WO2007034423A2 - Method for detecting single nucleotide variations in a nucleotide sequence using dry/lyophilized reagents. - Google Patents

Abstract

A new method for detecting single nucleotide variations in a nucleotide sequence is provided, which method provided strong and clear determinations of the nucleotide identity in a particular position of a known sequence. Additionally the method can easily be automatized and operated using commonly available laboratory equipment.

Description

METHOD FOR DETECTING SINGLE NUCLEOTIDE VARIATIONS IN A NUCLEOTIDE SEQUENCE USING DRY/LYOPHILIZED REAGENTS.

The present invention relates to a method for detecting single nucleotide changes in a known DNA sequence. In particular, the invention relates to a method for detecting single nucleotide mutations in the human globin gene leading the beta thalassamia. Further, the invention relates to a kit for performing said method.

Background of the invention

Single nucleotide polymorphism (SNP) detection is commonly performed by PCR, where the 3' end of one of the primers (probe) is complementary to the muta- tion of interest and the DNA polymerase used lacks 3' -5' exonuclease activity. Reaction conditions are selected such that PCR proceeds only when the primer perfectly matches the target sequence. In most appli^¬ cations, in order to discriminate between normal, ho- mozygous mutant and heterozygous sample, two PCR re^¬ actions are performed. In one reaction the primer used for discrimination of the alleles is complementary to the normal allele and in the other the primer is complementary to the mutant allele. PCR products are then detected by gel electrophoresis.

Nowadays there is an increased need for large- scale SNP genotyping, where more that one mutation must be detected simultaneously. In that case one shall perform either multiples PCR with primers de^¬ signed to result in products differing in size, in order to be well fractioned by gel electrophoresis, or different PCR reactions for each different muta^¬ tion analyzed. The formed products can also be as^¬ sayed by immunoimmobilization onto a solid support via an interaction of a label, which they carry and have obtained through primer labelling, with a tag already immobilized to the support. Multiplex (4-plex or more) is not always straightforward. Even when the selected conditions are stringent enough to allow the differentiation between normal and mutant allele, the performance of PCR is not always reproducible, espe- cially when tens or hundreds of fragments have to be produced. Non-specific product accumulation is a problem usually faced, especially at high number of cycles used in PCR.

WO 2004/018707 A2 discloses a method for de- tecting single nucleotide polymorphisms (SNP) in hu^¬ man DNA. It is disclosed to use primers having the SNP in the 3' nucleotide in order to limit amplifica^¬ tion of templates having the correct match for the 3' nucleotide of the primer. The primers may be tagged using biotin and the amplification products bound to an avidin coated microtiter plate for detection by hybridisation .

WO 2004/029288 A2 discloses a method for de^¬ tecting a mutation in a nucleic acid by using one PCR primer having a suspected mutation in the 3' end and a second primer in the opposite direction based on the wild type sequence. Formation of an amplification product is considered indicative of the presence of the mutation in the target DNA. A DNA polymerase without 3' -5' exonuclease activity is used.

EP 439 208 A2 discloses the detection of PCT amplified DNA labelled with biotin using a peroxi- dase-avidin complex.

Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC and Markham AF. Nucl . Acid. Res. 17:2503-2516 discloses the use of PCR primers capable of discriminating point mutations in DNA, where the point mutation is at the 3' end of the primer.

Hori K, Shin WS, Hemmi C, Toyo-oka T and Makino T, Curr Pharm Biotechnol. 2003 4(6): 477-84 describes the problem of mismatched priming of sequence spe^¬ cific primers being solved by using unidirectional PCR and detecting the amplification products using Fluorescence Correlation Spectroscopy.

It is known that a number of single nucleotide changes in the human globin gene leads to a predispo^¬ sition for beta-thallasemia .

Description of the invention

The present inventors have thoroughly investi^¬ gated the problem of detecting single nucleotide changes in a given DNA sequence and have thereby con- eluded the present invention providing an improved method for detecting single nucleotide changes.

The present method according to the invention has the benefit of high sensibility, low background and high reproducibility. Further, the method accord^¬ ing to the invention can easily be automatized and performed using commercially available laboratory equipment allowing high through put analysis of large numbers of specimens.

