotide present at said site or to one of the possible nucleotides present in the case of mutation at that site. If desired, the method may be carried out by use of two or more different reaction vessels one containing one labelled terminator nucleotide, and the other containing another labelled terminator nucleotide. However, by the use of different labels, two or more differently labelled terminator nucleotides may be contacted with the annealed product simultaneously and by the differential detection of the different labels the identity of the terminator nucleotide may be determined and hence the identity of the nucleotide at the putative mutation site.

Chain terminator nucleotides may be modified nucleotides in which the 3'-OH is so modified that once incorporated into an oligonucleotide primer no other nucleotide may be bound to this modified nucleotide by a reverse transcriptase or DNA polymerase enzyme. Examples may be nucleotides in which the 3 -OH has been modified. Such modification can be the replacement of the 3'-OH group by a suitable group such as H, SH, etc., or alternatively the binding of various substituent groups. By one embodiment of the invention the modified terminator nucleotides are dideoxynucleptides (ddNTP) or their analogues. ddNTP are any one of ddATP, ddCTP, ddGTP, ddTTP and ddUTP. The terminator nucleotide may carry any suitable label, such as a radioactive label, e.g. 32P, - - fluorescent labels, e.g. fluorescein, NBD (4-chloro-7-nitrobenzo-2-oxa-l- diazole), tetramethylrodamine, Texas red, rhodamine, and many others each one of which has a different absorbance and fluorescence spectrum. ddNTP carrying fluorescent labels are generally preferred in accordance with the present invention. Each ddNTP may carry a different fluorescent label, which labels may easily be distinguished from one another in a fluorimeter. Accordingly in each specific test all four of the labelled analogs may be added into a single reaction vessel and following the incorporation reaction it may easily be determined which of the specific analogs was incorporated.

The primers may consist of between about 10 to about 200 nucleotides, the longer the sequence the greater the annealing specificity of the primer to the nucleotide sequence being tested. Time and expense considerations tend to shift preference to shorter primers, in particular such having about 15 to about 20 nucleotides, this length being optimal to ensure high sequence specificity on the one hand whilst also ensuring rapid, easy and accurate preparation on the other hand.

By the use of two different primers each being of a different length and each being identical to a different sequence to be tested, it is possible, for example, to perform the simultaneous detection of two different sequences in a single gene, or in different genes. This may, for example, serve for simultaneous analysis of two different point mutations in a single gene or in different genes. In such a case the method will require to separate the extended primers on the basis of their different size, e.g. by gel electrophoresis, and the identity of the incorporated ddNTP on each extended primer, may then be determined directly on the gel.

The sample of genetic material being tested by the above method may be in the form of RNA or DNA. When the sample is RNA, then the enzyme to be employed for incorporating the terminator nucleotide - - into the primer will be a reverse transcriptase. When the sample is DNA, then a DNA polymerase will be used.

The present invention also provides a diagnostic kit for use in a method for the detection of the presence of a specific nucleotide sequence in a sample containing DNA or RNA, comprising: a) an oligonucleotide primer having a sequence complementary to said specific nucleotide sequence short of one nucleotide at its 3' end complementary to the nucleotide at the 5' end of said specific nucleotide sequence; b) a reverse transcriptase or a DNA polymerase; c) at least one labelled chain elongation terminator nucleotide; and optionally d) suitable buffers for the action of the reverse transcriptase or DNA polymerase and suitable wash solution for washing out all non-incorporated labelled terminator nucleotides.

The kit may be provided with instructions for use in the method of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS In the following description reference will be made at times to the annexed drawings in which: Fig. 1 is a schematic outline of features of a method in accordance with the invention for detecting point mutation in a known gene; Fig. 2A and 2B are schematic outlines of features of the method of the present invention when applied to detecting mutations in the CFTR gene, 2A - detection of point mutations and 2B - detection of small deletions; and Fig. 3 is the complete cDNA sequence of the CFTR gene.

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described in more detail with an emphasis made on a method for identifying point mutations in genes, which mutations are associated with genetic disorders. While this application of the method of the invention is presently preferred, this is by no means the only application of the invention as will no doubt be appreciated by the artisan. On the contrary, the method has various other applications such as detection of specific genetic sequences in a sample such as those associated with certain pathogenic microorganism, e.g. viruses, in a tested sample; in parenteral testing; in forensic medicine; etc.

Fig. 1 is a schematic illustration of an outline of the method of the invention in a particular case. In order to test for the occurrence of a point mutation of a gene having the bace A in the novel, i.e. non-mutative gene. A sample of genomic DNA or mRNA from cells of the tested subject are obtained and a specific primer having a sequence which is complementary to the sequence of the region immediately flanking the 3' end of the suspected mutation site, is annealed to the sample DNA or RNA. Labelled dideoxynucleotides are then added together with a DNA polymerase or a reverse transcriptase, as the case may be. Following the incorporation reaction the non-incorporated dideoxynucleotides are washed away and the primaries are tested for the presence of a label. In the case of a normal individual, both copies of its gene will have the base A in their respective site, and accordingly all primers will be labelled by dideoxythymidine and where the individual is homozygous or a mutation at that site, e.g. one in which the gene carries the base G instead of the base A in the respective site, all primers will be labelled by dideoxycytozine. Where the individual is heterozygous, i.e. one of its alleles is normal and the other being mutated, some primers will be labelled by dideoxythymidine and others by dideoxycytozine. If each of the dideoxynucleotides carries a different label, the - - incorporation and the subsequent label detection may all be carried out in a single reaction vessel.

