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WO2007035697A1 - Utilisation de pcr en temps réel pour la détection de l'expression allélique - Google Patents

Utilisation de pcr en temps réel pour la détection de l'expression allélique Download PDF

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Publication number
WO2007035697A1
WO2007035697A1 PCT/US2006/036411 US2006036411W WO2007035697A1 WO 2007035697 A1 WO2007035697 A1 WO 2007035697A1 US 2006036411 W US2006036411 W US 2006036411W WO 2007035697 A1 WO2007035697 A1 WO 2007035697A1
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Prior art keywords
allele
rna
expression
transcripts
gene
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Marjan Huizing
Enriko Klootwijk
Paul J.M. Savelkoul
Carla Ciccone
William Gahl
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US Department of Health and Human Services
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US Department of Health and Human Services
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    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates, e.g. , to a method for simultaneously detecting the levels of expression of a plurality of alleles of a gene of interest.
  • the method comprises the use of a plurality of distinguishable allele-specific reporter probes and real time PCR amplification kinetics. Kits for performing such assays and diagnostic methods are also disclosed.
  • allelic variation in gene expression is common in eukaryotic, including human, genomes. See, e.g., Lo et al. (2003) Genome Research 13 . , 1855-1862.
  • the phenomenon of imprinting, in which one allele is suppressed early during development while the other is expressed, is widespread. It is of interest to study experimentally the occurrence and the mechanisms of this and other types of allelic variation in non-pathological tissues.
  • a variety of disease conditions appear to be mediated by, or accompanied by, aberrant expression, such as over-expression, of one allele (often a mutant) of a gene. Such differences in allelic expression can serve as the basis for diagnostic tests for those conditions.
  • the ability to specifically silence the expression of detrimental alleles with agents such as siRNAs provides both a method to study the role of the allelic expression in the disease phenotype and, in some cases, a therapeutic method to treat the disease. It would be desirable to have a method to measure such differences in allelic expression, as well as to validate an agent for its ability to specifically and efficiently silence the expression of an allele of interest.
  • RFLP restriction fragment length polymorphism analysis
  • Figures 1A-1E show a validation of the allelic discrimination of dual-probe GNE Taqman assays.
  • endpoint fluorescent readings Rn, normalized reporter signal
  • ntc no template control
  • Fig. IA c.735C>T heterozygous in HIBM patient Hl
  • Fig. IB c.403G>T heterozygous in HIBM patient Hl
  • Fig.1C c.646T>C heterozygous in HIBM patient H2
  • Fig. ID C.2018OT heterozygous in HIBM patient H2
  • Fig. IE c.787G>T heterozygous in sialuria patient Sl.
  • Figures 2A-2C show standard regression curves for quantifying mutation-dependent allelic expression of GNE.
  • FIG. 3 A illustrate the Sialic acid pathway, GNE defect and siR ⁇ A design Fig. 3 A: Intracellular sialic acid metabolism.
  • the synthesis of sialic acid (Neu5 Ac) is initiated in the cytosol, where glucose undergoes several modifications to eventually become sialic acid.
  • the UDP-GIcNAc 2-epimerase (GNE)/ ManNAc kinase (MNK) enzyme is the central and rate-limiting enzyme in this cytosolic process.
  • GNE activity is feedback inhibited by the downstream product CMP-sialic acid.
  • Sialic acid is converted in the nucleus to CMP-sialic acid, which is utilized by the Golgi complex to sialylate oligosaccharides (OGS).
  • Fig.3B Schematic of the GNE gene structure (not to scale) and the allosteric site residing in exon 5, which codes for the epimerase enzymatic domain of GNE.
  • Sialuria patient S2 (Ferreirae ⁇ /. (1999) MoI Genet Metab 67, 131-7) presented with mildly coarse facies, slight motor delay, and urinary excretion of large quantities (>1 gram/day) of free sialic acid.
  • Fig. 3C Two siRNAs were designed, one targeting the normal allele (siRNA normal) and one specifically targeting the mutant allele (siRNA mutant).
  • the upper sequence is SEQ ID NO:23.
  • the lower sequence is SEQ ID NO:24.
  • Fig.3E Patients fibroblasts were transfected with siRNA (mock, normal and mutant) and cultured for 48 hours, after which total RNA was isolated and subjected to the quantitative real-time PCR assay. 2 ⁇ Ct values were determined and extrapolated on the regression curve to A-allele/G-allele ratios for both siRNA constructs. Transfection with a mock siRNA did not result in a significant difference in allelic expression.
  • This invention relates, e.g. , to a method for simultaneously determining, in a single container (such as a tube), the relative amounts of a plurality of different KNA transcripts expressed from a gene of interest.