Thus in one aspect the invention relates to a method for detecting single nucleotide changes in a nucleotide sequence comprising the following steps: a) Providing a DNA sample comprising the target DNA, b) Amplification of the target DNA by duplex PCR, c) Hybridization of two 5' -tagged primers to two aliquots of the amplified DNA of step b) , the 3' end of the 5' -tagged primers being complementary to the polymorphic nu^¬ cleotide, one primer is complementary to the mutant sequence, and the other is com^¬ plementary to the normal sequence, d) Extension of the hybridized primers in 2-8 cycles using an exonuclease negative ther^¬ mostable polymerase in the presence of the four triphosphonucleotides, of which at least one triphosphonucleotide is la^¬ belled, e) Immobilization of the extension products of step d) at a support having affinity for the tag on the primers, f) Determining if labelling has been immobil- lized on the support, g) Based on the presence of labelling decid^¬ ing whether the mutant sequence of the normal sequence was present in the DNA sample .

In the method according to the invention a duplex PCR is performed in step b) with unlabelled primers, where the conditions do not have to be ex^¬ tremely stringent since no allelic differentiation is required at this step. The differentiation step is the primer extension reaction, step d) , following the differentiation .

Allele specific primer extension reaction is preferred rather that hybridization with allele spe- cific oligonucleotides, because of better (up to 10 fold or more) discrimination results due to the se^¬ lectivity of the DNA polymerases (Pastinen T., Kurg A., Matspalu A., Peitonen L., Sycanen A-C-., (1997): Minisequencing : A specific tool for DNA analysis and dioagnostics on oligonucleotide arrays. Genome Res. 7:606-614) . Primer extension may be accomplished in two ways, either be single nucleotide addition or by full extension. In the first case, the primer compro^¬ mised has its 3' -end complementary to the nucleotide adjacent to the citation. Consequently four labelled nucleotides each carrying a different label, are needed for the detection of one mutation. If the label is the same, then four different reactions must be performed to detect one mutation. In cases where the primer is fully extended, only one modified nu^¬ cleotide is used, which is incorporated to the newly formed product . In the present description and claims the term "single nucleotide change" is intended to mean that the target DNA in one position contains a nucleotide differing from the corresponding nucleotide found in the same position in other alleles of the same se^¬ quence. It will be appreciated that the term corre^¬ sponds to other known expressions used in the scien^¬ tific literature such as single base mutation and single base polymorphism (SNP) . Even though the invention is mainly described using the terms "normal" and "mutant" for identifica^¬ tion of the two sequences the skilled person will ap^¬ preciate that the method according to the invention may be used for discriminating between two DNA se- quences differing by a single nucleotide, such as e.g. differentiation between two naturally occurring alleles of a nucleic acid sequence.

The DNA sample may, in principle, be any DNA specimen suspected of containing a single nucleotide change. The DNA specimen can be provided by any suit^¬ able technique for preparing DNA from a suitable bio^¬ logical sample. Generally such DNA preparation tech^¬ niques involve dissolution of the biological sample using chemicals, followed by precipitation of the nu- cleic acids using e.g. alcohol. In some embodiments it may even be possible to perform the PCR amplifica^¬ tion of step b) using a biological sample without previous isolation of DNA from the sample. Several such DNA preparation techniques are available for a skilled person.

The biological sample may be any DNA containing tissue. Examples of biological samples include a bi- opsy and a blood sample, when the method according to the invention is used for detecting single nucleotide changes in a mammal, including human beings.

In the present description and claims, the term "target DNA" is intended to mean the DNA sequence in which single nucleotide sequences is suspected to oc^¬ cur. The nucleotide sequence of the target DNA must be known in order to design suitable primers and per^¬ form the method according to the invention.

The amplification in step b) can be performed using well known methods for PCR amplification. The primers may be selected using well known procedures for primer design for amplification of a DNA fragment containing the nucleotide position of the suspected single nucleotide change.

The skilled person will further appreciate that more than one sequence may be amplified in this step, in which one primer pair is used for each amplified sequence. Amplification of more than one sequence al^¬ lows the simultaneous amplification of several posi^¬ tions each containing a possible SNP, which may be advantageous since one amplification reaction may subsequently be divided and used for detecting sev- eral SNP' s (steps c-g) .

In principle, there are no particular require^¬ ments for the amplified fragment, except that it must contain the nucleotide position of the suspected sin^¬ gle nucleotide change. However, it is usually pre- ferred that the amplified fragment is not too big or too small in order to avoid problems during the am^¬ plification. Thus, in one preferred embodiment the amplified fragment is in the range of 50-5000 nucleo^¬ tides, preferably in the range of 100-2000 nucleo^¬ tides, more preferred in the range of 200-1000 nu^¬ cleotides . The primers may be designed using well known procedures for design of PCR primers with due care of providing a suitable annealing temperature and avoid^¬ ing strong secondary structures and primer dimeriza- tions . The primers may be regular DNA primers contain^¬ ing solely deoxyribose nucleotide units, or the prim^¬ ers may additionally comprise one or more modified nucleotide units, as long as the primers are capable of priming in the PCR amplification. Thus, the prim- ers may be DNA, LNA, PNA or any other modified DNA binding primers .

