US20020187477A1 - Method for detecting single nucleotide polymorphisms (SNPs) and point mutations - Google Patents

Method for detecting single nucleotide polymorphisms (SNPs) and point mutations Download PDF

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US20020187477A1
US20020187477A1 US09/876,727 US87672701A US2002187477A1 US 20020187477 A1 US20020187477 A1 US 20020187477A1 US 87672701 A US87672701 A US 87672701A US 2002187477 A1 US2002187477 A1 US 2002187477A1
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nucleotide
primer
chain
reaction mixture
reaction
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Hong Xue
Jeffrey Wong
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Hong Xue
Wong Jeffrey Tze Fel
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6858Allele-specific amplification

Abstract

A method of genotyping single nucleotide polymorphisms (SNP) and point mutations in nucleic acid based on chain extension by polymerase. This invention is based on the fact that the nucleoside immediately 5′ adjacent to any SNP/point mutation site is known, and the neighboring sequence immediately 3′ adjacent to the site is also known. A primer complimentary to the sequence directly adjacent the 3′ side of the SNP in a target polynucleotide is used for chain elongation. The polymerase reaction mixture contains one chain terminating nucleotide having the base complimentary to nucleotide directly adjacent to the 5′ side of the SNP of the target polynucleotide. An additional dNTP may be added to produce a primer with the maximum of two base extension. The resultant elongation/termination reaction product are analysed for incorporation of the chain terminator nucleotide or for chain length extension of the primer.

Description

    FIELD OF INVENTION
  • The present invention relates to the analysis of nucleic acid sequences. In particular, the present invention relates to the detection of genetic polymorphisms. [0001]
  • BACKGROUND OF INVENTION
  • Single nucleotide polymorphism (SNP) and point mutations are the most abundant type of genetic variations. These variation sites are present at high density in genomes, making them powerful tools for mapping and diagnosing disease-related alleles. [0002]
  • Many methods have been described for the detection of these genetic polymorphisms. For example, U.S. Pat. No. 6,110,709 describes a method for detecting the presence or absence of an SNP in a nucleic acid molecule by first amplifying the nucleic acid of interest, followed by restrictions analysis and mobilizing the amplified product to a binding element on a solid support. Patent Publication WO9302212 describes another method for amplification and sequencing of nucleic acid in which dideoxy nucleotides are used to create amplified products of varying lengths. The varying length products are then separated and visualized by gel electrophoresis. Patent Publication WO20853 further describes a method of detecting single base changes using tightly controlled gel electrophoretic conditions to scan for conformational changes in the nucleic acid caused by sequence changes. [0003]
  • In order to screen a large number of different samples, there is a need to devise a new method with improved efficiency. It is therefore an object of the present invention to provide a novel method for scoring single nucleotide polymorphism. [0004]
  • SUMMARY OF INVENTION
  • Accordingly, the present invention is directed to a method of genotyping SNPs (including point mutations) in nucleic acids, which is based on the simple principle of chain/primer extension. This invention is based on the fact that the nucleoside immediately 5′ adjacent to any SNP/point mutation site is known, and the neighbouring sequence immediately 3′ adjacent to the site is also known. If primers employed axe complementary to sequences in the target polynucleotide where the next succeeding 5′-nucleotide of the target polynucleotide is a potential SNP/point mutation, polymerase reaction mixture containing one ddNTP complementary to the 5′ nucleotide adjacent the SNP of the target polynucleotide in combination with dNTPs should produce nucleic acid chains with a maximum of two-base extensions. [0005]
  • In one aspect to the present invention, a known SNP at a defined location of a known nucleic acid may be scored. The nucleic acid to be analyzed is referred to as the target polynucleotide molecule and contains a 3′ portion, a 5′ portion and a target nucleotide therebetween. This target nucleotide is the position in which the single nucleotide polymorphism is known to be located. The next step involves providing for hybridization a primer oligonucleotide molecule with a sequence complimentary to a section of the 3′ portion of the target polynucleotide molecule directly adjacent to the target nucleotide. The oligonucleotide and the target polynucleotide are added to a reaction mixture that is further provided with a chain extending agent capable of extending the 3′ end of the primer molecule such as a polymerase enzyme. The reaction mixture is also provided with chain terminating nucleotide having a base that is complimentary to the base of the nucleotide directly adjacent to the 5′ side of the target nucleotide. The above reagents are then reacted under appropriate conditions for primer extension in the presence or absence of an extender nucleotide, and the numbers of bases that are extended from the primer are scored using various techniques known in the art. [0006]