  • the different RNA transcripts maybe, e.g., RNAs expressed from different alleles of a gene of interest (e.g. from two alleles, wherein one copy is wild type and the other has a mutation or other variant sequence); splice variants; RNAs expressed from variant virus species; etc.
  • One of the transcripts may be the wild type counterpart of a variant of interest (e.g., a wild type splice form, or a wild type counterpart of an allelic mutation).
  • the different RNA transcripts contain "defining features," which are nucleic acid sequences that distinguish a variant form of an RNA transcript from its wild type counterpart, or from another RNA variant.
  • the defining feature can be, e.g., the site of an allelic variation, such as a mutation; a variant splice site; or the like.
  • allelic variation such as a mutation
  • variant splice site or the like.
  • gene refers to all forms of a gene, including allelic variants.
  • the method employs real-time PCR amplification in the presence of reporter probes which are specific for each of the RNA transcripts and which are distinguishable from one another (e.g. comprise different fluorophores).
  • a probe that is "specific for" an RNA transcript binds preferentially to that RNA transcript, in comparison to another RNA transcript, e.g. an RNA transcript expressed from the same gene (e.g., a wild type sequence, another allelic variant or another splice variant).
  • RNA transcript RNA transcript
  • reverse transcriptase reverse transcriptase
  • cDNA RNA transcript
  • reverse transcriptase reverse transcriptase
  • cDNA RNA transcript
  • quencher quencher
  • the fluorophore maybe on the 5' end and the quencher on the 3' end, or vice-versa.
  • Much of the discussion herein is directed to probes with the fluorophore on the 5' end and the quencher on the 3 1 end; but it is to be understood that probes with the fluorophore on the 3 ?
  • each sequence-specific (e.g. allele-specific) probe contains a different fluorophore, so the expression of the plurality of nucleic acid transcripts (e.g. allelic variants) can be detected independently.
  • the expression "comprising perfo ⁇ ning real time PCR” generally includes a step of converting the RNA to DNA before PCR amplification of the DNA (e.g., a method of reverse- transcriptase (RT)-PCR).
  • the assay is designed such that the labeling moieties on the 5' and/or 3' ends of each probe do not fluoresce unless PCR amplification of the sequence to which the probe binds has occurred, followed by hybridization of the probe to the amplified sequences, in which case fluorescence of the probe can be seen.
  • the labeling moieties on both ends of a probe are fluorescent molecules, which quench one another.
  • the labeling moiety on one end is sometimes referred to herein as a "fluorophore," and the labeling moiety on the other end ⁇ e.g., the 3' end) as a "quencher.”
  • the probe molecule is flexible and folds back partially on itself, so that the quencher and the fluorophore are close together; the quencher thus prevents the probe from fluorescing.
  • the two labeling moieties are close enough to one another to quench each other.
  • the released fluorescent emission is measured continuously during the exponential phase of the PCR amplification reaction. Since the exponential accumulation of the fluorescent signal directly reflects the exponential accumulation of the PCR amplification product, this reaction is monitored in real time (“real time PCR").
  • the relative amounts of the plurality of RNA transcripts are measured by comparison to a standard curve.
  • a standard curve is generated by mixing isolated DNAs representing each of the plurality of RNA transcripts (rather than using genomic DNA) in a series of ratios, then amplifying those mixtures by real time PCR in the presence of variant-specific probes.
  • Advantages of the method of the invention include that it is rapid, sensitive and accurate, can be performed simply and conveniently in one vessel ⁇ e.g., tube), and requires only small quantities of RNA ⁇ e.g., less than about 25 ng).
  • the method can be applied to small tissue samples ⁇ e.g. biopsies and tumor samples), and can be readily miniaturized and/or adapted for use in a high throughput format.
  • the method can be used for a wide variety of applications, including research purposes ⁇ e.g. to study X-chromosomal inactivation, genetic imprinting, epigenetics, dominant disorders, and cancer gene expression); to validate allele-specific inhibition ⁇ e.g. allelic silencing by RNAi techniques); and in diagnostic methods ⁇ e.g.
  • One aspect of the invention is a method for quantitating (quantifying) the relative amounts of a plurality of different RJSTA transcripts expressed from a gene of interest in a subject or cell, comprising performing real time quantitative PCR, in a single container, of cDNAs of the transcripts, in the presence of probes specific for each of the different transcripts ⁇ i.e., specific for a defining feature of each transcript), wherein the probes specific for each of the different transcripts are labeled with distinguishable fluorophores.
  • the RNA transcripts can be, e.g., RNAs expressed from two or more alleles of a gene of interest ⁇ e.g.