In principle, any thermostable DNA polymerase can be used for the amplification. It is preferred to use one of the commercially available polymerases for the amplification, for example the polymerases sold as Taq polymerase, Pfu polymerase, Vent polymerase or Vent exo^~ polymerase.

The amplification is performed under conditions suitable for the particular used polymerase as de- scribed by the manufacturer of the polymerase.

After the amplification, the amplified DNA may be isolated or purified using well known procedures, or the amplified DNA may be used directly without any purification step. In particular, if the polymerase used for the amplification possess 3' -5' exonuclease activity it is usually preferred to remove or inacti^¬ vate the polymerase before continuing the method ac- cording to the invention.

In step c) 5' -tagged primers are hybridized to the amplified DNA of step c) . In this connection, the term "tag" in the pre^¬ sent description and claims is intended to indicate any moiety connected to the 5' end of the primers, which moiety is capable of forming stable and spe^¬ cific connections with a binding partner. Thus is should be appreciated that the invention uses a tag in step c) and a binding partner in step e) capable of specifically forming stable connections with each other. Several such tag/binding partners are known within the area, for example biotin/streptavidine, biotin/avidine, antigen/antibody, lectin/carbohy- drate, etc. The tag may even be a nucleotide sequence and the binding partner the complementary nucleotide sequence .

The primers are designed so that the 3' posi- tion of the primers is the position corresponding to the single nucleotide change of the nucleotide se^¬ quence .

In this step a 5' -tagged primer having the natural nucleotide in the 3' nucleotide is hybridized to one aliquot of the amplified fragment of step b) , and a 5' tagged primer having the changed nucleotide in the 3' nucleotide is hybridized to a second ali^¬ quot of the amplified fragment of step b) .

The primers may, in principle, be designed in any convenient way using known methods and algorithms for primer design with due consideration of melting point, nucleotide composition and possible secondary structures .

The primers may be regular DNA primers contain^¬ ing apart from the 5' tag solely deoxyribose nucleo^¬ tide units, or the primers may additionally comprise one or more modified nucleotide units, as long as the primers are capable of binding specifically to the intended sequence and further capable of being ex^¬ tended in step d) provided that the 3' nucleotide is matching. Thus the primers may be DNA, LNA, PNA or any other modified DNA binding primers.

In step d) the hybridized primers of step c) is extended using a 3' -5' exonuclease negative DNA poly^¬ merase in a few steps in the presence of at least one labelled triphosphonucleotide .

In the present description and claims, the term a 3' -5' exonuclease negative DNA polymerase is in^¬ tended to mean a DNA polymerase modified in a way so that the 3' -5' exonuclease activity of the modified polymerase is significantly reduced compared with the corresponding natural non-modified enzyme. A skilled person will appreciate that 3' -5' exonuclease activ^¬ ity may be determined using assays well known within the area. The 3' -5' exonuclease activity is preferably reduced with at least 25%, more preferred reduced with at least 40%, more preferred reduced with at least 50%, more preferred reduced with at least 70%, even more preferred with at least 80%, and in a par- ticularly preferred embodiment the 3' -5' exonuclease activity is reduced by more than 90%.

DNA polymerases having reduced 3' -5' exonucle- ase activity may be provided by modification of natu^¬ ral polymerases using chemical reagents, by mutating cells producing the natural polymerase and selecting for mutants having a mutated DNA polymerase exhibit- ing reduced 3' -5' exonuclease activity, or by modify^¬ ing the gene encoding a DNA polymerase and expressing the modified DNA in a suitable host cell. Techniques for preparing a 3' -5' negative DNA polymerase is clearly within the capabilities of a skilled person. Preferably the 3' -5' exonuclease negative DNA polymerase is thermostable.

It is preferred to use a commercially available 3' -5' exonuclease negative DNA polymerase, for exam^¬ ple the polymerases sold under the name Vent exo^~. The labelling used in this step may, in princi^¬ ple, be any labelling that can be connected to a triphosphonucleotide and incorporated into a polynu^¬ cleotide by use of a polymerase, which subsequently allows detection of the incorporated label. One or more of the four trinucleotides is labelled. A skilled person will appreciate that an increasingly stronger signal may be achieved when two, three or all four triphosphonucleotides are labelled compared to when only one triphosphonucleotide is labelled. It may even be possible to use two or more dif^¬ ferent labels on two or more triphosphonucleotides respectively, in order to allow use of different de^¬ tection techniques.