  • Chain terminating nucleotides refer to nucleotides that have functional residues to allow the chain extending agent to polymerise and extend one end of the primer while blocking further extension of the primer after the chain terminating nucleotide has been incorporated. Extender nucleotide refers to any nucleotides that can be used by the chain extending agent for continual chain elongation without causing chain termination. For example, if DNA polymerase is used as the chain extending agent, then 2′-dideoxyribonucleoside 5′-triphosphate (dNTP) may be used as an extender nucleotide, while 2′,3′-dideoxyribonucleoside 5′-triphosphate (ddNTP) may be used as the chain terminating nucleotide. If RNA polymerase is used as the chain extending agent, then 3′-dioxyribonucleoside 5′-triphosphate can be used as the chain terminating nucleotide while ribonucleoside 5′-triphosphate (NTP) can be used as the extender nucleotide. [0007]
  • In one embodiment, the polynucleotide that is to be analysed for SNP is first isolated and amplified using techniques such as conventional polymerase chain reaction (PCR) using a pair of first and second PCR primers. The first and second primers are designed to amplify the region containing the SNP of interest (i.e. the target nucleotide). The amplified products (referred to as the amplified target polynucleotide) are then separated from the first and second primers. The purified amplified target polynucleotide is then reacted with a third primer. The third primer is designed to be complimentary to the 3′ portion of the amplified target polynucleotide directly adjacent to the target nucleotide. The elongation/termination reaction is then initiated by adding a chain terminating nucleotide to an appropriate reaction mixture. The appropriate chain terminating nucleotide contains a base that is complimentary to the base directly adjacent to the 5′ side of the target nucleotide. An extender nucleotide may also be added to the reaction mixture to potentially allow the third primer to be extended across the target nucleotide. The extended third primer, which would have a maximum of only 2 nucleotides extended, can be analysed using standard analysis techniques such as electrophoresis or mass spectroscopy. Other techniques including fluorescence spectroscopy, capillary electrophoresis (CE), high performance liquid chromatography (HPLC) can be used for detection. [0008]
  • Another embodiment of the invention is directed to detection of SNPs/point mutations in a solid-phase mode including DNA chip. In this case, oligomers of DNA, RNA, or PNA (peptide nucleic acid) in modified or unmodified forms with known sequences 5′-upstream to SNP sites of interest can be coated onto a solid surface, such as that of glass, metal, plastic, nylon, beads or any other suitable matrices. Molecules under investigation in the form of, e.g. purified PCR product, can then be hybridized to the oligomers, or primers, immobilized on a solid surface. In the presence of a polymerase reaction mixture containing an unlabelled dNTP and a labelled ddNTP complimentary to the base 5′ to the SNP, the PCR product will serve as polymeration template, according to which the immobilized primers will be extended for two residues if the dNTP present is complimentary to the SNP site on the template sequence. The primer will however be extended for only one nucleoside if dNTP is absent and the ddNTP present is in base pairing with the SNP site.[0009]
  • BREIF DESCRIPTION OF DRAWINGS
  • FIG. 1 shows a schematic drawing of one example of how to use the patent invention. [0010]
  • FIG. 2A show the result of chain length analysis of the reaction shown in FIG. 1. [0011]
  • FIG. 2B is the result of detection of incorporated label onto the primer in the reaction shown in FIG. 1 using labelled chain terminator nucleotide and without chain analysis. [0012]
  • FIGS. 3A and 3D show another example of how to practise the present invention. [0013]
  • FIG. 4A shows the result of chain length analysis of the example shown in FIGS. 3A and 3B. [0014]
  • FIG. 4B is the result of detection of incorporated label onto the primer in the reaction shown in FIGS. 3A and 3B using labelled chain terminator nucleotide and without chain length analysis. [0015]
  • DETAILS OF INVENTION
  • The present invention is applicable to any technology platforms that use polymerase-based reaction in conjunction with the use of ddNTP followed by chain length/mass or label analysis with or without prior separation of free ddNTP from incorporated ddNTP. The technique may be applied to genetic material of any organism, including prokaryotic and enkayotic organism. [0016]
  • In one embodiment, a one-step elongation/termination reaction followed by chain length analysis provides complete information on the SNP of interest. In this embodiment, a primer is provided having a sequence complimentary to the section of the target polynucleotide that is directly adjacent the 3′ side of the target nucleotide. The target nucleotide refers to the sequence location in which the SNP to be screened is known to be located. One ddNTP which is complementary to the nucleotide 5′ adjacent to the SNP/point mutation is also provided in the reaction mixture. The ddNTP may be in a labelled or unlabelled form, depending on the method used for product analysis in the subsequent step. There should be also present in the reaction mixture one dNTP. In the case where the potential SNP/point mutation site and the 3′ adjacent site in a target DNA accommodate bases of the same identity, the reaction could proceed without dNTP. [0017]