The genetic material to be analyzed may in principle be any RNA or DNA obtained from the tissues or fluids of humans, animals or plants or that is obtained from cultures of microorganisms or human, animal or plant cells. The genetic material may also be obtained from non-living sources suspected of containing matter from living organism sources, for example, such as may be the case when applying the method in forensic medicine for detecting and identifying specific nucleotide sequences present in or on samples of clothing, furniture, weapons and any other items found at the scene of a crime. In this instance the genetic material obtained is usually in the form of DNA, any RNA in such samples of clothing, furniture, weapons, etc., usually having been degraded by ribonucleases.

The specific application of the inventive method for the detection and identification of mutations and/or poly-morphisms in genes having a known sequence is a presently preferred embodiment. In this application the method may be used as a diagnostic assay to determine the specific mutation present in an individual suffering from or showing symptoms of a disease known to be caused by a change in a specific gene. The method may also be applied for screening healthy individuals to determine whether they are carriers, i.e. heterozygous for gene mutations linked to known diseases, which diseases occur when an individual is homozygous for the mutant gene. This is the case, for example in the well elucidated Tay-Sachs disease in which diseased individuals have mutations in both alleles encoding the hexoaminidase A gene, and carriers of the disease having a mutation in one allele encoding this gene. Furthermore, the method may also be applied for screening embryos, i.e. samples of amniotic fluid to determine whether they have mutations in one (i.e. carriers) or both (i.e. diseased) allele encoding a gene known to be involved in a specific disease.

In its simplest form, the method of the invention for identifying point mutation may be summarized as follows: (i) A sample of genetic material in the form of RNA or DNA, to be analyzed is obtained and annealed to a specific oligonucleotide primer having a sequence complementary to the sequence flanking the 3' end of the suspected mutation site so that the primer will anneal to the sequence in the RNA or DNA with the 3' end of the primer immediately before the putative mutation site; (ii) A set of one to four labelled chain elongation terminator nucleotides, for example, terminator nucleotides such as the dideoxynucleotides ddATP, ddCTP, ddGTP, ddTTP and ddUTP, or analogs thereof, are added to the mixture of (i); (iii) A DNA polymerase when the sample genetic material is DNA, or reverse transcriptase when the sample is RNA, is then added in an appropriate buffer, and an incorporation reaction of the terminator nucleotides is thereby initiated; (iv) After incorporation, a series of appropriate washes are carried out to remove the non-incorporated labelled terminator nucleotide; and (v) Labelling of the oligonucleotide primer is then determined by suitable analytical means, the labelling indicating the incorporation of a terminator nucleotide to the primer, which incorporation provides for indication of the specific nucleotide base- pair is present at the site of the suspected mutation.

- - In Table I below there is listed a sampling of the various diseases which are known to be the result of one or more mutations in a gene encoding a specific protein or enzyme, the sequence of which gene or gene region encoding the protein or enzyme is known. Most of these diseases are recessive diseases, i.e. the diseased individual has both alleles carrying the mutation, the mutation resulting in the protein being absent (gene not expressed), the protein being in an inactive state (having an altered amino acid sequence), or the protein being present in less than the required amounts (significantly reduced gene expression) - - TABLE I Disease Gene Hemophilia A Factor VIII Hemophilia B Factor IX Duchenne (DMD) and Becker (BMD) Muscular Dystrophy Dystrophin Lesch-Nyhan Syndrome HPRT L-ornithyl-carbamyl transferase deficiency OTCase Gaucher Glucocerebrosidase Cystic Fibrosis CFTR Osteogeneesis imperfection Collagen Hemoglobinophathies (e.g. β-thalassaemia, Sickle Cell Anemia) Globin Acute intermittant pyrphyria (AIP) Porphobilinogen deaminase (PBG) Phenylketonuria Phenylalanine hydroxylase gene (PAH) Tay-Sachs Hexosaminidase A (HEXA) Familial hypercholesterolemia (FH) LDL Pearson's marrow/pancreas syndrome Mitochondrial respiratory enzyme Neurofibromatosis NF 1 The ongoing research to determine the genetic basis for diseases and the advent of technologies such as the polymerase chain reaction (PCR) has resulted in the discovery and complete sequencing of more and more genes encoding structural protein or enzyme products, a mutation in which would lead to no expression or reduced expression of the gene or an altered gene product and thereby result in a disease. There is thus an ever expanding field of application of the above method of the invention.

The method of the invention besides having use in diagnosis of specific disease-linked mutations in known gene regions, may also be of use in testing for the presence of a specific sequence associated with blood typing, tissue classification, HLA-typing, sex determination or possible susceptibility of an individual to a certain disease. Tissue classifications, for example, may be determined by identifying polymorphisms in the HLA genes, such polymorphisms being specific for a particular individual. Screening these known HLA gene sequences by the present method may also be used as a diagnostic tool to determine whether the individuals in question are susceptible to certain diseases, e.g. various specific autoimmune diseases which are correlated with the specific HLA genes carried by the individual.

As noted above, the method of the invention may also be applied in the field of forensic medicine in which polymorphisms in specific genes, e.g. the β-globin gene cluster and the various known repeat sequences can be determined in, for example, blood or semen samples obtained at the scene of a crime and the results used to indicate whether or not a suspect was involved in the crime, e.g. murder or rape. Accordingly, the aforesaid determination of such polymorphisms may also be used to determine whether a certain individual is likely to be the father in cases of disputed parenthood.

- - More and more evidence now points to the possibility that certain cancers are the result of specific point mutations in the sequence of certain genes and accordingly, the present method may be used as an early diagnostic tool to screen the general population or those individuals considered most likely to develop such cancers.