A multitude of such labels are known within the area, including labels based on radioactivity, fluo^¬ rescing labels and antigens for subsequent detection using antibodies. It is preferred to use fluorescing labels such as Cy5 and FITC, where FITC is particu^¬ larly preferred.

Most commonly used labelled nucleotides are modified dUTP's because they are well recognized by DNA polymerases, in contrast to modified dCTPs. In the method according to the invention any modified nucleotide could be used. Fluirescein-dCTP (FITC- dCTP) is preferred.

In primer extension protocols described in the prior art, there is a requirement for purification of the amplicons, or treatment with shrimp alkaline phosphatase for the inactivation of the remaining nucleotides prior to the extensions, to avoid any pos^¬ sible interference with the incorporation of the Ia- belled nucleotide. In a preferred embodiment of the method according to the invention using the sensitive chemiliminiscence system, PCR products have to be di^¬ luted 5Ox-IOOx prior to the extension, so primers and nucleotide contained in this portion ale also di- luted. As a result, there is no need for a purifica^¬ tion step prior to the primer extension reaction, which would be time consuming.

The extension is performed in a few cycles, where each cycle consists of an extension, a denatu- ration where the extended product is released from the amplified DNA from step b) by heating followed by an annealing of more 5' tagged primers. In case the 3' -5' exonuclease negative polymerase is not thermo^¬ stable, additional polymerase must be added before each extension.

The extension takes place at a temperature from about the annealing temperature of the primer to ap- proximately 75°C, usually in the range of 60-75°C, preferably around 72 "C, in a time period of approxi^¬ mately 10 seconds or longer usually in the range of 10 s to 5 minutes, preferably in the range of 30 s to 3 minutes.

It may even be possible to dispense for the ex^¬ tension step provided that sufficient extension takes place during the annealing, in which case the extension cycle consists of an annealing and a denatura- tion. This will generally only be possible if the an^¬ nealing temperature is close to 72⁰C.

Denaturing takes place by heating the mixture to a temperature above 9O⁰C for at least a few sec^¬ onds, usually heating to a temperature in the range of 92-95⁰C in a period of 5 s to 2 minutes.

Annealing takes place essentially as described under step c) .

In one embodiment, step d) is ended after the extension or the denaturing in the last cycle. In an- other embodiment, the final extension cycle is fol^¬ lowed by a denaturation, whereafter the sample is placed on ice until the next step.

It has, surprisingly, been found that when the extension takes place in a few cycles, a satisfactory strong signal can be achieved with a simultaneous surprisingly low background. A higher number of cycles will provide a stronger signal, but also a stronger background. It is, therefore, an important feature of the invention that the number of cycles is selected with due consideration of both the strength of the signal and the background signals. Preferably, the number of cycles according to the invention is selected between 2 and 10, preferably in the range of 2-8, more preferred 3-6, and most preferred 4.

In step e) the extension products of step d) is immobilized on a support having affinity for the 5' tag on the primers.

The support may, in principle, be any support known within the area provided with a binding partner as described in connection with step c) . The binding partner is attached to the support using techniques well known within the area.

Generally, the support is made of a polymeric material shaped into a particular form, such as a bead.

In one preferred embodiment the support is in the form of a microtiter plate, where the bottom of the wells is provided with binding partners for the actual tag. This embodiment has the advantage that many samples may be handled essentially simultane^¬ ously using equipment adapted to the handling of mi^¬ crotiter plates, such as multichannel pipettes, plate readers, etc. Additionally this embodiment also has the advantage of being easily automatizable using e.g. commercially available pipetting robots etc.

After the primer extension, the reaction products are transferred to microtiter plates and the de^¬ tection assay is performed. Most genotyping methods use photometry or fluorescence as the detection sys- tern. Other techniques have also been used, such as gel-based capillary electrophoresis and mass spec^¬ troscopy. Bio-chemiluminometric methods are gradually introduced to nucleic acid analysis. For SNP detec^¬ tion, bioluminescence has been described using fire^¬ fly luciferase to detect pyrophosphate produced in the primer extension reaction (BAMPER) . Chemilumi- nometric methods, despite the relatively low quantum yields, offer higher detectability and dynamic range than spectrophotometric and fluorimetric ones, be^¬ cause of the low background signals achieved. The method according to the invention exploit the bene- fits arising from chemiluminometric detection, espe^¬ cially the broad dynamic range, which results in bet^¬ ter assay performance, definite discrimination be^¬ tween signals derived from extension of probe comple^¬ mentary to the mutant allele. In that way the analy- sis leads to genotypes well assigned, without ambigu^¬ ous results.

