Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

• the present invention relates generally to a method for determining the presence of a target nucleic acid that may be present in a sample and more specifically to an assay for detecting or determining the genotype of an amplification product at two or more allelic sites in a nucleic acid.
• Nucleic acid amplification has become an increasingly popular technique in many medical and research settings. The ability to detect and identify a specific genotype is pivotal to understanding disease and protein function. Many diseases, such as cystic fibrosis, occur as a result of a single nucleotide polymorphism (SNP) or greater genetic alteration, such as a deletion, affecting one or more base pairs within a gene. Germline mutations affecting one or both alleles of the affected gene are found throughout the cell population making the target sequences more abundant and thus technically less challenging to identify. However, even heterozygous samples have previously required complex methods to reliably elucidate a genotype.
• SNP single nucleotide polymorphism
• Somatic mutations are even more difficult since the mutation may be present in a small percentage of the starting material, such as a mutation within a tumour. Sensitive genotyping solutions for such mixed populations are often inadequate and unable to obtain the sensitivity required for detection of a low abundance mutated sequence in a high background of wild type sequence.
• PCR Polymerase Chain Reaction
• competing fluorescently labelled probes are often used.
• the method requires amplification of the DNA sequence encompassing the mutation, with the same primers also amplifying the wild type sequence.
• Two or more probes labelled with spectrally distinct fluorophores are designed in the same position on the resultant amplicons such that the mutation is reflected in the probe sequence of one probe and the alternative probe pairs to the wild type sequence. When bound or in solution the fluorescence of the probes is quenched by a quencher molecule bound in close proximity to the fluorescent molecule itself.
• Taq polymerase not only drives the nucleic acid amplification but also contains 5' nuclease activity that cleaves the bound probe as it extends the complimentary sequence. This releases the fluorophore from the quencher and thus fluorescent emission can be detected. This assay is sometimes known as the 5' nuclease assay.
• a 5' nuclease assay can include the following steps.
• a nucleic acid amplification reaction is performed on a target sequence using a nucleic acid polymerase having 5' ⁇ 3' nuclease activity.
• Forward and reverse primers are also used that can hybridise to a target sequence in the presence of an oligonucleotide probe which is capable of hybridising to the target sequence that is amplified by primers.
• the binding site of the oligonucleotide probe which can be sited on either strand is located downstream relative to the binding site for either of the forward or reverse amplification primer.
• the nucleic acid polymerase extends the forward and reverse amplification primers.
• the polymerase During extension of the primer, the polymerase encounters the probe hybridized to the target sequence and degrades the probe. Digestion of the probe can result in the formation of a detectable signal from the probe. This creates a signal that is indicative of the extension of the primer and hence the amplification of the target sequence.
• the invention described herein seeks to satisfy this need.
• Nucleic acid probes have been designed to have a predicted melting temperature that is lower than conventionally used in the art and is closer to the predicted melting temperature of the primers used in the assay.
• the probe(s) can be used together with an at least two stage cycling protocol in which the second stage of the cycling protocol utilises an annealing temperature that is higher than conventionally used in the art and is optimum for probe discrimination.
• the probe(s) can achieve enhanced sensitivity in the detection of nucleic acids and/or enhanced levels of discrimination between nucleic acids. This can permit highly sensitive quantification of mutations.
• a method for determining the presence of a target nucleic acid in a sample comprising: (i) performing a nucleic acid amplification on the sample using a reaction mixture comprising: (a) a nucleic acid polymerase; (b) one or more sets of forward and reverse primers capable of hybridising to the target nucleic acid; and (c) one or more oligonucleotide probes for detecting the target nucleic acid when present in the sample and wherein the oligonucleotide probe(s) has a predicted melting temperature that is higher then the predicted melting temperature of the one or more sets of forward and reverse primers(s); and wherein the cycling conditions during the nucleic acid amplification reaction comprise at least two different sets of cycling parameters, the first set of cycling parameters including an annealing temperature in the nucleic acid amplification reaction that is less than or about the same as the temperature corresponding to the predicted melting temperature of the lowest melting temperature probe; and the second set of cycling parameters including
• the predicted melting temperature of the nucleic acid probes is less than or equal to about 4 °C higher than the melting temperature of the nucleic acid primers.
• the predicted melting temperature of the nucleic acid probes is less than about 3 °C, 2 °C or 1 °C higher than the melting temperature of the nucleic acid primers.
• the probes have a predicted melting temperature range of between about 62 to 65 °C or 62 to 64 °C , with the proviso that the predicted melting temperature is less than or equal to about 4 °C higher than the melting temperature of the nucleic acid primers
• the primers have a predicted melting temperature range of between about 58 to 60 °C. In one embodiment, the first and/or second set of cycling parameters are repeated at least 5 or more times.
• the probe produces a detectable signal when the target nucleic acid is present in the sample.
• the probe(s) comprises a fluorescence moiety and a quenching moiety.
• the second set of cycling parameters is repeated enough times for fluorescence data collection to occur for detection of a fluorescence spectrum.
• the probe is chemically modified to increase the effective melting temperature of the probe.
• the method includes the further step, after step (i), of treating said reaction mixture under conditions that allow for the hybridisation of the oligonucleotide probe(s) to the target nucleic acid thereby resulting in the production of a detectable signal.
• the nucleic acid polymerase has 5' to 3' nuclease activity.
• said method includes the further step, after step (i) of treating said reaction mixture under conditions that allow for the digestion of the oligonucleotide probe(s) via the nuclease activity of the polymerase when said oligonucleotide probe(s) is hybridised to the target nucleic acid.
• the annealing temperature in the first cycling stage is between about 58 °C to about 60 °C.
• the annealing temperature in the second cycling stage is between about 64 °C to about 66 °C.
• a method for genotyping a sample comprising, or suspected of comprising two or more nucleic acid sequences that differ from each other by at least one base position, the method comprising: (i) performing a nucleic acid amplification on the sample using a reaction mixture comprising: (a) a nucleic acid polymerase; (b) one or more sets of forward and reverse primers capable of hybridising to the nucleic acid; and (c) two or more oligonucleotide probes for detecting each of the two or more sequences when present in the amplified nucleic acid, wherein each of the oligonucleotide probes generate a different detectable signal, and wherein the two or more oligonucleotide probes have a predicted melting temperature that is higher then the predicted melting temperature of the primers; and wherein the cycling conditions during the nucleic acid amplification reaction comprise at least two different sets of cycling parameters, the first set of cycling parameters including an annealing temperature in the nucle
• each of the two or more oligonucleotide probes is for distinguishing between two or more substantially homologous sequences, the two or more substantially homologous sequences differing from each other by at least one nucleotide, each probe in the set perfectly matching one of two or more substantially homologous sequences.
• the two or more oligonucleotide probes are allelic probes for genotyping a first allelic site and a second allelic site, suitably, wherein the probes differ from each other by at least one nucleotide.
• the probes each produce distinguishable fluorescence signatures for the different genotypes being detected.