Another application of the present method, as noted above, is for the detection of microorganisms in a sample on the basis of the presence of specific sequences in the sample. For example, an individual suspected of being infected by a microorganism, for example a bacteria, or virus can be tested by using such a specific oligonucleotide that anneals only with a specific bacterial or viral DNA sequence and not with sequences present in the individual. One example of such an application is in the screening of individuals for the presence of the AIDS virus. Moreover, by application of the present method the specific strain of virus, e.g. HIV-I, HIV-II or HIV-III may also be determined in a sample. Similarly, different species or strains of bacteria in a sample may be distinguished one from the other, e.g. the presence of Shigella vs. Salmonella bacteria in a sample which are difficult to distinguish from one another by standard techniques.

For all of the aforementioned applications of the present method the sample of genetic material, in the form of either RNA or DNA, to be tested, can be obtained by any of the well known standard techniques for RNA or DNA preparation. Once the genetic material has been obtained and before it is to be annealed with the specific oligonucleotide primer, the genetic material may be treated in a number of ways for the purpose of optimizing the annealing reaction and the subsequent labelled chain elongation terminator nucleotide incorporation to provide an amount of labelled test oligonucleotide product that can be detected rapidly and easily by any of the standard means for detecting the particular label with which the terminator nucleotides were labelled.

The following are examples of the manner of preparing samples of genetic material for testing by the method of the invention: The genetic material to be tested may be in the form of total RNA or mRNA, obtained from a cell lysate using any one of a large number of known techniques. One particularly rapid and easy method for carrying this out is a one step RNA extraction procedure^ using magnetic beads coated with oligo(dT)25.

Once the RNA sample has been prepared this can be used, as detailed below, directly in the method of the invention by adding to this sample one or more specific oligonucleotide primers, labelled chain elongation terminator nucleotides and a reverse transcriptase enzyme for incorporating the terminator nucleotides into the primer using the RNA as a template. In this procedure it is important to ensure that any possible ribonuclease activity is reduced to a minimum, which can be achieved by any of the standard precautionary methods or RNase inhibitors as are well known in the art. As RNA is usually abundant in living tissue material, this method is particularly applicable when it is desired to test genetic material originating from body fluids, blood samples, cell cultures and the like.

When it is not possible to obtain suitable amounts of RNA or it is otherwise desired to use DNA, then the above obtained RNA sample can be used as a template to obtain cDNA by standard techniques, thereby obtaining a variety of cDNA molecules corresponding to essentially all of the various RNA molecules originally present in the extracted mRNA fraction.

The initially synthesized cDNA molecules may then be amplified using the now standard polymerase chain reaction (PCR) with the Taq I polymerase and appropriate DNA primers(8,9) so that the entire cDNA sequence of the gene region to be determined will thus be amplified. In situations where the gene region of interest is very long or where it is - - desirable to amplify more than one gene region, then the PCR amplification may be by way of the recently described multiplex PCR procedure(9) where a number of appropriate primers corresponding to various different regions within a long gene region or corresponding to different gene regions will be used. This multiplex DNA amplification procedure has been described for Duchenne muscular dystrophy (DMD). Application of such a procedure is particularly useful, for example, when it is desired to test a particular individual for the presence of a mutation which may occur at a number of different sites within a specific gene, or when it is desired to determine whether the individual has mutations in a number of different gene regions or has a number of different specific sequences that are being sought.

The aforesaid PCR procedure may be optimized, if desired, by employing the Biotin-Streptavidine system(10,u), which facilitates the separation of the DNA strands for better annealing and for simplifying the washing required in each step of the procedure.

The genetic material to be analyzed may also originally be in the form of DNA, such as for example DNA extracted using standard procedures from cell or tissue samples or samples such as those obtained at the scene of a crime carrying minute amount of cellular material attached to objects such as furniture, weapons, clothing or the like, in which case it is highly likely that only DNA may be obtained from such samples, the RNA therein having been degraded by ribonucleases. The DNA starting material may also be amplified using the aforesaid PCR procedure to provide specifically enriched fractions of the actual gene regions of interest. Owing to its high sensitivity, it is possible to carry out the method of the present invention also without amplification of the genetic material.

The annealing of the primers may be carried out on purified DNA or RNA on a sample genetic material in situ.

- - The oligonucleotide primer should preferably have a sequence of at least about 15 nucleotides, although it is possible also to use primers of longer length. By current oligonucleotide synthesis techniques it is possible to prepare reliably nucleotides of specifically desired sequence having up to about 200 nucleotides. The longer the specific oligonucleotide primer, the greater the specificity of the annealing reaction is and accordingly the less chance of any undesired non-specific annealing. However, constraints of time and costs in preparing large oligonucleotides render those of about 15-20 nucleotides preferably.

It should be noted that even when more than one site in a certain gene region or when more than one gene region is to be analyzed for the presence of a specific sequence or point mutation, the analysis of both sites or regions may be carried out in one reaction, i.e. simultaneously. This is achieved by using more than one specific primer, each having a different length, e.g. one is 15 nucleotides long, another is 20 nucleotides long, etc. Following a terminator nucleotide incorporation, the different primers may be separated on, for example, a polyacrylamide gel, and each primer is then analyzed to determine which terminator nucleotide, was incorporated therein, for which purpose the primers may first be separated from one another on the basis of their different size.

It should be noted that in situations where the gene region being tested in the RNA or DNA sample is one having a certain sequence similarity with other known sequences which may be present in the same sample RNA or DNA, it is desired to provide conditions in which only highly specific annealing can take place.