Chemiluminometric methods are also advantageous over other methods because of the simple instrumenta^¬ tion needed. Generally, the immobilization is performed by well known procedures, for example comprising con^¬ tacting the sample of step d) with the support having binding partners attached for at certain period of time, where after the sample now depleted for the ex- tension product is removed, and the support with the immobilized extension products is optionally rinsed using a suitable liquid.

In principle, the support may be any solid in^¬ ert material whereto the binding partner can be at- tached. Examples of suitable materials include, but are not limited to, polyethylene, polystyrene and polycarbonate. The support may be formed in particu- late form allowing the formation of a bed of the sup^¬ port having properties allowing a fast and reliable flow of liquid through the bed. Alternatively, the support is in form of a larger shaped object allowing attachment of the binding partner to the surfaces thereof, such as reaction tubes or microtiter plates. Preferably the support is a microtiter plate where the binding partner it attached to the bottom of the wells .

In step f) the presence of an extension product on the support is detected using means and methods corresponding to the particular selected label of step d) . It is within the skills of the average prac- titioner to select suitable detection means depending on the label.

In particular, if the label is an antigen or a moiety that may serve as an antigen, e.g. FITC, the detection of the label can be performed by incubation with a suitable antibody conjugated with a detection moiety e.g. an enzyme, which in a further step may be detected using well known procedures. In addition to being a suitable detection method, this method will also provide a certain amplification of the signal, which may be useful in order to obtain a strong signal .

In a preferred embodiment the label is FITC, which allows detection based on fluorescence, pro^¬ vided that the signal is sufficiently strong, or by incubation with anti-FITC antibody conjugated with e.g. horseradish peroxidase, followed by incubation with a chemiluminescence substrate for the horserad- ish peroxidase, whereby the signal can be detected.

In a preferred embodiment of the invention only conventional instrumentation is used, such as a PCR machine and a luminometric plate reader. The proce- dure is rapid, a whole plate analysis lasts about 3 Vi hours, 2 hours for PCR, 15 minutes for primer exten^¬ sion and 65 minutes for the detection assay. Antibody against FICT labelled with horseradish peroxide (HRP) is preferred, instead of the commonly used alkaline phosphatase because the incubation time needed for the reaction of HRP with its substrate (3.5-5 min^¬ utes) is much shorter than the incubation time needed for the reaction of ALP with its corresponding substrate (30 minutes) and that makes the whole proce- dure faster. Microtiter wells allow many samples to be analysed in parallel in a single plate. As a con^¬ sequence, microtiter well-based assay formats are highly automatable and suitable for high throughput genotyping. In case a support in the form of a microtiter plate is used, the detection means is conveniently a microtiter plate reader adapted to the particular label.

Following the detection of the presence of ex^¬ tension products in step f) the presence of the changed sequence in the sample can be determined. If an extension product is detected only for the primer having the native nucleotide in the 3' position, it can be concluded that the sample only contains the natural sequence. If an extension product is detected only for the primer having the changed nucleotide in the 3' position, it can be concluded that the sample only contains the SNP sequence. If extension products are detected for both primers, it can be concluded that the sample contains both the natural and the SNP sequence, for example in case the donor of the speci^¬ men is heterozygous for the sequence.

A skilled person will appreciate that all the procedures of the invention are known within the area, but that the particular combination of the pro^¬ cedures and the surprising benefits of this particu^¬ lar combination is novel. It is, therefore, within the skills of the average practitioner to perform each individual step of the procedure and, if neces- sary, supplement the teachings herein with the gen^¬ eral knowledge within the area.

The invention also relates to a kit comprising the essential reagents for performing the method ac- cording to the invention. In this connection the term "kit" is intended to mean a collection comprising all the essential reagents needed for the analysis ac^¬ cording to the invention. In this connection essential reagents are intended to mean the reagents hav- ing specificities for the particular SNP to be de^¬ tected, i.e. the two 5' tagged primers for use in step c) . However, it is preferred that the kit addi^¬ tionally contains one or more of the following re^¬ agents and items: primers for the duplex PCR amplifi- cation of step b) , reaction buffer, nucleotides and polymerase for the duplex PCR amplification of step b) , labelled and unlabelled triphosphonucleotide for step c) , polymerase and reaction buffer for extension in step d) and the support having attached binding partners for the tag on the 5' tagged primers.