• the nucleic acid amplification reaction is performed using the polymerase chain reaction.
• the method is a 5' nuclease polymerase chain reaction amplification method.
• the method includes the further step, after step (i), of treating said reaction mixture under conditions that allow for the hybridisation of the oligonucleotide probe(s) to the target nucleic acid thereby resulting in the production of a detectable signal.
• the nucleic acid polymerase has 5' to 3' nuclease activity.
• said method includes the further step, after step (i) of treating said reaction mixture under conditions that allow for the digestion of the oligonucleotide probe(s) via the nuclease activity of the polymerase when said oligonucleotide probe(s) is hybridised to the target nucleic acid.
• the predicted melting temperature of the nucleic acid probes is less than or equal to about 4 °C higher than the melting temperature of the nucleic acid primers.
• the predicted melting temperature of the nucleic acid probes is less than about 3 °C, 2 °C or 1 °C higher than the melting temperature of the nucleic acid primers.
• the probes have a predicted melting temperature range of between about 62°C to 65 °C or 62°C to 64 °C , with the proviso that the predicted melting temperature is less than or equal to about 4 °C higher than the melting temperature of the nucleic acid primers
• the primers have a predicted melting temperature range of between about 58 to 60 °C.
• the first and/or second set of cycling parameters are repeated at least 5 or more times.
• the probe produces a detectable signal when the target nucleic acid is present in the sample.
• the probe(s) comprises a fluorescence moiety and a quenching moiety.
• the second set of cycling parameters is repeated enough times for fluorescence data collection to occur for detection of a fluorescence spectrum.
• the probe is chemically modified to increase the effective melting temperature of the probe.
• the annealing temperature in the first cycling stage is between about 58 °C to about 60 °C. In one embodiment, the annealing temperature in the second cycling stage is between about 64 °C to about 66 °C.
• a method for determing the genotype of a sample comprising, or suspected of comprising two or more nucleic acid sequences that differ from each other by at least one base position, the method comprising: (i) performing a nucleic acid amplification on the sample using a reaction mixture comprising: (a) a nucleic acid polymerase; (b) one or more sets of forward and reverse primers capable of hybridising to the nucleic acid; and (c) two or more oligonucleotide probes for detecting each of the two or more sequences when present in the amplified nucleic acid, wherein each of the oligonucleotide probes generate a different detectable signal, and wherein the two or more oligonucleotide probes have a predicted melting temperature that is higher then the predicted melting temperature of the primers; and wherein the cycling conditions during the nucleic acid amplification reaction comprise at least two different sets of cycling parameters, the first set of cycling parameters including an annealing temperature in
• a method for determining the presence of a target nucleic acid in a sample comprising: (i) performing a nucleic acid amplification on the sample using a reaction mixture comprising: (a) a nucleic acid polymerase having a 5' to 3' nuclease activity; (b) one or more sets of forward and reverse primers capable of hybridising to the target nucleic acid; and (c) one or more or more oligonucleotide probes for detecting the target nucleic acid when present in the sample and wherein the oligonucleotide probe(s) has a predicted melting temperature that is higher then the predicted melting temperature of the one or more sets of forward and reverse primers(s); and wherein the cycling conditions during the nucleic acid amplification reaction comprise at least two different sets of cycling parameters, the first set of cycling parameters including an annealing temperature in the nucleic acid amplification reaction that is less than or about the same as the temperature corresponding to the predicted melting temperature of
• a method for genotyping a sample comprising, or suspected of comprising two or more nucleic acid sequences that differ from each other by at least one base position, the method comprising: (i) performing a nucleic acid amplification on the ssample using a reaction mixture comprising: (a) a nucleic acid polymerase having a 5' to 3' nuclease activity; (b) one or more sets of forward and reverse primers capable of hybridising to the nucleic acid; and (c) two or more oligonucleotide probes for detecting each of the two or more sequences when present in the amplified nucleic acid, wherein each of the oligonucleotide probes generate a different detectable signal, and wherein the two or more oligonucleotide probes have a predicted melting temperature that is higher then the predicted melting temperature of the primers; and wherein the cycling conditions during the nucleic acid amplification reaction comprise at least two different sets of cycling parameters, the
• a method for genotyping a sample comprising, or suspected of comprising two or more nucleic acid sequences that differ from each other by at least one base position, the method comprising: (i) performing a nucleic acid amplification on the ssample using a reaction mixture comprising: (a) a nucleic acid polymerase having a 5' to 3' nuclease activity; (b) one or more sets of forward and reverse primers capable of hybridising to the nucleic acid; and (c) two or more oligonucleotide probes for detecting each of the two or more sequences when present in the amplified nucleic acid, wherein each of the oligonucleotide probes generate a different detectable signal, and wherein the two or more oligonucleotide probes have a predicted melting temperature that is higher then the predicted melting temperature of the primers; and wherein the cycling conditions during the nucleic acid amplification reaction comprise at least two different sets of cycling parameters, the
• Figure 1 illustrates the fluorescence output obtained from a PCR reaction using the standard cycling conditions described in the Examples using a wild type probe with signal detected on a first channel and a SNP probe with signal detected on a second channel.
• Figure 2 illustrates the fluorescence output obtained from a PCR reaction using annealing and elongation temperatures for all cycles of 60 °C and 66 °C.
• Figure 3 illustrates the fluorescence output obtained from a PCR reaction using the standard cycling conditions described in the Examples ( Figure 3A) and the two stage cycling conditions described in the Examples ( Figure 3B). In each case a wild type probe with signal detected on a first channel and a SNP probe with signal detected on a second channel are used
• Figure 4 illustrates the flourescence output from a probe on homologous and non-homologous templates when used with a competitor probe or alone, in conjunction with standard cycling conditions (Figure 4A), and with the two stage cycling with the higher anneling and extention temperature ( Figure 4B).
• Figure 5 illlustrates the flourescence output obtained in a multiplex reaction where one wild type probe and multiple mutant probes have been included in the same reaction.
• the wild type probe only gives a fluorescent signal when the wild type template is present whilst no wild type signal is observed on mutant templates ( Figure 4A).
• the mutant probes all give a signal when the homologous mutant template is present whilst giving no signal on the wild type template.
• Figure 6 illustrates the fluorescence output obtained from a PCR reaction using the standard and two stage cycling conditions described in the Examples with a mixed nucleic acid sample at a frequency as low as 5%.
• the top figure in Figure 6A relates to results obtained using standard cycling conditions described in the Examples; an enlarged view of this top figure is shown in the bottom figure; the top figure in Figure 6B relates to results obtained using the two stage cycling conditions described in the Examples; the bottom figure is an enlarged view of the top figure.
• melting temperature Duplex stability between complementary nucleic acid molecules is expressed by the duplexes "melting temperature", which is often abbreviated as Tm.
• the melting temperature indicates the temperature at which a duplex nucleic acid dissociates into single-stranded nucleic acids.
• the melting temperature is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the random coil states.