  • The identity of the base at the potential SNP/point mutation site of the target DNA can be determined by checking for chain extension after the elongation/termination reaction. Fully informative results can be obtained if different reaction mixtures are used, each containing a different dNTP. A dideoxy polymerization that gives no chain extension suggests that the base of the target nucleotide is not complementary to any dNTP present in the reaction mature, while one base extension suggests the base is complementary to the ddNTP present. The production of oligonucleotide chains with two-base extension indicates that the target nucleotide contains a base that is complementary to the specific dNTP added to the reaction. For example, if in the presence of ddCTP and dATP, but not any other nucleotides, a two-base extension occurred in the elongation/termination reaction, the sequences of the target polynucleotide at the potential SNP/point mutation site and 5′ adjacent site should read as T and G in DNA or U and G in RNA respectively. [0018]
  • In one specific preferred embodiment, a set of three reaction and a control are carried out in parallel. The four reactions mixes are different from one another in the content of dNTP. For example, the first has dATP, the second dTTP, the third dGTP, and the fourth, or the control, has no dNTP, while all four reactions contain ddCTP. The occurrence of incorporation of ddCTP in labelled and unlabelled form in any of the reactions indicates that a base complementary to the dNTP present in the reaction occupies the position of potential SNP/point mutation site in the target DNA. If chain extension occurred even in the absence of any dNTP, as in the case of the control reaction, the base of the target nucleotide is complimentary to that of the ddCTP in the reaction (i.e. the target nucleotide contains in base G). In this case, the chain/primer will extent for only one base but not two. [0019]
  • In the case where two or three dNTPs are present in the reaction mixture, the base at the targeted SNP/point mutation site can be ambiguously identified to be complementary to one of the two, or three dNTPs. This kind of experimental design can be meaningful in some special cases. [0020]
  • In another embodiment, the entire procedure or some of the reactions may be performed in solid phase. A typical procedure for the implementation of a solid-phase mode will include essential the following steps: template amplification, quenching/purification, probe binding, probe extension, quenching/purification and detection, where each step can be varied and simplified. [0021]
  • The principles described above are illustrated by way of the following examples: [0022]
  • EXAMPLE ONE
  • Referring first to FIG. 1, the target polynucleotide molecule is a human genomic DNA sequence and contains an SNP with the base A (for ease of description, the location of the SNP on the target polynucleotide is also referred to as the target nucleotide). As a further example, it is known that among the human population, the bases C, G or T also occur at this site at certain frequencies. The sequences flanking this target nucleotide are known. The user in this example is given an unknown sample, so he does not know that the identity of the base of the target nucleotide is an A, and the following description describes how the method according to the present invention may be applied to identify the base at this specific location for this unknown sample. [0023]
  • The first step in the process is the purification of the human genomic DNA and the separation therefrom of other contaminating material such as cell debris. After DNA purification, a primer, as indicated in FIG. 1, is provided for use in the detection method according to the present invention. The primer contains a sequence that is complimentary to the section of the target DNA that is directly adjacent to the 3′ portion of the SNP. In this particular example, only the two bases flanking each side of the SNP are indicated for ease of description. The other bases are indicated as Ys in the target DNA and the complimentary bases are indicated as Xs in the primer. In this example, 2 bases TT are present directly adjacent the 3′ side of the SNP. For ease of description, a 5-mer is used as an example for the primer, and contains the sequence AAXXX as shown in FIG. 2. [0024]
  • During the elongation/termination reaction, an appropriate polymerase enzyme is added to the reaction mix under suitable reaction condition for the extension of the primer such as the presence of appropriate substrates and cofactors. For DNA polymerase, ATP and Mg[0025] ++ are provided in the reaction mix. The components of the reaction mixtures are shown in the top row of FIG. 2. For convenience, co-factors and reaction conditions, are not specified. It is understood that the appropriate reaction conditions have to be provided for chain extension to occur. These are known in the art and may be obtained from standard laboratory manuals such as Sambrook et al. in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, CSH, NY, 1989. Since the nucleotide directly adjacent to the 5′ side of the SNP is known to the user (in this case a G), the dideoxy nucleotide that is selected is ddCTP. In order to determine the base at the target location i.e. the SNP, three reaction mixes are used as shown in row 1 of FIG. 2A. Each of these reaction mixes contains ddCTP, the primer and the target DNA. As shown in row 1 of FIG. 2A, reaction mixtures (a), (b) and (c) contain the nucleotides dATP, dTTP and dGTP respectively, i.e. each reaction mix contains a nucleotide with a unique base. The last column of FIG. 2A shows the bases that can possibly be found at the SNP and two flanking bases on either side of the SNP site. The base at the SNP site is highlighted within the rectangle.