In case that the labelling requires development for detection, the kit may additionally comprise re^¬ agents for this development, for example if the la^¬ belling is an antigen coupled to the triphosphonu- cleotide, the kit may comprise antibodies against said antigen and reagents for detecting said anti- body.

The kit will usually be provided with an in^¬ struction for use thereof.

The reagents for the method may be provided in a lyophilized form comprising some or all reagents ready for use upon reconstitution . Preferably, all the reagents are mixed and provided in lyophilized form in a reaction tube, so that the reaction can be performed by reconstituting the reaction mixture with water and an aqueous solution of the sample DNA fol^¬ lowed by initiating the heating regimen.

The lyophilized reagents are prepared using well known techniques for lyophilization . Generally the reaction mixture is prepared and a stabilizing agent is added, the mixture is distributed to reac^¬ tion tubes and lyophilized.

In a preferred embodiment the kit according to the invention contains: 1. PCR mix lyophilized in separate 0.2 ml tubes, DNA polymerase and the appropriate buffer are not included in the mix, but added together with the template DNA by the user for the reconstitution of the mix. In another con- tept, though, they could also be included in the lyophilized mixture. 2. Primer extension mix lyophilized in 96-well PCR plate. DNA polymerase lacking 3' -5' exonu- clease activity and the appropriate buffer are not included in the mix, but added by the used for the reconstitution of the mix. In another concept, though, they could also be included in the lyophilized mixture.

3. Microtiter plate with 96 polystyrene white wells containing immobilized dried strepta- vidine . 4. anti FITC-HRP antibody lyophilized in amounts sufficient for a 96-well plate, or liq^¬ uid anti-FITC-HRP antibody in appropriate sta^¬ bilizer solution. 5. Chemiluminescent substrate for HRP. 6. Concentrated wash buffer.

Lyophilization of the mixtures for PCR and primer extension results in more stable reagents that can be stored for longer time than liquids. Separate tubes containing PCR mixture ready to use, minimize the handling operations by the final used and as a consequence eliminate the risk of contamination, which is the major drawback of amplification reactions. Since, the biotinylated primers are already in the lyophilized primer extension mixture, the used does not have to make 2n different mixtures for the detection of n different mutations per sample, but just one mixture per sample and use it for the recon- stitution of the lyophilized primer extension mixtures .

In one embodiment, the method according to the invention is performed in order to detect SNPs in the human globin gene leading to a predisposition for beta-thalassemia . These SNPs may be already identi^¬ fied SNPs, for example the SNPs in the human beta globin gene disclosed in table 2, or it may be not previously described SNPs.

Other preferred embodiments of the method ac^¬ cording to the invention include detection of SNPs in CYPs, apoE, kRAS, cystic fibrosis and factor V.

In a preferred embodiment, the method for de^¬ tecting single nucleotide changes in a nucleotide se^¬ quence is used for detecting single nucleotide changes in the human globin gene leading to a predis- position for beta-thalassamia comprising the following steps : a) Providing a DNA sample derived from a human individual, b) Amplification of a fragment of the human glo- bin gene by duplex PCR, c) Hybridization of two 5' biotinylated primers to two aliquots of the amplified DNA of step b) , the 3' end of the 5' -biotinylated primers being complementary to the polymorphic nu- cleotide, one primer is complementary to the mutant sequence, and the other is complemen^¬ tary to the normal sequence, d) Extension of the hybridized primers in 4 cy^¬ cles, using Vent exo-polymerase in the pres^¬ ence of fluorescein-dCTP (FITC-dCTP) and unlabelled dATP, dGTP and dTTP, e) Immobilization of the extension products of step d) in a micritoter plate having strepta- vidine coated wells, f) Determining the presence of FITC labelling in the microtiter wells by incubating with an horseradish peroxidase conjugated anti-FITC antibody, adding a chemiluminiscence sub^¬ strate for the horseradish peroxidase and measuring the chemiluminiscence, g) Based on the measured chemiluminiscence de- termining the presence of the mutant sequence in the DNA sample.

In another preferred embodiment, all the re- agents needed for all the steps of the above method for detecting single nucleotide changes in the human globin gene leading to a predisposition for beta- thalassamia are provided in a kit in a lyophilized form. In conclusion, the method according to the invention used for SNP genotyping is highly accurte, rapid, reproducible, low cost, and amenable to auto^¬ mation .