The annealing reaction is carried out by taking the sample RNA or DNA and treating it appropriately, if necessary, to be ready for annealing to the oligonucleotide primer, then adding the specific primer followed by addition of the labelled terminator nucleotides, e.g. dideoxy- - - nucleotides and the reverse transcriptase or DNA polymerase to initiate the incorporation step. When the sample is DNA, treatment of this sample prior to the annealing reaction may consist of heating the DNA to bring about melting of the double strand structure to obtain single strands, whereupon the primer is added and the mixture is allowed to cool whereby the primer is annealed at the specific region on one of the strands of the sample DNA. Heating, may be for a short period, for example 2 min, at temperatures of up to about 75°C, (at times a temperature of about 65°C may be sufficient to provide the desired single stranded DNA). When the sample is an oligo-dT purified mRNA, usually no special treatments are needed before the annealing step, as the mRNA is single-stranded, but care must be taken to prevent RNase activity as mentioned above. The primer should be added in a sufficiently large amount to increase the likelihood of annealing of the primer with the corresponding region of the DNA sample. Cooling of the mixture to allow for the annealing step is usually to about 37°C, which is an optimal temperature for the subsequent action of a DNA polymerase, such as the T7 DNA polymerase. No such cooling is necessary if the heat stable Taq I DNA polymerase is used.

Another way of converting the sample DNA into suitable single stranded form for the annealing step with the specific oligonucleotide primer, is by sodium hydroxide treatment, which may be performed at room temperature. The sodium hydroxide treatment procedure is generally preferable when the DNA sample is immobilized on a substrate, for example on the aforementioned Biotin-Steptavidin-magnetic beads. Such sodium hydroxide treatment is followed by appropriate washing and neutralization of the sample before addition of the specific oligonucleotide primer. Once the specific oligonucleotide primer has been added and has annealed to the immobilized single DNA strand, the mixture can then be heated to about - - 37°C to provide optimal conditions for the subsequent DNA polymerase mediated incorporation of the terminator nucleotides.

Following the annealing step, the labelled terminator nucleotides, e.g. dideoxynucleotides, will be added together with the DNA polymerase or reverse transcriptase. The terminator nucleotides may be labelled with any suitable detectable label, for example a radioactive label or a fluorescent label, or they may be modified to carry one part of an immuno-or chemical- detection couple, the other part of this couple to be provided in the subsequent detection procedure to determine which specific terminator nucleotide was incorporated.

The terminator nucleotides (chain-elongation terminator nucleotides) may be any of those described hereinabove. Particularly useful terminator nucleotides are the widely-available dideoxynucleotides (ddNTPs). The following description relates specifically to the use of dideoxynucleotides, it being understood that this is not intended as meaning to limit the invention only to such use.

When each of the dideoxynucleotides, i.e. ddATP, ddCTP, ddGTP, ddTTP and ddUTP are labelled with the same or a very similar label such that they cannot be distinguished one from the other on the basis of their label, it is then necessary to carry out four separate incorporation reactions on the annealed RNA or DNA mixture, each reaction carried out with only one of the so-labelled dideoxynucleotides. This is the usually the case when the dideoxynucleotides are radiolabeled, e.g. each has an a-32P label, making each indistinguishable from the others in the detection assay.

Recently, a set of four chain-terminating dideoxynucleotides, i.e. ddATP, ddCTP, ddGTP, ddTTP, each carrying a different succinyl-fluorescein dye have been developed(2_4) for automatic sequencing methods. These dyes are distinguishable one from the other by differences in their absorption and emission spectra and can therefore be detected by standard - - fluorometry methods. It is thus possible to add in one reaction mixture all four of these fluorescently-labelled dideoxynucleotides and then to detect which specific one was incorporated by fluorometry. This approach is therefore a preferred one in that it is much simpler to carry out, it cuts down on sample processing time and saves on the amounts of sample RNA or DNA, specific oligonucleotide primer, and reaction reagents necessary for the procedure.

Immuno-chemical labelling and detection systems for the different dideoxynucleotides may also be employed, in which case, for example, different highly specific monoclonal antibodies may be prepared against each one of the dideoxynucleotides and then the detection of which specific antibody is bound to the specific incorporated dideoxynucleotide can be carried out by any standard immunological or chemical assay. Further, it is also possible to bind to each of the different dideoxynucleotides a different chemical substrate and then to determine which specific dideoxynucleotide was incorporated by using standard chemical or enzyme-linked chemical reactions which detect the presence of the specific substrate. Another possibility is to use the biotin-avidin system for labelling the dideoxynucleotides. In this case one member of the couple, e.g. biotin, is labelled and conjugated to the dideoxynucleotide. Preferably, several such members each carrying a different label are used, and each such labelled member is bound to a different dideoxynucleotide. After incorporation, the primer carrying the so-labelled ddNTP can be readily separated by attachment to the other member of the couple allowing rapid and reliable determination.

In the DNA polymerase-mediated incorporation of the dideoxynucleotides, any suitable DNA polymerase may be used including active DNA polymerase fragments on condition that these have at least the 5'→3' chain-elongation synthesis activity of the DNA polymerase, i.e. are capable of adding a dideoxynucleotide to the 3' end of the oligonucleotide primer using the sample DNA as a template. The widely available T7 DNA polymerase is one such preferred enzyme to be used with the method of the present invention, this polymerase, amongst its other properties, is capable of carrying out the aforesaid dideoxynucleotide incorporation very effectively even at room temperature.

When RNA, ie. mRNA is the template then reverse transcriptase is used in the incorporation reaction. The reverse transcriptase may be any of the widely available kinds used in standard cDNA preparation procedures, and the reaction conditions are likewise the same.