• the melting temperature can depend on variables including the length of the molecule, the specific nucleotide sequence composition of that molecule and the molarity of the salt and the nucleic acid in the solution.
• Duplex stability and melting temperature are important in nucleic acid amplification where thermocycling may be involved.
• the temperature is raised high enough above the melting temperature so that duplexes of the target nucleic acid and its complement are dissociated.
• the temperature is brought to below the melting temperature such that duplexes of the target nucleic acid and primer(s) and/or probe(s) are able to form, while still remaining high enough to avoid non-specific hybridization.
• Melting temperature and how to measure it is thoroughly described in the art. See for example, Nucleic Acids Research (1990) 18, 6409-6412 and Bioinformatics, V. 12(12), pp. 1226-1227.
• Theoretical or empirical models that relate duplex stability to nucleotide sequence can be used to predict melting temperatures for nucleic acids.
• the predicted melting temperature refers to the theoretically calculated melting temperature of an oligonucleotide that has not been conjugated with any moieties. It is usually an estimate of the actual melting temperature of an oligonucleotide and is based on the sequence of the oligonucleotide typically without considering the effect of any labels and any associated linkers and the like. Given the exact sequence of an oligonucleotide, the melting temperature of the oligonucleotide can be predicted as discussed above. The predicted melting temperature according to the present invention is calculated, in one embodiment, using the nearest neighbour model.
• the predicted melting temperature according to the present invention is calculated using the nearest neighbour thermodynamic theory using SantaLucia values (SantaLucia, J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. (1998) 95, 1460-1465).
• the SantaLucia values are contained in various computer programs that can be used to predict melting temperatures.
• the "Beacon Designer" software package is used.
• the SantaLucia values are used with the following reaction conditions; nucleic acid concentration of 0.25 nM; monovalent ion concentration of 50 mM; free magnesium ion concentration of 5mM; total Na + equivalent of 332.84mM; and temperature for free energy calculations of 25 °C.
• the same software program or methodology is desirably used, suitably, with its default settings, to ascertain the predicted melting temperatures of the primer(s) and the probe(s) used in the present invention.
• Amplification refers to any method that results in the formation of one or more copies of a nucleic acid molecule (for example, exponential amplification) or in the formation of one or more copies of only the complement of a nucleic acid or polynucleotide molecule (for example, linear amplification).
• PCR polymerase chain reaction
• PCR is a primer extension reaction that provides a method for amplifying specific nucleic acids in vitro. PCR can produce million fold copies of a DNA template in a single enzymatic reaction mixture within a matter of hours, enabling researchers to determine the size and sequence of target DNA.
• General procedures for PCR are taught in U.S. Patent Nos. 4,683,195 and 4,683,202.
• the amplification method is an amplification method that incorporates the use of a polymerase which has 5' nuclease activity, suitably 5' exonuclease activity.
• the amplification method is the polymerase chain reaction.
• the amplification method is or is based on a 5' nuclease polymerase chain reaction.
• Nucleic acid This term refers to a compound or composition that is a polymeric nucleotide or nucleic acid polymer.
• the nucleic acid may be a natural compound or a synthetic compound.
• the nucleic acid can have from about 10 to 5,000,000 or more nucleotides. The larger nucleic acids are generally found in the natural state.
• the nucleic acid can have about 30 to 50,000 or more nucleotides - such as about 100 to 20,000 nucleotides or about 500 to 10,000 nucleotides.
• the nucleic acid can include nucleic acids, and fragments thereof, from any source in purified or unpurified form including DNA (dsDNA and ssDNA) and RNA, including tRNA, mRNA, rRNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA/RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomes of biological material - such as bacteria, yeasts, viruses, viroids, moulds, fungi, plants, animals, humans, and the like.
• the nucleic acid may be cleaved to obtain a fragment that contains a target nucleotide sequence, for example, by shearing or by treatment with a restriction endonuclease or other site specific chemical cleavage method.
• the nucleic acid may be at least partially denatured or single stranded or treated to render it denatured or single stranded.
• treatments are well known in the art and include, for instance, heat or alkali treatment, or enzymatic digestion of one strand.
• dsDNA can be heated to a high temperature for a period of about 1 to 15 minutes to produce denatured material.
• Oligonucleotide This refers to a nucleic acid that is usually single stranded and is usually a synthetic nucleic acid but may be a naturally occurring nucleic acid. Oligonucleotide(s) are generally comprised of at least about 5 contiguous nucleotides, suitably, about 10 to 100 contiguous nucleotides, more suitably, about 20 to 50 nucleotides, more suitably 10 to 30 nucleotides, more suitably, 20 to 30 nucleotides, more suitably, 22 to 26 nucleotides, more suitably, 23 to 25 nucleotides in length. In some embodiment, the oligonucleotides are about 23 or about 25 nucleotides in length.
• the precise length will depend to some extent on the required melting temperature of the oligonucleotide.
• Various techniques can be employed for preparing an oligonucleotide that include biological or chemical synthesis.
• the oligonucleotide can be synthesised using standard methods such as those used in commercial automated nucleic acid synthesisers. Chemical synthesis of DNA on a suitably modified glass or resin can result in DNA covalently attached to the surface. This may offer advantages in washing and sample handling. For longer sequences standard replication methods employed in molecular biology can be used.
• Oligonucleotide primers are employed in a chain extension on a nucleic acid template such as in, for example, the amplification of nucleic acid.
• the oligonucleotide primer is usually a synthetic nucleotide that is single stranded, containing a sequence at its 3'-end that is capable of hybridising with the target nucleic acid.
• an oligonucleotide primer has at least 80%, preferably 90%, more preferably 95%, most preferably 100% complementarity to a target nucleic acid sequence or primer binding site.
• the number of nucleotides in the oligonucleotide primer should be such that stringency conditions used to hybridize the oligonucleotide primer will prevent excessive random non-specific hybridisation.
• the number of nucleotides in the oligonucleotide primer will be at least about 5 contiguous nucleotides, suitably, about 10 to 100 contiguous nucleotides, more suitably, about 20 to 50 nucleotides, more suitably 10 to 30 nucleotides, more suitably, 20 to 30 nucleotides, more suitably, 22 to 26 nucleotides, more suitably, 23 to 25 nucleotides in length.
• the oligonucleotides are about 23 or about 25 nucleotides in length. The precise length will depend to some extent on the required melting temperature of the primer.
• primer extension amplification primers hybridise to, and are extended along (chain extended), at least the target nucleotide sequence within the target polynucleotide and, thus, the target sequence acts as a template.
• the target sequence usually lies between two defined sequences.
• the primers hybridise with the defined sequences or with at least a portion of such target polynucleotide, usually at least a ten-nucleotide segment at the 3'-end thereof and preferably at least 15, frequently a 20 to 50 nucleotide segment thereof.
• Oligonucleotide probe This refers to an oligonucleotide employed to bind to a portion of a polynucleotide such as another oligonucleotide or a target nucleotide sequence.