  • As mentioned above, the base at the SNP site for this particular example is actually an A. Therefore, as shown in row [0026] 2, the results is that reaction mix (b) will contain a product having a two-base extension of the primer, while no base extension is found in reaction mixtures (a) and (c).
  • To fully illustrate the power of the present invention, the expected results of chain length analysis for each reaction mixture if other bases are found at the target nucleotide site are shown in rows [0027] 3-5 of FIG. 2A. In each row, the number of bases expected to be extended and the actual sequences of the extended primer are shown. As shown in row 3, a 7-mer would be found in reaction mixture (a) if the target nucleotide has a base T; no base extension is expected for tubes (b) and (c). If the target nucleotide contains a base C, two-base extension (7-mer) would be expected in reaction mix (c) while no reaction (i.e. only 5-mer) would be expected in reaction mixtures (a) and (b). Finally, if the target nucleotide contains a base G, one base is expected to be added to the primer (i.e. 6-mer) in each of the three tubes as shown in row 5 of FIG. 4.
  • From this example, it can be clearly seen that using a parallel set of three elongation/termination reactions, the sequence of a target nucleotide in a single nucleotide polymorphism can be readily identified. The technique used for the chain length or label analysis of the extended primer may be any technique that is available in the art, including electrophoresis or mass spectroscopy. The length of the primer is dependent on many factors, including the base composition (which affects the melting temperature T[0028] m value) of the sequence, reaction temperature and hybridisation stringency required, and may be any length as determined by the user. For detection, the dideoxy nucleotide may be unlabelled or labelled either with a radioactive isotope, with a fluorescence molecule or with an enzyme that can be used for colour-based analysis. For a radioactive isotope system, autoradiography may be performed after gel separation of the primer and its extended products. If capillary electrophoresis or mass spectroscopy is used, chain length may be determined without labelling of the dideoxy nucleotide. Alternatively, fluorescence labelling may be used.
  • If label analysis alone is performed without chain length analysis, the result would simply be a yes or no answer to the question of whether there was chain elongation of the primer. FIG. 2B shows such analysis. It is clear that using the present invention, fully informative results may be obtained even without chain length analysis. [0029]
  • EXAMPLE TWO
  • Example two is similar to example one except that an amplification reaction is used before the elongation/termination reaction for the SNP determination. In this example, a very small quantity of target double stranded genomic DNA is isolated from a human and polymerase chain reaction (PCR) amplification step is first used to amplify the purified DNA sample. In this case, two primers axe first used for the PCR reaction after genomic DNA purification, referred to as primer [0030] 1 and primer 2. As shown in FIG. 3A, primers 1 and 2 are complimentary to sequence 1 and sequence 2C within the target DNA and the antisense strand respectively.
  • Sequence [0031] 1 is 3′ downstream of the target DNA. Sequence 2C is located on the complimentary nucleic acid strand. Sequence 2 is complimentary to sequence 2C and identical to primer 2, and is located 5′ upstream of the SNP. For ease of description, the relative positions of the primers are described in relation to only one strand (the target DNA) of the double-stranded nucleic acid (i.e. in relation to sequence 1 and sequence 2). It is understood that if the PCR amplification reaction is required to amplify the signal, the primers are complimentary to opposing strands of the double-stranded nucleic acid.
  • After PCR amplification reaction under appropriate conditions known to the person of the ordinary skilled in the art, the amplification product is purified from the unreacted primers [0032] 1 and 2 as shown in step 2 of FIG. 3A, using conventional methods such as size exclusion chromatography
  • During the elongation/termination reaction (shown in step [0033] 3 of FIG. 3B), the amplification product also referred to as the amplified target DNA containing the SNP of interest is hybridised with primer 3. Primer 3 is complimentary to the 3′ sequence (referred to as sequence 3) directly adjacent the SNP target nucleotide. For ease of description, only two bases (TT) directly adjacent to the 3′ side of the SNP is shown in sequence 3. The other bases are only indicated as Xs. The Ys shown in primer 3 are bases that are complimentary to the corresponding Xs. In this example, the bases directly adjacent to the 5′ side of the target SNP location is a C. Therefore, the dideoxy nucleotide that should be used for chain termination is ddGTP. Also in this example, the base at the target nucleotide location is an A. The reaction mixtures that are provided to give completely informative results include a set of three parallel reactions. These three reaction mixtures are shown in FIG. 4A. The elongation/termination reactions are similar to those described in example one. FIG. 3B shows the elongation/termination reaction that would occur in the presence of ddGTP and dTTP. The normal dTTP would be added to the 3′ end of primer 3 followed by the addition of ddGTP. Upon the addition of this dideoxy nucleotide, chain termination would occur and 2-base extension occurs in this reaction mixture. In this example, the dideoxyl nucleotide ddCTP is radioactively labelled with 32P. The incorporation of the radioactive nucleotide would light up the extended primer and can be detected after separation by gel electrophoresis or autoradiography.