In the dideoxynucleotide incorporation reaction only one specific dideoxynucleotide can be added by the reverse transcriptase or the DNA polymerase to the oligonucleotide primer, as the incorporated dideoxynucleotide causes chain termination. Thus, the incorporated dideoxynucleotide is the one that corresponds to the nucleotide present in the sample RNA or DNA immediately adjacent to the sequence that annealed with the specific oligonucleotide primer. The incorporation of the dideoxynucleotide gives rise simultaneously to two indications: (a) that the RNA or DNA sequence corresponding to that of the primer exists in the RNA or DNA sample; and (b) the identity of the base immediately adjacent the 3' end of this sequence.

The incorporation reaction is usually carried out at temperatures ranging from about 20°C (room temperature) to about 37°C, but where desired and depending on the reverse transcriptase or the DNA polymerase used this temperature may be substantially higher, e.g. 72°C for Taq I DNA polymerase.

After the dideoxynucleotide incorporation reaction all the non-incorporated dideoxynucleotides are removed, only the incorporated labelled dideoxy-nucleotide remaining in the tube in the vessel in which the - - incorporation reaction took place. This washing procedure should be carried out carefully in order to ensure that no unincorporated labelled dideoxy- nucleotides remain in the reaction vessel as this clearly would give rise to false-negative or false-positive results as the case may be and this irrespective of whether a single specific dideoxynucleotide was added to one of four separate reaction tubes or whether all four were added to the same reaction tube, as described above.

Following washing the samples are then analyzed to identify which specific dideoxynucleotide was incorporated into the primer. For detection of the label procedures generally known per se may be followed. For example, where the dideoxynucleotides each carry a different fluorescent label, then the identification of exactly which dideoxynucleotide was incorporated will be carried out by fluorimetry in an apparatus which can differentiate between the four different fluorescent dyes.

A number of possible variations of the aforesaid method of the invention may be employed. One example is the use of two specific oligonucleotide primers for each specific gene region being tested. In this situation each specific oligonucleotide primer will be designed to correspond to one of the strands of a sample DNA in such a way that when annealed with the sample DNA each specific oligonucleotide primer will be annealed to one of the single DNA strands of the sample DNA and the 3' end of each specific primer will be immediately adjacent to the specific site in the sample DNA that is being tested. For example, if in the sample DNA the site in question was composed of an A-T base pair then one of the specific oligonucleotide primers will anneal to the strand carrying the adenosine (A) and the other one will anneal to the strand carrying the thymidine (T) such that the 3' end of the one oligonucleotide is immediately adjacent to the A and the other is immediately adjacent to the T. Accordingly, during the incorporation step a dideoxythymidine (ddTTP) will be incorporated into the - - one oligonucleotide primer while a dideoxyadenine (ddATP) will be incorporated into the other. In the determination step the results will show that both ddT and ddA were incorporated, meaning the existence of an A-T base-pair at the site. This by itself will not give information which strand carries one base and which strand the other, for which purpose additional steps will be required such as first separating the two primers, e.g. on an acrylamide gel on the basis of different sizes of the two primers.

Thus, gene regions corresponding to all of those set forth in Table I above and many others, may be analyzed for the presence of one or more point mutations at any number of sites within the gene region, or the existence of polymorphisms for any specific allele, or whether the individual being tested is homozygous for a specific base pair mutation, heterozygous therefor (i.e. a carrier) or whether the individual is normal for this specific base pair (i.e. carrying two normal genes).

The present multimutation detection or screening method can be a very effective alternative for the traditional mutation detection methods which use radioactive material, different hybridization or PCR conditions for every mutation, special gels or an expensive automated sequencer. The present method enables a large-scale diagnostic procedure for multimutation detection with the possibility of screening many different samples in a short period of time. Furthermore, the present method provides a means for population screening of multimutations in a wide range of inherited diseases and genetic disorders such as genetic cancers and the like, and can also be easily adapted for screening genetic polymorphisms such as those in HLA genes, or detecting for the presence of pathogenic RNA or DNA, e.g. that of bacterial or viral origin in a sample, and also distinguishing between different related strains of such bacteria or viruses.

The invention will now be further illustrated by the following examples: - - EXAMPLE 1 a) Detecting and identifying mutations in the cystic fibrosis gene region The cystic fibrosis (CF) gene has been cloned and the cDNA thereof has been completely sequenced(12_14). The CF gene is more than 250 kb in length and encodes a transcript of about 6.5 kb in length, the transcript- encoding sequences of the gene being divided amongst 27 exons. The protein encoded by the gene, i.e. translated from the aforesaid transcript is 1480 amino acids in length having a molecular weight of about 168 Kd. Due to its putative role in the regulation of ion transport across the membrane the CF protein and hence the CF gene has also been renamed the cystic fibrosis transmembrane conductance regulator (CFTR protein and CFTR gene).

Since the elucidation of the complete cDNA sequence many patients suffering from cystic fibrosis have been tested for the presence of mutations within the CFTR gene in an attempt to understand the molecular basis for the disease. From these studies it has been observed that a deletion of three base pairs within exon No. 10, which results in the loss of a single codon, No. 508, encoding a phenylalanine residue (hence this mutation called A-F508) is the most frequent mutation amongst cystic fibrosis patients and causes cystic fibrosis with pancreatic insufficiency. To date more than 170 additional mutations, each of low frequency in the studied populations have been reported.

Existence of such a large number of different mutations of low frequency has made it difficult to detect and identify mutations in cystic fibrosis patients. All of the aforementioned mutations were identified after the laborious procedure of isolating and sequencing the CFTR genes of cystic fibrosis patients. Accordingly, to positively diagnose a suspected cystic fibrosis patient and to identify the exact mutation in the CFTR gene causing this condition has up to now been an arduous process. The method - - of the present invention, as detailed above overcomes difficulties encountered with prior art methods and provides a much more rapid and efficient screening procedure to determine whether or not the mutation occurred at a specific site.