The invention will now be described further in the following examples, which are provided for illus^¬ tration and should not be considered limiting for the invention .

Example

Detection of single nucleotide polymorphisms in the human globin gene.

This example discloses the invention used for detection of SNP' s in the human globin gene leading to beta-thallasemia . The sequence of the human globin gene is disclosed in figure 3 and can further be found in public available databases.

The mutations detected in this example are in- dicated in figure 2. The mutations of beta-globin gene detected in this example, include the seven most common mutations in the human beta globin gene, which together account for more that 90% of beta- thallasemia in the Mediterranean area, and seven less common mutations but yet met in the Mediterranean area. Also the mutation responsible for HbS (leading to sicle cell anaemia) is detected.

The method is composed of following operations:

1. Amplification by duplex PCR of the target DNA sequence containing the mutations.

2. Hybridisation of two (for each mutation) 5' - biotinylated primers at a portion of the PCR product. The 3' -end of the primers is comple^¬ mentary to the polymorphic nucleotide, one primer is complementary to the normal and the other is complementary to the mutant sequence. The allele specific primers are extended in 4 cycles, in the presence of fluorescein-dCTP (FITC-dCTP) , using Vent exo^~ DNA polymerase. Several molecules FITC-dCTP are incorporated in the newly formed products by DNA polymerase only if the biotinylated primer is perfectly matched at the 3 '-end. 3. Subsequently, the product formed in the previ- ous step are immobilized to streptavidin- coated microtiter plate wells and incubated with anti-FITC horseradish peroxidase antibod^¬ ies. Finally substrate for the horseradish peroxidase is added, chemiluminescence is measures and the genotypes are assigned ac^¬ cording to the ratios of the signals.

Polymerase chain reaction

The gene regions containing the mutations of in^¬ terest are amplified in a duplex PCR. The amplifi^¬ cation products are of sizes 670 bp and 754 bp, which covers the whole beta-globin gene besides a region of 452 bases into the intron IVSII, where none of the analysed mutations is located. Also a region of 189 bases before and another 293 basses after the coding region are included at the prod^¬ ucts. The 754 bp product contains 11 mutations and the 670 bp , 4 mutations. PCR is performed in 60 μl reactions containing 75 mM TrisHCl (pH 9.0), 50 mM KCl, 20 mM (NH₄) ₂SO₄, 2 mM MgCl₂, 200 μM de- oxynucleotide triphosphates, 2.5 U of thermostable Tth polymerase (BIOTools) , 15 pmoles of each primer (HBB-FF, HBB-IR, HBB-IF, HBB-FR, Table 1) and 100-250 ng template DNA.

PCR mixture, besides DNA polymerase, DNA poly- merase buffer and the template DNA, is provided lyophilized. For the lyophilization all the re^¬ agents needed for PCR are mixed at a final volume of 20 μl, containing dextrane 7% and lyophilized overnight at a Lyophlex 0.8 Edwards lyophiliser. Amplification is carried out as follows: 1 cycle of 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 45 s, and 1 cycle of 72⁰C for 5 min. The above amplification program was performed either as the Mastercycler gradient (Eppendorf) or at the PCRSprint cycler (Thermo) .

Primer extension reaction

After the amplification reaction, PCR products are subjected to primer extension (PE) reaction. PE reaction is performed at a final volumen of 20 μl containing 10 mM KCl, 10 mM (NH₄) ₂SO₄, 20 mM

TrisHCl pH 8.8, 2 mM MgSO₄, 0.1 % Triton X-100, 5 μM dATP, 5 μM dTTP, 5 μM dGTP, 3.5 μM dCTP, 1.5 μM FITC-dCTP, 3 % dextrane, 1.5 pmol biotinylated primer, 0.5 U Vent exo^~ DNA polymerase and 0.5-1.5 μl of the PCR product.

The biotinylated primers for use in the extension step are shown in table 1. PE mixture, besides Vent exo^~ DNA polymerase and its appropriate buffer, is provided lyophilized.

For the lyophilisation all the reagents needed for PE are mixed at a final volume of 10 μl, contain^¬ ing 7 % dextrane and lyophilized over night at a Lyophlex 0.8 Edwards lyophiliser.