  • To further illustrate how the present invention can be used to analyse sequences of different bases, FIG. 4A shows the expected results when three reaction mixes containing nucleotides having bases differently from that of the dideoxy nucleotide are used for the reaction. This analysis is similar to that shown in FIG. 3. Briefly, the base of the SNP and the flanking region around is shown in the right most column. The expected oligomers to be found within the three separate tubes are indicated in the space below each reaction mixture. Briefly, a two-base extension in the mixture (b) indicates that the sequence of the target nucleotide is an A while a two-base extension in the (c) reaction mixture indicate that the SNP nucleotide is a G. If one base extension is found in all three reaction mixtures, then the sequence nucleotide is a C. If two bases extension is found in the (a) reaction mixture, then the sequence of the target nucleotide is a T. [0034]
  • Alternatively, the incorporation of labled ddGTP into polynucleotide can be used as an indication that extension has occurred in a particular reaction, without the need for chain length analysis. Expected results are shown in FIG. 4B and are similar to those found in FIG. 3B. [0035]
  • EXAMPLE THREE
  • Example three is similar to Example two except that the 5′ end of primer [0036] 3 is immobilized onto a solid surface. After PCR amplification similar to the one described in Example two, the reaction mixture is quenched with SAP (shrimp alkaline phosphatase) and Exol, or purified by gel electrophoresis to remove dNTPs and amplification primers. The amplified template is then denatured and annealed with the immobilized primer 3.
  • The elongation and termination reaction is initiated by adding the appropriate dideoxynucleotide to an extension mixture. In this case, the base directly adjacent to the 5′ side of the SNP is a C and ddGTP is provided in the elongation/termination reaction. In addition, dATP is added to allow the polymerase to extend the primer across the target nucleotide if the target nucleotide is a T. In order for the reaction to be fully informative even if four nucleotides are possibly found in the target nucleotide sequence, three consecutive extension reactions may be performed using different dideoxynucleotides, with a quenching step after each extension reaction. [0037]
  • As an illustration, the first extension reaction contains all the reagents shown in column A of FIG. 4A. In this case, a two-base extension will occur only if the target nucleotide is a T. A one-base extension will occur if the target nucleotide is a G. A detector sensitive to labelled molecules e.g. a fluorescence array scanner or reader can then be used to detect the chain extension product. [0038]
  • If no chain extension is detected, a second extension reaction may be performed. Before the second extension reaction is performed, the solid surface may be purged or rinsed to eliminate free ddGTP and dATP. This is followed by the second extension reaction in which dTTP and additional ddGTP are added to the reaction mixture. This allows the extension of the immobilized primer [0039] 3 if the base of the target nucleotide is an A. The reaction is again followed by detection for chain extension.
  • If no chain extension is detection in the second reaction, a further third extension reaction may be performed. This is again preceded by purging or removal of any free nucleotides. This third extension reaction is performed using dCTP and additional ddGTP. Results would be similar to those found in FIGS. 4A and 4B, except that the three reactions are performed consecutively rather than in parallel. In this way, only one DNA chip is needed to give a completely informative result. [0040]
  • It is clear from the description above that many reaction combinations may be designed based on the present invention. Thus, while the present invention is specifically described with reference to the afore-mentioned examples, it should be understood that these examples are for illustration only and should not be taken as limitation on the invention. It is contemplated that many changes and modifications may be made by one of ordinary skilled in the art without departing from the spirit and the scope of the invention described. The primers used in the examples are extremely short for ease of illustration. As discussed above, the length of the primers may vary according to the user's needs. [0041]
  • For example, although the second example describes PCR reaction as an amplification step, followed by nucleic acid purification to separate the primers from the amplified products, it should be understood that should priers [0042] 1 and 2 be designed in such a way as to be distinguishable from primer 3, then it becomes unnecessary to have the purification step. For example, if primers 1 and 2 are of a length that is substantially longer than primer 3 and dideoxy chain termination occurs directly after polymerization of primers 1 and 2, then even with a 2 base-extension of primer 3 in the subsequent dideoxy polymerization step, a technique that is capable of distinguishing between the three different primers and their dideoxy extension products would be able to produce informative data without the purification step after DNA amplification. In general, if size detection is used to distinguish the dideoxy extension products, then a primer 3 of less than 50 bases would be preferred, as most mass spectroscopic method work well only with DNA fragments not much longer than 50 base pairs. If capillary electrophoresis is used as the separation method for analysing the length of the extension products, then a primer of less than 100 bases in length may be used. Thus the optimal length of primer 3 depends on the method of detection used and can be determined by the end user. As a non-limiting example, a primer of about 20 bases in length may be used for the chain elongation/termination reaction.