. CFTR gene analysis is carried out as follows (this is a preferred procedure, but any of the alternative procedures as set above is also applicable): A sample of genetic material which may be mRNA or total RNA or genomic DNA, is first extracted from a crude blood lysate from the patient.

As noted previously, RNA may be analyzed directly using the same primers and procedures set forth hereinbelow with respect to analyzing DNA, the only major difference in procedure being the use of reverse transcriptase instead of a DNA polymerase. When it is not desired to use such RNA, a one-step first strand cDNA is then synthesized by reverse transcription using the extracted RNA as a template. This can then be followed by a multiplex PCR procedure using the appropriate primers so as to amplify essentially the entire cDNA sequence. The reverse transcription and the PCR reaction may be done in one reaction vessel by first carrying out the reverse transcription in an appropriate buffer at temperatures of about 37°C and after a suitable incubation period to ensure the synthesis of full length cDNA molecules the reverse transcription reaction may be terminated by heating the reaction mixture to about 65°C thereby inactivating the reverse transcriptase enzyme and bringing about separation of the cDNA from its mRNA template. At this higher temperature the appropriate PCR primers and the heat stable Taq I DNA polymerase may be added in a suitable buffer to initiate the PCR reaction using the cDNA as template. Depending on the reaction conditions and the PCR primer used it is possible to amplify the entire CFTR cDNA or only specific regions of the CFTR - - cDNA sequence. If desired the PCR primers may be biotinylated whereby the synthesized DNA can be separated by the use of Streptavidin-coated magnetic beads.

The sample genetic material being tested may thus contain the entire CFTR gene sequence in the form of complete full sequence molecules or in the form of a sequential or an overlapping series of molecules each member of the series corresponding to a specific fragment of the entire CFTR gene sequence. In any event, the genetic material sample may be simultaneously tested for the presence or absence of a mutation at any one of the number of possible mutation sites along the CFTR gene. In this case the sample is divided into a number of aliquots, each to be tested with a different specific oligonucleotide primer, or if desired, different specific oligonucleotide primers of different lengths may be added to the sample in one reaction mixture, as noted above.

It should also be noted, as set forth hereinabove, that an in situ procedure may also be carried out by applying the oligonucleotide primers directly to tissue or cell sample material from an individual, such that the primers anneal directly to genomic DNA. Such a method is useful when the terminator nucleotides, e.g. dideoxynucleotides are labelled with highly detectable labels, e.g. fluorescent labels having high levels of fluorescent emission.

For each specific region of the CFTR gene to be analyzed an appropriate specific oligonucleotide primer is prepared having a length of 15-20 nucleotides (15-20 mer oligonucleotide).

Table II below lists eight of the most common mutations in the exons of the CFTR gene, next to each mutation appearing the specific test oligonucleotide to be used to detect this mutation, next to which is listed the labelled dideoxynucleotide which would be incorporated at the 3' end of the specific test oligonucleotide in a normal individual or in an individual having the mutation. Also listed in Table II are three common mutations in the introns of the CFTR gene, the specific test oligonucleotide that may be used to detect and determine the mutation, and the labelled terminator nucleotide, e.g. dideoxynucleotide which would be incorporated at the 3' end of the test oligonucleotide in normal or mutant individuals.

- - TABLE II Mutation Site Specific oligomer primer Labelled ddNTP incorpo¬ (15-mer) ration at 3' end of oligomer primer (i) in EXONS Normal Mutant Δ508 5' ATCATAGGAAACACC 3' ddATP ddGTP Δ507 5' GGAAACACCAAAGAT 3' ddGTP ddATP 542 5' GTGATTCCACCTTCT 3' ddCTP ddATP 551 ' 5' ATTCTTGCTCGTTGA 3' ddCTP ddTTP 553 5' AAGAAATTCTTGCTC 3' ddGTP ddATP 560 5' TCTTTGTATACTGCT 3' ddCTP ddGTP 1282 5' TCCAAAGGC iTCCT 3' ddCTP ddTTP 1303 5' TTCATAGGGATCCAA 3' ddGTP ddCTP (ii) in INTRONS 621 + 1 5' TTGATTTATAAGAAG 3' ddGTP ddTTP 711 + 1 5' AACAAAT1TGATGAA 3' ddGTP ddTTP 1717 - 1 5' TGCCAACTAGAAGAG 3' ddGTP ddATP - - It should be noted that instead of using the specific oligonucleotide primers noted in Table II above, each of which is capable of annealing with the RNA or with only one of the two DNA strands of the CFTR gene at the specific site, it is also possible, when the sample is in the form of DNA, to use an alternative primer specific for the same CFTR gene site but which is complementary to the other DNA strand. This alternative is illustrated in Fig. 2A wherein use of specific test oligonucleotide No. 1 of sequence 5^.CTTCTTGCTCGTTGA...3' in the above method will anneal to CFTR gene strand 1 and incorporate either labelled ddCTP in the normal condition or labelled ddTTP if a mutation has occurred at the site in question. Use of primer No. 2 of sequence (15-mer) 5'...TCACACTGAGTGGAG...3' will anneal to CFTR gene DNA strand No. 2 and incorporate at the site in question either labelled ddGTP in normal samples or labelled ddATP in mutant samples. As noted above, it is at times advantageous to use such primers simultaneously in which case they should preferably have different lengths.