• the oligonucleotide probe is able to produce a detectable signal that is indicative that the nucleic acid that it is designed to detect is present in the sample.
• the number of nucleotides in the oligonucleotide probe will be at least about 5 contiguous nucleotides, suitably, about 10 to 100 contiguous nucleotides, more suitably, about 20 to 50 nucleotides, more suitably 10 to 30 nucleotides, more suitably, 20 to 30 nucleotides, more suitably, 22 to 26 nucleotides, more suitably, 23 to 25 nucleotides in length.
• the oligonucleotides are about 23, 24 or 25 nucleotides in length.
• each of the oligonucleotide probes is able to produce a different or a distinct detectable signal - such as spectrally distinct signal.
• the oligonucleotide probe includes at least one fluorescer and at least one quencher which can be digested by the 5' nuclease activity of a polymerase in order to detect any amplified target oligonucleotide sequences.
• the oligonucleotide probes will have a sufficient number of phosphodiester linkages adjacent to its 5' end so that the 5' ⁇ 3' nuclease activity employed can efficiently degrade the bound probe to separate the fluorescer(s) and quencher(s), for example.
• the fluorescer can be any molecule capable of generating a fluorescence signal.
• the quencher molecule can be any molecule capable of absorbing the fluorescence energy of the excited fluorescer, thereby quenching the fluorescence signal that would otherwise be released from the excited fluorescer.
• the quencher In order for a quencher molecule to quench an excited fluorescer, the quencher must generally be within a minimum quenching distance of the excited fluorescer at some time prior to the fluorescer releasing the stored fluorescence energy.
• each of the oligonucleotide probes is able to produce a spectrally distinct fluorescent signal.
• Target nucleotide sequence This refers to the nucleotide sequence that is to be amplified and/or detected and can contain one or more mutations or polymorphisms to be detected.
• a nucleotide sequence to which primers and/or probes can anneal is considered to be a target nucleotide sequence.
• the nucleotide sequence of a nucleic acid is described from the 5'-end to the 3'-end of the sense strand.
• the target nucleotide sequence can include not only the sense strand but also the nucleotide sequence of the complementary strand thereof, i.e. the antisense strand.
• a sample can include biological or clinical samples that may contain or may be suspected of containing nucleic acids or fragments of nucleic acids to be detected or genotyped.
• the sample may be a complex mixture, a partically purified sample or a purified sample.
• the sample may be a partically purified sample or a purified sample of nucleic acid.
• a sample may be or may be derived from a biological sample that can include any tissue or material derived or derivable from a living or dead mammal or organism, including, without limitation, blood cells, saliva, blood, plasma, serum, mucous and biopsies. Samples may comprise in vitro cell culture constituents.
• the sample may be treated to physically or mechanically disrupt tissue or cells to release intracellular nucleic acids into a solution which may contain enzymes, buffers, salts, detergents and the like, to prepare the sample for analysis.
• the methods exclude the physical step of obtaining a sample from a subject.
• Mutation refers to a difference (eg. one or more differences or two or more differences) in the nucleotide sequence of a nucleic acid and is typically observed among individual organisms of the same origin. With respect to multicellular organisms, differences observed among organs, cells, and so on within an individual are also included as mutations. Mutations can arise from point mutations, deletions, insertions, duplications, inversions, and translocations. Further, mutations may occur within gene regions - such as protein coding regions, intron sequences or expression regulatory regions, which can include promoters or enhancers and the like. Differences in nucleotide sequences found in other genomic sequences are also considered as mutations.
• Polymorphism Among the above mutations, those that are present at a frequency of 1 % or more within a certain population are particularly referred to as "polymorphisms". Among polymorphisms, polymorphisms consisting of displacement, insertion, or deletion of a single nucleotide are particularly referred to as single nucleic polymorphisms (or "SNPs").
• homology refers to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules compared by sequence alignment.
• the degree of homology between two discrete polynucleotide sequences being compared is a function of the number of identical, or matching, nucleotides at comparable positions.
• the degree of similarity expressed in terms of percent identity may be determined by visual inspection and mathematical calculation.
• the percent identity of two polynucleotide sequences may be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res.
• GAP program includes: (1 ) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
• Various programs known to persons skilled in the art of sequence comparison can be alternatively utilized.
• One of the improvements in the methods described herein relates to the predicted melting temperature of the nucleic acid probe that is used in the assay as compared to the predicted melting temperature of the nucleic acid primers.
• the probe is allowed to anneal to all of the nucleic acid targets present in order to saturate the sample, without the primers annealing at the same time. This permits sequential binding of the probe followed by the primers when ramping down to the annealing temperature used in the amplification reaction. This step is therefore conventionally performed at a temperature that is too high for the primers to anneal, but low enough for the probes to anneal.
• the primers When the temperature is at the annealing temperature, which will be below the melting temperature of the primers, the primers will extend and in certain embodiments can cleave off the probe. It is not desirable for the probe to come off the target nucleic acid simply because of temperature. Instead, it is desired in some embodiments that the probe comes off the target because it was removed by the action of the 5' nuclease. Conventionally therefore, the probe will have a predicted melting temperature that is greater than the predicted melting temperature of the primers by about 5-10°C. The predicted melting temperature of each of the primers will be very similar to each other, if not about the same.
• the present invention utilises one or more probes that have a melting temperature that is less than about 5 °C higher than the melting temperature of the nucleic acid primer(s).
• a mismatch in the probe will be of greater discriminating effect when the melting tempearture of the probe has been lowered.
• this modification increases specificity since a mismatched probe has less time to bind to the wrong sequence before primer extension removes the probe binding site from this cycle of the nucleic acid amplification reaction.
• the probe(s) has a predicted melting temperature that is only about 4 °C higher than the melting temperature of the nucleic acid primer(s). In one embodiment, the probe(s) has a predicted melting temperature that is only about 3 °C higher than the melting temperature of the nucleic acid primer(s). In one embodiment, the probe(s) has a predicted melting temperature that is only about 2 °C higher than the melting temperature of the nucleic acid primer(s). In one embodiment, the probe(s) has a predicted melting temperature that is only about 1 °C higher than the melting temperature of the nucleic acid primer(s). In one embodiment, the probe(s) has a predicted melting temperature that is only about 0.5 °C higher than the melting temperature of the nucleic acid primer(s). In one embodiment, the probe(s) has a predicted melting temperature that is only about 0.1 °C higher than the melting temperature of the nucleic acid primer(s).
• the probe(s) has a predicted melting temperature that is between about 0.1 and about 4 °C higher than the melting temperature of the nucleic acid primer(s). In one embodiment, the probe(s) has a predicted melting temperature that is between about 0.5 and about 4 °C higher than the melting temperature of the nucleic acid primer(s). In one embodiment, the probe(s) has a predicted melting temperature that is between about 1 and about 4 °C higher than the melting temperature of the nucleic acid primer(s). In one embodiment, the probe(s) has a predicted melting temperature that is between about 2 and about 4 °C higher than the melting temperature of the nucleic acid primer(s).