  • The primers used in examples one and two are very short simply for the sake of ease of illustration. It is clear that primers of different lengths may be designed and that the length and location of the PCR primers are dependent on the sequences of the target nucleotide and the hybridization conditions used. In the example shown in FIG. 3A, there are only 7 bases shown between sequence [0043] 1 and a SNP, and 6 bases between sequence 2 and the SNP for ease of illustration. It should be understood that the position of sequence 2 may be any distance 5′ upstream of the SNP as long as the base directly adjacent to the 5′ side of the SNP is also amplified, and the amplified product has sufficient length for primer 3 to anneal and for the polymerase to transcribe therefrom ding the subsequent elongation/termination reaction. Preferably, there should be a minimum of 100 bases between sequence 2 and the SNP, and more preferably more than 100 bases therebetween.
  • The PCR reaction can be symmetrical, meaning the two amplification primers are at roughly equal molar concentration, or it can be asymmetrical, in which one of the primers is at about 100 molar excess and this primer can be modified at its 5′ end for the seek of immobilization in the late steps. [0044]
  • The position of sequence [0045] 1 should be sufficiently 3′ distal from the SNP to accommodate a third primer therebetween during the subsequent elongation/termination reaction, Primer 3 may be, for example, 15-55 bases long.
  • Also, as shown in example one, if sufficient DNA can be obtained from the source material, it is possible to do the elongation/termination reaction without prior DNA amplification. Furthermore, if a sufficiently sensitive technique is used to detect the primers after dideoxy chain extension, it is contemplated that the reaction can be scored without prior amplification even with the isolation of a small amount of DNA. In addition, DNA purification is not an essential step, if specific amplification can be performed using highly specific primers and PCR conditions. [0046]
  • DNA and the use of dNTP and ddNTP are used in the aforementioned examples. It is clear that the polymerase may be DNA polymerase or RNA polymerase, or any other nuleic acid extender that may be available to one in the art. Different nucleic acid extenders may prefer different nucleotides for chain extension. It is understood that the appropriate nucleotide co-factors and reaction conditions for use for chain extension would be provided, and that the appropriate chain terminating nucleotide would be provided for chain termination. Thus, the term extender nucleotide is used in the claims to refer to nucleotides that are used for chain extension including 2′-deoxyribonucleoside 5′-triphospate (dNTP), and ribonucleoside 5′-triphosphate (NTP) to distinguish them from the chain terminating nucleotide. [0047]
  • It is also clear that under certain specific conditions, a single reaction mixture may provide informative results, such as a case in which the target nucleotide and its immediate 5′ neighbour both contain the same base. For example, both are Ts. In this case, the dideoxy nucleotide ddATP would give either a one-base or two-base extension product. Furthermore, not all four bases are necessarily found in every SNP polymorphism. Thus, if a particular polymorphism consists of only two bases, then it is possible that a reduced number of parallel reaction mixes is required to provide a completely informative result. [0048]
  • For instance, if only the frequency of one specific base of an SNP site of the target nucleotide is required to be scored, e.g., the base G with a 5′ adjacent C, a single reaction contra dCTP and a ddGTP should in principle be sufficient to provide the required information. However, parallel reactions containing dATP, dTTP, and ddGTP together with the ddGTP could be included for confirmation purposes. A control reaction with no dNTP could also be included. Alternatively, one of the reaction mixes may be omitted from the elongation/termination reaction to save costs while retaining ability to give fully informative results. For example, reaction mixture (c) in example 2 may be omitted, such that no chain extension in either mixes (a) or (b) implies that the target nucleotide is a G and the sequence around the SNP is CGGTT (FIG. 4A). In other instances, the answer that is required may be a yes or no answer, in which a particular base in a polymorphic site is indicative of an important disease. In this case, reduced number of parallel reaction mixes may again be possibly used to reduce the cost of the screening. [0049]
  • In the case of an immobilized probe, an array of, for example, 96 or 384 probes may be immobilized onto the solid surface at individually defined locations. Bach extension reaction (in Example 3 above, the elongation/termination reaction is cared out in three consecutive extension reactions) may be carried out in a single cycle or, preferably, carried out in multiple thermal cycles to increase sensitivity. For example, 25 thermal cycles may be carried out such that all the immobilized probes are properly extended. [0050]