Fig. 2B illustrates the testing of the presence of a AF-508 mutation in a CFTR gene. A primer having the sequence 5'...ATCATAGGAAACACC...3' is annealed to the template DNA and then following an incorporation reaction the identity of the incorporated ddNTP in the extended primer is tested. The normal gene contains a T triplet at the tested site and hence the incorporated ddNTP will be a ddATP, and in a mutated gene, where this triplet has been deleted, the incorporated ddNTP will be ddGTP. Incorporation of only ddATP will indicate a normal subject; incorporation of only ddGTP will indicate a subject which carries two alleles of the mutated gene, i.e. homozygous; and incorporation of both ddATP and ddGTP will indicate that the subject is heterozygous for this mutation.

In Fig. 3 there is illustrated the complete nucleotide sequence of the cDNA encoding the CFTR protein together with the deduced amino - - acid sequence thereof. The numbers on the left-hand side of the sequence denote the base positions while the numbers on the right-hand side of the sequence denote the amino acid residue positions. Also indicated on the left-hand side of the sequence with Roman numerals are the exon numbers from which the mRNA was transcribed. The arrows indicate the transcription initiation site, while the vertical lines indicate the positions of the exon junctions. The boxed amino acids denote potential membrane-spanning segments of the protein. Underneath the nucleotide sequence, or where appropriate above the amino acid sequence there is indicated the position of the eight mutations, listed in Table II above, which occur in the CFTR gene exons and the 5' to 3' direction of the specific oligonucleotide primers to be used to detect and identify the mutation at each of these positions. Also shown are the three mutations, listed in Table II above, which occur in the introns of the CFTR gene and the appropriate specific oligonucleotide primers to be used for screening for the presence of these mutations.

EXAMPLE 3 A diagnostic kit for screening or detecting mutations A diagnostic kit for carrying out the methods detailed above may contain the following constituents: a) one or more specific oligonucleotide primers, each specific primer or appropriate pairs of specific primers designed to be specific for a specific gene or specific region in a gene that is to be screened for the presence thereof in a sample or that is to be screened for the presence or absence of a mutation at a specific site in the gene; b) suitable buffers and wash solutions for carrying out at least the labelled dideoxynucleotide incorporation step of the method and the subsequent washing step; - - c) a set of four labelled chain-terminating dideoxynucleotides or any other suitable chain elongation terminator nucleotides; and d) a suitable DNA polymerase or reverse transcriptase for carrying out the dideoxynucleotide incorporation step of the method.

When the kit is to be used for CFTR gene screening, then it may contain any one or all of the specific oligonucleotide primers listed in Table II above for screening or detecting the most common mutations occurring in this gene. When the kit is to be used for screening for the presence of one or all of the various known genetic diseases, e.g. those listed in Table I above, then it may contain any number, as appropriate, of the specific oligonucleotide primers, each specific oligonucleotide primer for screening for a specific mutation in a particular disease-related gene and in cases where a particular disease-related gene may have one or more mutations, e.g. the CFTR gene, then the kit should contain the specific oligonucleotide primers for screening the most common of the mutations. When the kit is to be used for blood typing or tissue typing analysis then it may contain any number of the specific oligonucleotide primers each designed for identifying a particular blood or tissue type. Depending on the circumstances all of the kits may also contain an additional oligonucleotide primer for determining the presence or absence of a DNA sequence corresponding specifically to the presence of a pathogen, for example the presence of the AIDS virus or a specific strain of such virus, e.g. HIV-I, HIV-II or HIV-III. Accordingly, one kit may be used for testing any number of genes or gene sites within a single gene, and this only requires that the kit contain a number of the specific oligonucleotide primers, all the other components of the kit being the same in all cases.

As indicated above, the preferred terminator nucleotides are dideoxynucleotides of which the preferred ones are those which are fluorescently labelled, in particular those in which each one of the different dideoxynucleotides carries a different fluorescence label. However, as mentioned hereinabove, any suitable terminator nucleotide may be used, which terminator nucleotide may be labelled with any one of the various labels available; the most useful being those in which each set of terminator nucleotides has each member labelled in a detectably different manner! Further, as also mentioned above the preferred DNA polymerase of the kit is the widely available and reliable T7 DNA polymerase. When reverse transcriptase is included in the kit any suitable such enzyme as is commercially available may be used. According to which DNA polymerase or reverse transcriptase is to be incorporated into the kit so the appropriate buffer and wash solution included in the kit will be determined and this in line with the enzyme manufacturer's instructions.

- - LIST OF REFERENCES 1 Sanger, F. (1981), Science 214, 1205-1210. 2. Chehab, F.F. et al. (1989), Proc. Natl. Acad. Sci (USA) 86, 9178- 9182. 3. Prober, J.M. et al. (1987), Science 238, 336-341. 4. Smith, L.M. et al. (1980), Nature 321, 674-678. 5. Kuppuswamy, M.H. et al. (1991), Proc. Natl. Acad. Sci.

(USA) 88, 1143-1147. 6. Singer-Sam, J. et al. (1992), in PCR Methods and Applications, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, U.S.A. pp 160-163. 7. Homes, E. et al. (1990), Genet. Anal. Tech. (1990), 2 (6), 145-150. 8. Fuqua, S.A.W. et al. (1990), Biotechniques 9, 206-211. 9. Chamberline, J.S. et al. (1988), Nuc. Acid. Res. 16, 11141- 11156. 10. Wahlberg, J. et al. (1990), Mol. Cell Probes 4 (4), .285-297. 11. Lundeberg, J. et al. (1990), DNA and Cell Biol. 9, 287-292. 12. Rommens, J.M. et al. (1989), Science 245, 1059-1065. 13. Riordan, J.R. et al. (1989), Science 245, 1066-1073. 14. Kerem, B. et al. (1989), Science 245, 1073-1080.