• the probe(s) has a predicted melting temperature that is between about 3 and about 4 °C higher than the melting temperature of the nucleic acid primer(s).
• the full benefit of the use of probes that have a melting temperature that is less than about 5 °C higher than the melting temperature of the nucleic acid primer(s) is fully realised when probe discrimination is allowed to occur, for at least a period of time, at a higher than normal annealing temperature in the nucleic acid amplification reaction than would otherwise have been used.
• the present inventors have found that the enhanced specificity of the probe for its cognate template is intrinsic to the design specification of that particular probe and to the cycling parameters used in the method.
• the methods of the prior art require the use of multiple competing probes to synergise stringent signalling since they will compete against each other for the range of possible binding sites.
• Each one will preferentially bind to its cognate template having a higher melting temperature for that target than any other probes in the mix which will all differ by at least one base. Sequestration of alternative binding sites greatly reduces the potential for mis-binding on non cognate targets.
• very high stringency can be achieved with a single probe.
• the probe when a mismatch is present, the probe may bind, but with such a weak interaction that it is preferentially strand displaced by the polymerase, rather than degraded to produce a fluorescent signal.
• the method of the present invention adds a new level of probe specificity to the nucleic acid amplification reaction since a mismatched probe, even when used alone, can allow substantially complete exclusion of mis-hybridisation to non-complimentary target sequences.
• a single probe can advanatgeously be used to achieve highly stringent probe binding between sequences that differ by just one nucleotide base.
• the probes have a predicted melting temperature range of between about 62°C to 65 °C or about 62°C to 64 °C with the proviso that the predicted melting temperature is less than or equal to about 4 °C, about 3 °C, about 2 °C, about 1 °C, about 0.5 °C or about 0.1 °C higher than the melting temperature of the nucleic acid primers.
• the probes have a predicted melting temperature range of between about 62°C to 65 °C or about 62°C to 64 °C with the proviso that the predicted melting temperature is less than or equal to about 0.1 and about 4 °C higher than the melting temperature of the nucleic acid primer(s), about 0.5 and about 4 °C higher than the melting temperature of the nucleic acid primer(s), about 1 and about 4 °C higher than the melting temperature of the nucleic acid primer(s), about 2 and about 4 °C higher than the melting temperature of the nucleic acid primer(s), about 3 and about 4 °C higher than the melting temperature of the nucleic acid primer(s).
• the cycling protocol is separated into two different cycling stages.
• the first cycling stage allows for efficient amplification of the target sequence since the annealing temperature is optimal for efficient nucleic acid priming and amplification.
• These early cycles can be used to initiate efficient nucleic acid amplification in a complex biological mixture to increase the abundance of the target nucleic acid sequence.
• Efficient nucleic acid amplification typically requires that the annealing and extension temperature are less than or similar to the melting temperature of the primers and so the early cycles are expected to take place at about or around the optimal predicted melting temperature for primer annealing to occur.
• the amount of probe cleaved in each cycle will normally be insufficient to raise the signal produced by probe cleavage to above the background level. Probe discrimination and signal detection are not therefore relevant to these early amplification cycles and data need not normally be collected during this first cycling stage.
• the annealing temperature used in the first cycling stage of the nucleic acid amplification reaction is equal to or less than about 2 °C of the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature used in the first cycling stage of the nucleic acid amplification reaction is equal to or less than about 1 °C of the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature used in the first cycling stage of the nucleic acid amplification reaction is equal to or less than about 0.5 °C of the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature used in the first cycling stage of the nucleic acid amplification reaction is equal to or less than about 0.1 °C of the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature used in the first cycling stage of the nucleic acid amplification reaction is equal to or less than about 2 °C lower than the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature used in the first cycling stage of the nucleic acid amplification reaction is equal to or less than about 1 °C lower than the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature used in the first cycling stage of the nucleic acid amplification reaction is equal to or less than about 0.5 °C lower then the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature used in the first cycling stage of the nucleic acid amplification reaction is equal to or less than about 0.1 °C lower than the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature in the nucleic acid amplification reaction is substantially the same as the predicted melting temperature of the lowest melting temperature primer.
• the annealing temperature in the nucleic acid amplification reaction is the same as the predicted melting temperature of the lowest melting temperature primer.
• the first cycling stage comprises activating the enzyme used for amplification - such as a DNA polymerase - for a period of time followed by repeat cycles of melting and annealing/extension.
• the activation step is carried out at about 95 °C for between about 5 to 10 minutes, suitably about 10 minutes.
• the melting step is carried out at a temperature of about 95 °C for about 10 seconds.
• the annealing/extension is carried out a temperature of between about 58 °C and about 60 °C - such as at about 58 °C, 59 °C or 60 °C.
• the number of repeat cycles used in the first cycling stage can be at least about 5 cycles or at least about 10 cycles, or at least about 15 cycles, or at least about 20 cycles, or at least about 25 cycles, or at least about 30 cycles.
• One exemplary first cycling stage comprises an activation step at about 95 °C for about 10 minutes, followed by about 15 or more repeat cycles of about 95 °C for about 10 seconds and about 60 °C for about 60 seconds.
• the second cycling stage it is intended that efficient probe discrimination occurs to thereby give maximum discrimination between the probes which can ensure binding only to nucleic acid sequences with which they share 100% identity.
• probe discrimination is improved at higher predicted melting temperatures due to the fundamental kinetics of binding. At temperatures close to the effective melting temperature of the probe, bound probes are destabilised and a mismatch has a greater perturbing effect on binding and hence mis-probing on the wrong template is reduced.
• the second cycling conditions are predicated on the first stage cycles since it is understood that if a high annealing temperature was used from the outset, the nucleic acid amplification would probably fail to amplify efficiently leading to a failed amplification. The method is therefore understood to depend on the shifting thermodynamic properties of primer binding as the target copy number increases throughout the nucleic acid amplification reaction.
• the target copy number in the reaction is exponentially increased from the starting copy number.
• the target copy number present in the reaction following the first cycling stage will typically be between about 1 to 20 million copies (depending on the number of first stage cycles and the input copy number).
• the effective melting temperature of the primers will be greatly increased allowing efficient nucleic acid amplification at annealing temperatures far higher than the predicted melting temperature of the primers and far higher than would be possible at the start of the nucleic acid amplification reaction.
• the annealing temperature is higher than the predicted melting temperature of either the primers or the probe(s) which is understood to result in maximum probe discrimination without compromising the efficiency of the amplification.
• the annealing temperature used in the second cycling stage of the nucleic acid amplification reaction is equal to or greater than about 0.1 °C higher than the predicted melting temperature of the highest melting temperature probe.
• the annealing temperature used in the second cycling stage of the nucleic acid amplification reaction is equal to or greater than about 0.5 °C higher than the predicted melting temperature of the highest melting temperature probe.
• the annealing temperature used in the second cycling stage of the nucleic acid amplification reaction is equal to or greater than about 1 °C higher than the predicted melting temperature of the highest melting temperature probe.