  • Post extension treatment by shrimp alkaline phosphatase (SAP) or calf intestinal phosphatase (CIP) will remove unincorporated ddNTPs after each thermal cycle. Post-extension treatment with phosphatase is needed in cases, e.g. where incorporation of fluorescence labelled ddNTP is monitored by capillary electrophoresis, since left untreated, the unincorporated fluorescent ddNTP's will comigrate with the fragment(s) of interest. Removal of the 5′ phosphoryl groups by phosphatase treatment alters the migration of the incorporated fluorescent ddNTP's and thus prohibits interference. This treatment is to be done before the detection of extension products, and is not needed for every thermal cycle. [0051]
  • The extension probe may be immobilized after the extension reaction instead of prior to the reaction, if proper capture methods are employed. If a single strand probe is immobilized before extension reaction, there should be no need for purification of amplified template. In case that the extended probe is to be immobilized after extension reaction, isolation of the extended probe from unincorporated ddNTP and un-extended probe can be readily achieved without additional post-extension treatment. [0052]
  • It is also within the scope of the present invention to provide a SNP scoring method in which multiple probes are immobilized onto a single chip such that multiple SNPs representing different polymorphisms may be scored simultaneously for genetic material from the same individual or isolate. This may be performed using repetitive extension reactions, an example of which was described in Example Three above. [0053]
  • From the description above, it is clear that the present invention has many advantages: [0054]
  • Use of only one ddNTP, but not four as in single-base primer extension methods, reduces the cost on labeled ddNTPs, which can be significant in large-scale and fluorescence labeled tests. This feature also makes applicable methods accommodating only one labeling, such as most radioactive or enzyme-linked color labels. [0055]
  • Detection of the incorporation of only one kind of chain terminating nucleotide is required, which makes the methods more generally applicable than single-base extension methods, which typically requires four labeled ddNTPs. Two-base extension methods can be used on technology platforms less suitable for simultaneous multiple signal detections. The detection can be carried out on a solid surface such as a 96-well plastic plate or a chip. It can also be performed in solution phase, for example using high performance liquid chromatography or capillary electrophoresis. A reaction mixture according to the present invention refers to reaction in any of these environments. [0056]
  • Relative to the nucleic acid products of single-base primer extension methods, products with two-base extension are more readily to be detected by length/mass-based or length/mass-sensitive methods. [0057]
  • Methods derived from this invention have most potential being developed into widely distributable genotyping kits as a result of its simplicity in detection as well as reduced costs in labelling with substances such as fluorescence dye or radioactive material. [0058]
  • Two-base extension methods can also be developed to suit the needs of determination of SNP allele frequencies and association studies in pooled DNAs, which is more sensitive to quantitative experimental errors. Since only one ddNTP, therefore no more than one label is used in a two-base extension method, quantification of incorporated ddNTP should subject to less experimental variations resulted from, e.g., differences among fluorescent dyes in physical-chemical properties affecting quantitative detections of labeled nucleic acid products. [0059]
  • Since the reactions different in dNTP content are carried out separately, e.g., in separate tubes, two-base extension methods are less demanding on separation techniques. Only separation of one kind of incorporated nucleotide from unincorporated ddNTP, but not four as in case of single-base extension methods, may be required. [0060]
  • In case that the methods such as fluorescence anisotropy, or mass spectroscopy, capable of detecting in a heterogeneous mode are employed, there should be no separation needed prior to the one-step detection method. [0061]

Claims (14)

1) a method of analysing the base identity of a target nucleotide in a target polynucleotide molecule, said target polynucleotide molecule having a 3′ portion, a 5′ portion and said target nucleotide therebetween, said method comprising:
(a) reacting said target polynucleotide molecule with a primer oligonucleotide, said primer oligonucleotide having a sequence complimentary to a section of said 3′ portion directly adjacent to said target nucleotide;
(b) providing a chain termination nucleotide having a base that is complimentary to the base of the nucleotide directly adjacent to the 5′ side of said target nucleotide;
(c) elongating said primer across said target nucleotide under appropriate polymerisation condition using a chain extending agent, said chain terminating nucleotide having functional residues to allow said extending agent to incorporate said chain terminating nucleotide onto the 3′ end of said primer, said chain terminating nucleotide further terminating said elongation reaction by blocking further extension of said primer after said chain terminating nucleotide is incorporated;
(d) analysing said elongation/termination reaction for primer oligonucleotides containing incorporated chain terminator nucleotides.