Primer extension is carried out at the Mastercy- cler gradient (Eppendorf) as follows: 1 cycle of 95°C for 2 minutes, followed by 4 cycles of 95°C for 20 s, 68°C for 10 s and 72°C for 10 s. After the PE reaction is completed, 30 μl of buffer con^¬ sisting of 100 mM Tris (pH 7.6), 150 mM NaCl and 0.1% Tween-20 are added to each PE product.

Detection

50 μl of the previous step are transferred in wells with immobilized streptavidin and incubated with constant shaking for 30 min at room temperature. The wells are washed three times with 300 μl wash buffer (100 mM Tris pH 7.6, 150 mM NaCl and 0.1 % Tween-20). 50 μl of anti-FITC-HRP 0.11-0.22 μg/ml are added and incubated with constant shak^¬ ing for 30 min at room temperature. Wash is performed again as above and 50 μl chemiluminescent substrate is added in each well. The wells are in^¬ cubated with constant shaking for 3.5-5 min at room temperature and chemiluminescence is meas^¬ ured.

Streptavidin coated wells are provided dried. Coating and drying is performed as follows: 100 μl streptavidin 2 μg/ml is added to each well and in- cubated overnight at room temperature. The follow^¬ ing day the wells are washed three times with 300 μl wash buffer (100 mM Tris pH 7.6, 150 mM NaCl, and 0.1% Tween-20), to remove excess streptavidin that has not been absorbed. 100 μl sucrose 5% is added in each well and incubated for 90 min at room temperature. Sucrose is then aspired and fi- nally wells are dried at 37⁰C for 90-120 min.

Claims

1. A method for detecting single nucleotide changes in a nucleotide sequence comprising the fol- lowing steps : a) Providing a DNA sample comprising the target DNA, b) Amplification of the target DNA by duplex PCR, c) Hybridization of two 5' tagged primers to two aliquots of the amplified DNA of step b) , the 3' end of the 5' tagged primers being comple^¬ mentary to the polymorphic nucleotide, one primer is complementary to the mutant se- quence, and the other is complementary to the normal sequence, d) Extension of the hybridized primers in 2-8 cycles using an exonuclease negative thermo^¬ stable polymerase in the presence of the four triphophonucleotides, of which at least one triphosphonucleotide is labelled, e) Immobilization of the extension products of step d) at a support having affinity for the tag on the primers, f) Determining whether labelling has been immo- billized on the support, g) Based on the presence of labelling deciding whether the mutant sequence of the normal se^¬ quence was present in the DNA sample.

2. The method according to claim 1, wherein the target DNA is the human globin gene and the sin- gle nucleotide change to be detected is a change leading to a predisposition to beta-thalassamia .

3. The method according to claim 1 or 2, wherein the tag used in step c) and the support hav^¬ ing affinity to the tag is a support having strepta- vidine attached to an inert matrix.

4. The method according to claim 3, wherein the support is a microtiter plate having the wells coated with streptavidine .

5. The method according to any of the claims 1-4, wherein the extension in step d) is performed in 4 cycles.

6. The method according to any of the claims 1-5, wherein at least one labelled triphosphonucleo- tide is labelled with fluorescein (FITC) .

7. The method according to claim 6, wherein at least one labelled triphosphonucleotide is FITC- dCTP.

8. The method according to any of the claims 1-7, wherein the exonuclease negative thermostable polymerase is Vent exo- DNA polymerase.

9. The method according to any of claims 6-8, wherein step f) is performed by incubating with horseradish peroxidase conjugated anti-FITC antibod^¬ ies; adding a chemiluminiscent substrate for the horseradish peroxidase and measuring the chemilumi- niscence .

10. Kit comprising all the essential reagents for the method according to claim 1.

11. Kit according to claim 10, comprising PCR mix, primer extension mix and a microtiter plate having streptavidine coated wells.

12. Kit according to claim 10 comprising fol^¬ lowing components:

-PCR mix lyophilized in separate 0.2 ml tubes, DNA polymerase and the appropriate buffer are not included in the mix, but added together with the template DNA by the user for the re- constitution of the mix,

-Primer extension mix lyophilized in 96-well PCR plate. DNA polymerase lacking 3' -5' exonu- clease activity and the appropriate buffer are not included in the mix, but added by the used for the reconstitution of the mix, -Microtiter plate with 96 polystyrene white wells containing immobilized dried strepta^¬ vidine, -anti FITC-HRP antibody lyophilized in amounts sufficient for a 96-well plate, or liquid anti- FITC-HRP antibody in appropriate stabilizer so^¬ lution, -Chemiluminescent substrate for HRP, and -Concentrated wash buffer.