• the annealing temperature used in the second cycling stage of the nucleic acid amplification reaction is equal to or greater than about 2 °C higher than the predicted melting temperature of the highest melting temperature probe.
• the annealing temperature used in the second cycling stage of the nucleic acid amplification reaction is equal to or greater than about 3 °C higher than the predicted melting temperature of the highest melting temperature probe.
• the annealing temperature used in the second cycling stage of the nucleic acid amplification reaction is equal to or greater than about 4 °C higher than the predicted melting temperature of the highest melting temperature probe.
• the annealing temperature used in the second cycling stage of the nucleic acid amplification reaction is equal to or greater than about 5 °C higher than the predicted melting temperature of the highest melting temperature probe.
• the higher annealing temperature in the second cycling stage is believed to be important for discrimination since this will lead to very stringent probe hybridising conditions wherein the probe can only hybridise to its complimentary template and subsequently be cleaved to produce a detectable signal.
• the second stage comprises repeat cycles of activation and annealing/extension at a temperature that is higher than in the first cycling stage.
• the melting step is carried out at a temperature of about 95 °C for about 10 seconds.
• the annealing/extension is carried out a temperature of greater than or equal to about 64 °C.
• the annealing/extension is carried out a temperature of between about 64 °C and about 66 °C - such as about 64 °C, 65 °C or 66 °C.
• the melting temperature is about 95 °C and the annealing/extension is carried out a temperature of between about 64 °C to about 66 °C.
• the melting temperature is about 95 °C and the annealing/extension is carried out a temperature of about 64 °C. In one embodiment, the melting temperature is about 95 °C and the annealing/extension is carried out a temperature of about 65 °C. In one particularly suitable embodiment, the melting temperature is about 95 °C and the annealing/extension is carried out a temperature of about 66 °C.
• the number of repeat cycles used in the second cycling stage can be at least about 5 cycles, or at least about 10 cycles, or at least about 15 cycles, or at least about 20 cycles, or at least about 25 cycles, or at least about 30 cycles, or at least about 35 cycles, or at least about 40 cycles, or at least about 45 cycles or at least about 50 cycles.
• One exemplary second cycling stage comprises 30 or more repeat cycles of about 95 °C for about 10 seconds and between about 64 °C and 66 °C for about 60 seconds.
• One exemplary second cycling stage comprises 30 or more repeat cycles of about 95 °C for about 10 seconds and about 64 °C for about 60 seconds.
• One exemplary second cycling stage comprises 30 or more repeat cycles of about 95 °C for about 10 seconds and about 65 °C for about 60 seconds.
• One exemplary second cycling stage comprises 30 or more repeat cycles of about 95 °C for about 10 seconds and about 66 °C for about 60 seconds.
• the first and second cycling stages may be carried out sequentially or substantially sequentially.
• amplification of nucleic acids by PCR involves repeated cycles of heat- denaturing the DNA, annealing two primers to sequences that flank the target nucleic acid segment to be amplified, and extending the annealed primers with a polymerase.
• the primers hybridize to opposite strands of the target nucleic acid and are oriented so that the synthesis by the polymerase proceeds across the segment between the primers, effectively doubling the amount of the target segment.
• each successive cycle essentially doubles the amount of target nucleic acids synthesized in the previous cycle.
• a typical conventional PCR thermal cycling protocol comprises 30 cycles of (a) denaturation at a range of 90°C to 95°C for 0.5 to 1 minute, (b) annealing at a temperature ranging from 50°C to 65°C for 1 to 2 minutes, and (c) extension at 68°C to 75°C for at least 1 minute.
• Other protocols including but not limited to universal protocol as well as fast cycling protocol can be performed.
• a variant of the conventional PCR is a reaction termed "Hot Start PCR". Hot Start PCR techniques focus on the inhibition of polymerase activity during reaction preparation.
• Hot Start PCR By limiting polymerase activity prior to PCR cycling, nonspecific amplification is reduced and the target yield is increased.
• Common methods for Hot Start PCR include chemical modifications to the polymerase, inhibition of the polymerase by a polymerase-specific antibody, and introduction of physical barriers in the reaction site to sequester the polymerase before the thermal cycling takes place.
• the reagents necessary for performing Hot Start PCR are conveniently packaged in kits that are commercially available.
• Another variation of the conventional PCR that can be performed with the subject probes is "nested PCR" using nested primers. The method is preferred when the amount of target nucleic acid in a sample is extremely limited for example, where archival, forensic samples are used.
• the nucleic acid is first amplified with an outer set of primers capable of hybridizing to the sequences flanking a larger segment of the target nucleic acid. This amplification reaction is followed by a second round of amplification cycles using an inner set of primers that hybridizes to target sequences within the large segment.
• Another variant is ligase chain polymerase chain reaction (LCR-PCR).
• LCR-PCR ligase chain polymerase chain reaction
• the method involves ligating the target nucleic acids to a set of primer pairs, each having a target-specific portion and a short anchor sequence unrelated to the target sequences. A second set of primers containing the anchor sequence is then used to amplify the target sequences linked with the first set of primers. Procedures for conducting LCR-PCR are well known in the art.
• the methods can exploit the 5' to 3' nuclease activity of a polymerase - such as when the polymerase is used in conjunction with PCR.
• a polymerase During extension of the primer, the polymerase encounters the probe hybridised to the target nucleic acid first displaces the 5' end of the probe and then degrades it.
• the probe(s) can be added concomitantly with the primer(s) at the start of PCR, and the signal generated from hydrolysis of the labelled nucleotide(s) of the probe provides a means for detection of the target nucleic acid sequence during its amplification.
• a method for determining the presence of a target nucleic acid in a sample comprising: (i) performing a nucleic acid amplification using a reaction mixture comprising: (a) a nucleic acid polymerase having a 5' to 3' nuclease activity; (b) one or more sets of forward and reverse primers capable of hybridising to the target nucleic acid; and (c) one or more or more oligonucleotide probes for detecting the target nucleic acid when present in the sample and wherein the oligonucleotide probe(s) has a predicted melting temperature that is higher then the predicted melting temperature of the one or more sets of forward and reverse primers(s); and wherein the cycling conditions during the nucleic acid amplification reaction comprise at least two different sets of cycling parameters, the first set of cycling parameters including an annealing temperature in the nucleic acid amplification reaction that is less than or about the same as the temperature corresponding to the predicted melting temperature of the
• a method for genotyping a sample comprising, or suspected of comprising two or more nucleic acid sequences that differ from each other by at least one base position, the method comprising: (i) performing a nucleic acid amplification using a reaction mixture comprising: (a) a nucleic acid polymerase having a 5' to 3' nuclease activity; (b) one or more sets of forward and reverse primers capable of hybridising to the nucleic acid; and (c) two or more oligonucleotide probes for detecting each of the two or more sequences when present in the amplified nucleic acid, wherein each of the oligonucleotide probes generate a different detectable signal, and wherein the two or more oligonucleotide probes have a predicted melting temperature that is higher then the predicted melting temperature of the primers; and wherein the cycling conditions during the nucleic acid amplification reaction comprise at least two different sets of cycling parameters, the first set of cycling parameters including an
• Non-limiting examples include DNA polymerases - such as E. coli DNA polymerase I, Thermus thermophilus (Tth) DNA polymerase, Bacillus stearothermophilus DNA polymerase, Thermococcus littoralis DNA polymerase, and Thermus aquaticus (Taq) DNA polymerase and polymerases extracted from the thermostable bacteria Thermus flavus, Thermus ruber, Thermus thermophilus, Bacillus stearothermophilus, Thermus lacteus, Thermus rubens, Thermotoga maritima, Thermococcus littoralis, and Methanothermus fervidus.