2) A method according to claim (1), wherein step (d) involves analysing said elongation/termination reaction for oligonucleotides that have zero, one or two bases longer than said primer.
3) A method according to claim (2) wherein said chain terminating nucleotide is a 2′,3′-dideoxyribonucleoside 5′-triphosphate and said extending agent is DNA polymerase.
4) A method according to claim (2) wherein said target polynucleotide molecule is amplified nucleic acid.
5) A method according to claim (1) wherein a second reaction mixture is provided, said second reaction mixture containing all the reagents as those mentioned in claim (1), said second reaction mixture further containing an extender nucleotide with a second base having an identity different from said base of said chain terminating nucleotide, said extender nucleotide capable of supporting further chain elongation after incorporation into said oligonucleotide, said method further including the steps of elongating said primer oligonucleotide in said second reaction mixture using said extending agent under appropriate reaction conditions, and analysing said second reaction mixture for primer oligonucleotides containing incorporated chain termination nucleotide.
6) A method according to claim (1) wherein one, two or three additional reaction mixtures are provided, each said additional reaction mixture containing all the reagents as those mentioned in claim (1), each said additional reaction mixture further containing an extender nucleotide with a unique base having an identity different from said base of said chain terminating nucleotide, each said extender nucleotide capable of supporting further chain elongation after incorporation into said primer oligonucleotide, said method further including the step of elongating said primer oligonucleotide in said additional mixture using said extending agent under suitable reaction conditions, and analysing said additional reaction mixture for oligonucleotides containing incorporated chain terminator nucleotide.
7) A method according to claim (5) wherein said primer oligonucleotides are analysed for zero, one or two bases extension after said elongation/termination reaction.
8) A method according to claim (6) wherein said primer oligonucleotides are analysed for zero, one or two bases extension after said elongation/termination reaction.
9) A method of detecting single nucleotide polymorphism comprising:
(a) obtaining double-strand nucleic acid from a source for which said single nucleotide polymorphism analysis method is to be performed;
(b) amplifying a portion of said nucleic acid containing said single nucleotide polymorphism by polymerase chain reaction using a first primer and a second primer;
(c) separating said first and second primers from said amplification product;
(d) mixing said amplification product with a reaction mixture, said amplification product having a 3′ portion, a 5′ portion and a target nucleotide therebetween, said target nucleotide having said single nucleotide polymorphism;
(e) providing in said reaction mixture a third primer oligonucleotide molecule having a sequence complimentary to a section of said 3′ portion directly adjacent to said target nucleotide;
(f) providing in said reaction mixture a polymerase enzyme capable of extending the 3′ end of said third primer;
(g) providing in said reaction mixture a chain terminating nucleotide, said chain terminating nucleotide having functional residues to allow said polymerase to polymerise said chain terminating nucleotide onto the 3′ end of that primer while blocking further extension of said primer after said chain terminating nucleotide is incorporated; said chain terminating nucleotide further having a base that is complimentary to the base of the nucleotide directly adjacent the 5′ side of said target nucleotide;
(h) reacting said polymerase with said third primer and said amplification product in said reaction mixture under appropriate reaction conditions;
(i) analyzing said reaction mixture for oligonucleotides that have zero, one or two bases longer than said third primer.
10) A method according to claim (9) wherein a second reaction mixture is provided, said second reaction mixture containing all the reagents in said second reaction mixture, said second reaction mixture further containing an extender nucleotide containing a second base having an identity different from said base of said chain terminating nucleotide, said extender nucleotide capable of supporting further chain elongation after incorporation into said third primer by said polymerase, said method further including the steps of reacting said polymerase in said second reaction mixture, and analysing said second reaction mixture for oligonucleotides that have zero, one or two bases longer than said primer.
11) An oligonucleotide for detecting single nucleotide polymorphism located at a target site on a target polynucleotide, said oligonucleotide having a sequence complimentary to a sequence directly adjacent to the 3′ side of said target site.
12) An oligonucleotide according to claim (12) wherein the length of said oligonucleotide is between 15-55 bases.
13) The method according to claim (1) wherein the 5′ end of said primer oligonucleotide is attached to a solid surface.
14) A method according to claim (9) wherein said third primer oligonucleotide molecule is attached to a solid surface.
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