Claims (21)

1. CLAIMS: 1.

2. A method for the detection of the presence of a specific nucleotide sequence in a sample containing DNA or RNA comprising: a) providing an oligonucleotide primer having a sequence complementary to said specific nucleotide sequence short of one nucleotide at its 3' end complementary to the nucleotide at the 5' end of said specific nucleotide sequence; b) contacting said primer with the sample under conditions in which said primer will anneal to said nucleotide sequence if present in the sample; c) contacting the annealed product obtained in step b) with reverse transcriptase or DNA polymerase, as the case may be, and with one or more chain elongation terminator nucleotides carrying a detectable label; d) incubating for a time sufficient for incorporation of a terminator nucleotide complementary to the nucleotide at the 5' end of said specific nucleotide sequence into the 3' end of the primer to obtain an extended primer; e) removing all non-incorporated terminator nucleotides; and f) determining the presence of a label on the extended primer, labelling being an indication of the presence of said nucleotide sequence in said sample. 2i A method according to Claim 1, further comprising providing two or more different oligonucleotide primers in step (a), each primer having a different sequence and a different length each of which is complementary to a different specific nucleotide sequence in said RNA or DNA sample short of one nucleotide at the 5' end of the specific nucleotide sequence, and wherein said determining in step (f) further comprises a step of first separating the different primers on the basis of their different lengths and then determining the label on each of said primers.

3. A method according to Claim 1 or Claim 2, wherein the oligonucleotide primers have a length of between about 15 to about 200 nucleotides.

4. A method according to any one of Claims 1 to 3, wherein said method is applied for the detection of point mutations at a specific site in a specific gene and said oligonucleotide primer has a complementary sequence to the sequence of the gene immediately flanking the 3' end of the putative mutation site.

5. A method according to Claim 4 for detecting one or more specific mutations at one or more sites in the cystic fibrosis (CFTR) gene, wherein said specific nucleotide sequences to be tested are those included in the sequence of Fig. 3, and wherein the one or more oligonucleotide primers have sequences complementary to the sequences in the gene immediately flanking the 3' end of a putative one or more mutation site in said CFTR gene.

6. A method according to Claim 5, wherein said mutation sites to be detected in the CFTR gene are selected from those listed in Table II and said oligonucleotide primers for detecting each said mutation site have a nucleotide sequence as set forth in Table II.

7. A method according to any one of Claims 1 to 4, wherein the specific nucleotide sequence in said sample is selected from any one of the genes listed in Table I, and said oligonucleotide primer has a sequence complementary to said nucleotide sequence short of one nucleotide at the 5' end of said specific nucleotide sequence.

8. A method according to any one of Claims 1 to 7, wherein the chain elongation terminator nucleotides are the dideoxynucleotides ddATP, ddCTP, ddGTP, ddTTP and ddUTP.

9. A method according to Claim 8, wherein each of said dideoxynucleotides carries a different label.

10. A method according to Claim 9, wherein said dideoxynucleotides each carry a different fluorescent label.

11. A method according to Claim 10, wherein said fluorescent label is selected from the group comprising fluorescein, NBD (4-chloro-7-nitrobenzo-2-oxa-l-diazole), tetramethylrhodamine, Texas red, and rhodamine.

12. A diagnostic kit for detecting the presence of a specific nucleotide sequence in a sample containing RNA or DNA, comprising: a) an oligonucleotide primer having a sequence complementary to said specific nucleotide sequence short of one nucleotide at its 3' end complementary to the nucleotide at the 5' end of . said specific nucleotide sequence; b) a reverse transcriptase or a DNA polymerase; c) at least one labelled chain elongation terminator nucleotide; and optionally d) suitable buffers for the action of the reverse transcriptase or DNA polymerase and suitable wash solution for washing out all non-incorporated labelled terminator nucleotides.

13. A kit according to Claim 12, wherein said nucleotide sequence to be detected is selected from any one of those contained in the genes listed in Table I, and said oligonucleotide primer has a sequence complementary to said nucleotide sequence short of one nucleotide at the 5' end of said specific nucleotide sequence.

14. A kit according to Claim 12 or Claim 13, further comprising two or more different oligonucleotide primers each having a different sequence and different length, each primer being complementary to a different specific nucleotide sequence in said RNA or DNA sample, short of one nucleotide at the 5' end of the specific nucleotide sequence.

15. A kit according to Claim 14, wherein each of said oligonucleotide primers has a length of between about 15 to about 200 nucleotides.

16. A kit according to any one of Claims 12 to 15, for the detection of point mutations at a specific site in a specific gene, wherein said oligonucleotide primer has a complementary sequence to the sequence of the gene immediately flanking the 3' end of the putative mutation site.

17. A kit according to Claim 16 for detecting one or more specific mutations at one or more sites in the cystic fibrosis (CFTR) gene, wherein said one or more oligonucleotide primers have sequences which are complementary to the sequences immediately flanking the 3' end of putative mutation site in said CFTR gene.

18. A kit according to Claim 17, wherein said one or more oligonucleotide primers have sequences selected from those set forth in Table II.

19. A kit according to any one of Claims 12 to 18, wherein the chain elongation terminator nucleotide is at least one of the dideoxy-nucleotides (ddNTPs) ddATP, ddCTP, ddGTP, ddTTP. and ddUTP.

20. A kit according to Claim 19 containing more than one labelled ddNTP each of which being labelled with a different label.

21. A kit according to any one of Claims 12 to 20, wherein the chain elongation terminator nucleotides carry a fluorescent label. For the Applicants, DR. REMOLD COHN AND PARTNERS By: 85772s pc.IR/prg/l.7.1992