• DNA polymerases such as E. coli DNA polymerase I, Thermus thermophilus (Tth) DNA polymerase, Bacillus stearothermophilus DNA polymerase, Thermococcus littoralis DNA polymerase, and Thermus aquaticus (Taq) DNA polymerase
• Nucleic acid amplification can be performed with polymerases that exhibit strand-displacement activity (also known as rolling circle polymerization). Strand displacement can result in the synthesis of tandem copies of a circular DNA template, and is particularly useful in isothermal PCR reaction.
• Non-limiting examples of rolling circle polymerases include but are not limited to T5 DNA polymerase, T4 DNA polymerase holoenzyme, phage M2 DNA polymerase, phage PRDI DNA polymerase and Klenow fragment of DNA polymerase I.
• Nucleic acid amplification is generally performed with the use of amplification reagents.
• Amplification reagents typically include enzymes, aqueous buffers, salts, primer(s), probe(s), target nucleic acid, and nucleoside triphosphates.
• amplification reagents can be either a complete or incomplete amplification reaction mixture.
• the assay may be a homogeneous assay.
• a target nucleic acid is detected and/or quantified without the requirement of post-assay processing to record the result of the assay.
• a homogeneous PCR reaction can be carried out in a closed sample holder and no further addition or removal of reagents is necessary to record the result once the assay is started.
• Homogeneous assays allow recordation of the result of the assay in real time.
• the result of the assay can be continuously recorded as the assay progresses in time or recorded intermittently at one or more point during the assay or upon completion of the assay.
• homogeneous assays can be multiplexed, such that more than one target nucleic acid can be detected in one assay.
• a multiplex assay two or more specific nucleic acid probes, which differ in the nature of their covalently attached dyes, are added to the mixture to be assayed. The dyes are chosen to produce distinguishable signals from each specific nucleic acid probe. The signals of the different dye combinations of the nucleic acid probes can be recorded simultaneously to detect and/or quantify the corresponding target nucleic acids. Multiplexing greatly reduces the cost of analysis and can tremendously increase throughput in high volume settings.
• the method is a multiplexed amplification method.
• the method described herein can be used for the detection of low abundance target sequences in the presence of a high background of alternative substantially homologous sequence(s).
• a target sequence that is present at as little as about 5% of a mixed population of nucleic acid sequences can be accurately identified.
• the method described herein can be used to detect or determine mutations, including SNPs. Methods are provided that can detect a single mismatch or more between the probe sequence and a target nucleic acid sequence. Another application of the method described herein is for determining the genotype of a sample of nucleic acid at two or more different allelic sites.
• the two or more different allelic sites may be on a single strand of nucleic acid or may be on different strands of nucleic acid.
• the two or more different allelic sites may be amplified by a single amplification primer, for example when the allelic sites are on the same strand of nucleic acid and adjacent each other, or by multiple different amplification primers.
• this assay can be performed under competitive conditions, since multiple probes to the same allelic site can be present in the same reaction vessel.
• Part of the discrimination against a mismatch is that the probe that is perfectly matched functions to prevent the mismatched probe from binding because of the perfectly matched probe's stable hybridisation to the sequence being amplified.
• primers for use in nucleic acid amplification will depend on the target nucleic acid sequence. In general, the following factors are considered in primer design: a) each individual primer of a pair preferably does not self-hybridize in an amplification reaction; b) the individual pairs preferably do not cross-hybridize in an amplification reaction; c) the selected pair preferably have the appropriate length, and sequence homology in order to anneal to two distinct regions flanking the nucleic acid segment to be amplified; and d) the selected primers should have the predicted melting temperature.
• the primer sequence need not reflect the exact sequence of the target nucleic acid.
• a non-complementary nucleotide fragment may be attached to the 5 end of the primer with the remainder of the primer sequence being complementary to the target.
• non-complementary bases can be interspersed into the primer, provided that the primer sequence has sufficient complementarily with the target for annealing to occur and allow synthesis of a complementary nucleic acid strand.
• the oligonucleotide primers and/or probes can comprise DNA, RNA, single-stranded or double- stranded and any chemical modifications thereof, such as PNA and LNA.
• Modifications include, but are not limited to, those that provide other chemical groups that incorporate additional charge, polarisability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications include, but are not limited to, modified bases such as 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitution of 5-bromo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping. The presence of phosphorothioates and methylphosphonates is also contemplated.
• the primers have a predicted melting temperature range of between about 58 to 60 °C. In one embodiment, the primers have a predicted melting temperature of about 58, about 59 °C or about 60 °C.
• the assay utilises one or more labelled probes to identify a target nucleic acid.
• the label may be detetacable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. When two or more probes are used then each of the probes may be separately or distinctly detectable.
• the assay utilises one or more fluorescer-quencher probes to identify a target nucleic acid. In another embodiment, the assay utilises one or more fluorescer-quencher probes with spectrally distinct fluorophores to identify a target nucleic acid.
• the assay utilises combinations of fluorescer-quencher probes to identify members of a first set of two or more substantially homologous sequences that may be present, or suspected of being present, in a sample of nucleic acid and a second set of fluorescer-quencher probes in the same reaction to identify which members of a second set of two or more substantially homologous sequences are also present in the sample, or suspected of being present.
• Each of the fluorescer-quencher probes can have spectrally distinct fluorophores to separately identify the sets of sequences.
• the assay can be performed in a single reaction containing both the first and second sets of probes. The assay enables one to determine which members of the first set of substantially homologous sequences are present in the sample while simultaneously enabling one to determine which members of the second set of substantially homologous sequences are present in the sample.
• each of the two or more oligonucleotide probes is for distinguishing between two or more substantially homologous sequences, the two or more substantially homologous sequences differing from each other by at least one nucleotide, each probe in the set perfectly matching one of two or more substantially homologous sequences.
• the two or more oligonucleotide probes are allelic probes for genotyping a first allelic site and a second allelic site, suitably, wherein the probes differ from each other by at least one nucleotide.
• the probes each produce distinguishable signals or signatures - such as fluorescent signals or signatures for each of the different genotypes being detected.
• the number of probes that are used in the assay can vary depending upon the application of the method. Thus, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, or 20 or more probes can be used in each assay.
• One or more of the probes may differ by one or more nucleotides - such as two or more or three or more nucleotides.
• Each of the probes may differ by one or more nucleotides - such as two or more or three or more nucleotides. In certain embodiments, it may be desirable to determine only that a mutation is present rather than identifying the specific type of mutation.
• the method can be used in a multiplex assay format using multiple probes, each of which differs by at least one nucleotide.
• the probes produce the same or a different detectable signal.
• each of the probes produces the same or a different detectable signal.
• exemplary fluorescer-quencher probes are molecular beacons.
• Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore whose fluorescence is restored once bound to a target nucleic acid sequence.
• the loop portion is a probe sequence complementary to a target nucleic acid sequence.
• the stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence.
• a fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm.
• the stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer so that it is unable to fluoresce.
• the probe encounters a target nucleic acid sequence, it forms a hybrid that is longer and more stable than the stem and its rigidity and length preclude the simultaneous existence of the stem hybrid.
• the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart, and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence.
• the probe is molecular beacon.
• the probe dye and quencher are separated without the requirement for a polymerase enzyme with 5'-3' nuclease activity.
• Molecular beacons are described in WO90/03446.
• the method includes the step of treating said reaction mixture under conditions that allow for the hybridisation of the oligonucleotide probe(s) to the target nucleic acid thereby resulting in the production of a detectable signal.
• the assay involves the digestion of an oligonucleotide probe containing a fluorescer and quencher during a nucleic acid amplification reaction to evidence nucleic acid amplification.
• the oligonucleotide probe can include at least one fluorescer and at least one quencher.
• the polymerase encounters the probe hybridised to the target nucleic acid and degrades the probe via the nuclease activity of the polymerase.
• digestion of the probe results in release of the fluorescer (or quencher) from the probe. This causes the fluorescer and quencher on the probe to become spatially separated from each other, thereby creating a change in fluorescence in the sample to indicate the extension of the primer and hence the amplification of the target nucleic acid.
• the fluorescers are fluorescent organic dyes derivatised for attachment to the terminal 3' carbon or terminal 5' carbon of the probe via a linking moiety.
• Methods also exist for linking both fluorescers and quenchers to internal moieties within the oligonucleotide.
• quencher molecules can include organic dyes, which may or may not be fluorescent. Whether the quencher molecule is fluorescent or simply releases the transferred energy from the fluorescer by non-radioactive decay, the absorption band of the quencher should substantially overlap the fluorescent emission band of the fluorescer.
• the amplification reaction can be carried out as an automated process.
• the present invention also provides a reaction mixture useful for amplification of a target nucleic acid target comprising: (i) a nucleic acid polymerase, suitably, having a 5' to 3' nuclease activity;
• oligonucleotide probe(s) for detecting the target nucleic acid when present in the amplified nucleic acid, wherein the oligonucleotide probes generate a detectable signal, and wherein the oligonucleotide probes have a predicted melting temperature that is higher than the predicted melting temperature of the primers; and optionally (iv) reagents and buffers required to carry out the amplification reaction.
• the present invention also provides a kit useful for amplification of a target nucleic acid comprising: (i) a nucleic acid polymerase, suitably, having a 5' to 3' nuclease activity; (ii) one or more sets of forward and reverse primers capable of hybridising to the target nucleic acid; and
• oligonucleotide probes for detecting the target nucleic acid when present in the amplified nucleic acid, wherein the oligonucleotide probes generate a detectable signal, and wherein the oligonucleotide probes have a predicted melting temperature that is higher than the predicted melting temperature of the primers; and optionally (iv) reagents and buffers required to carry out the amplification reaction.
• the reaction mixture or the kit includes at least two sets of probes where each set of probes is for distinguishing between two or more substantially homologous sequences, the two or more substantially homologous sequences differing from each other by at least one nucleotide, and each probe in the set perfectly matching one of two or more substantially homologous sequences, and wherein the oligonucleotide probes have a predicted melting temperature that is higher than the predicted melting temperature of the primers.
• the reaction mixture or the kit includes at least a first set of allelic probes for genotyping a first allelic site and second set of allelic probes for genotyping a second allelic site.
• Each set of probes includes at least two probes which are capable of hybridizing to the allelic site but differ from each other by at least one nucleotide. The at least two sets of probes are selected so as to produce distinguishable fluorescence signatures for the different genotypes being detected.
• the reaction mixture or kit may also include sample nucleic acid which can serve as a control in the assay.
• the reaction mixture or kit may also include a fluorescent material for use as a passive internal standard.
• the reaction mixture or kit may also include buffer or other reagents for performing the method.
• Reagents can be supplied in a solid form or dissolved/suspended in a liquid buffer suitable for inventory storage, and later for exchange or addition into the reaction mixture when the hybridization assay is performed.
• Suitable individual packaging is normally provided and instructions for users are generally supplied as well. Diagnostic or prognostic procedures using the kits of this invention can be performed by clinical laboratories, experimental laboratories, practitioners, or private individuals.
• melting temperatures are derived from the "Beacon Designer version 6.02" software package (Premier Biosoft, Palo Alto, CA). The melting temperature calculations within this software are based on the nearest neighbour thermodynamic theory using SantaLucia values (SantaLucia, J., Jr. (1998) "A Unified View of Polymer, Dumbbell, and Oligonucleotide DNA Nearest-neighbour Thermodynamics", Proc. Natl. Acad. Sci. U.S.A. 95, 1460-1465.)
• Real time PCR reactions are performed on an ICycler IQ5 or an lllumina ECO thermocycler. Each assay contains 300nM (final concentration) of forward and reverse primer as well as 300nM (final concentration) of both the wild type (WT) specific probe and the SNP specific probe.
• the PCR reaction is driven by the use of 0.5 Units of thermostable Taq DNA polymerase in an appropriate buffer containing 5mM MgCI 2 . dNTPs are included at a final concentration of 0.15mM each.
• Target DNA is added as either synthetic vector template (present at 10 4 copies per reaction) or as gDNA at 10ng per reaction.
• the nucleic acid primers and probes used are shown in Table 1 . Cycling protocols are as follows: Standard cycling conditions:
• FIG. 6A depicts that genotype quantification can be achieved through analysis of end point fluorescence since the fluorescent signal output is directly related to the number of probe binding and cleavage events.
• the false probe signal on its non-cognate channel is observed at 60°C (see Figure 6A and B) but not when using the two stage cycling protocol (see Figures 6B and 4D). This allows considerably improved detection limits down to 5% of the starting sample.
• Figure 6B is an enlarged section of Figure 6A where at 60°C no discrimination between false signal and low abundance signal can be made whereas the use of low melting temperature probes with two stage cycling conditions gives very clear genotype identification when the template is present at a frequency of 5% of the starting sample - see Figure 6B.
• unbroken line 100% probe target template; dotted line: 100% non cognate sequence; dashed and dotted lines: fluorescent signal from probe target template present at 5% of starting DNA sample.

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