  • RNA viral genomes from related species can also be detected by methods of the invention.
  • the relative amounts of the different transcripts is quantitated by comparison to a standard curve generated from isolated DNAs representing each of the plurality of transcripts.
  • TherPCR is carried out by selecting PCR primers which flank a defining feature of the RNA transcript (or a cDNA thereof) (i. e., a feature which distinguishes a variant RNA from its wild type counterpart, or from other RNA variants).
  • the PCR primers can flank the site of a mutation which distinguishes an allelic variant from its wild type counterpart.
  • the PCR primers can flank the splice site of an alternatively spliced variant.
  • the flanking PCR primers are selected such that they amplify a suitable length of the cDNA template. Factors to be considered for determining a suitable amplicon length are discussed elsewhere herein.
  • a preferred embodiment of the invention is a method for quantitating the relative amounts of the two transcripts expressed from two alleles of a gene of interest (allelic RNAs) in a subject or cell, comprising performing real time quantitative PCR, in a single container, of cDNAs of each of the transcripts, in the presence of probes which are specific for each of the two allelic transcripts, wherein the probes for each transcript are labeled with distinguishable fluorophores, and wherein the relative amounts of expression of the two transcripts is determined by comparison to a standard curve generated from isolated DNAs representing each of the two alleles.
  • a cDNA copy is generated from RNA expressed from each of the alleles, and the cDNA is amplified by real time PCR.
  • the two alleles differ by a single nucleotide.
  • a standard curve is used to quantitate the relative amounts of the plurality of RNA transcripts.
  • the curve may be generated by various methods, each of which employs isolated DNAs representing the RNA transcripts (one of which may be a wild type counterpart of a particular variant of interest).
  • An isolated DNA which "represents" an RNA transcript is a DNA which comprises a defining feature of the transcript, or a complete complement thereof.
  • the DNA may be a full-length clone of a gene, or it may be a fragment of the gene, provided that the fragment comprises the defining feature of the transcript, such as a mutation or other allelic variation.
  • the standard curve is generated by expressing RNA from cloned DNA encoding each of the RNA transcripts; generating cDNA from each of the RNAs; and amplifying mixtures comprising different ratios of the cDNAs by real time quantitative PCR in the presence of distinguishable, variant-specific probes.
  • the PCR primers and probes for amplifying and detecting a DNA representing an RNA transcript of interest are the same as those used to amplify and detect the variant transcript which is being quantitated.
  • non-mammalian cDNA such as insect or E.
  • the standard curve is generated by providing mixtures comprising different ratios of isolated DNAs representing each of the RNA transcripts.
  • amplicons as described above which comprise defining features of the RNA transcripts can be used.
  • each amplicon can be cloned and then excised for use in preparing a standard curve.
  • the mixtures of DNA are then amplified by real time PCR in the presence of distinguishable, variant-specific probes.
  • kits for quantitating the relative amounts of RNA expression from a plurality of alleles of a gene of interest may be a wild type counterpart of a variant(s) ⁇ e.g., a wild type splice form, or a wild type counterpart of an allelic mutation).
  • the kit comprises one or more of the following elements:
  • reagents for reverse transcribing the RNAs expressed from the alleles to produce cDNAs (e.g., reverse transcriptase and, optionally, suitable buffers, dNTPs, or the like);
  • primers and fluorophore-containing probes for amplifying and detecting the cDNAs, wherein the primers and probes are preferably in a single container;
  • c the components for generating a standard curve of the invention, comprising varying ratios of isolated DNAs representing each of the plurality of alleles.
  • the gene of interest is mammalian
  • DNAs used to generate the standard curve are generated by reverse transcribing RNAs expressed from the alleles
  • non-mammalian cDNA may be included in the kit to be included in this reverse transcription reaction.
  • a method of the invention may be used for a variety of applications.
  • a method of the invention can be applied to validating a method for specifically inhibiting expression of an allele of interest, e.g. using an siRNA.
  • One aspect of the invention is a method for detecting the inhibition of RNA expression in a cell or subject from a first allele of a gene by a putative inhibitor of gene expression which is specific for that first allele (an "allele- specific" inhibitor or silencer), e.g. an siRNA, compared to the expression from a second allele of the gene, using a method of the invention to quantitate the relative expression of the two alleles in the presence ofthe putative inhibitor.
  • the amount of expression of each allele is determined by comparison to a standard, curve generated from isolated DNAs representing each ofthe two alleles. An illustration of such a method is shown in Example IV and Figure 3.
  • Kits for validating that an siRNA is specific for an allelic variant of interest and that it is able to inhibit ⁇ e.g., silence) expression of that allele are also included.
  • the primers and fluorophore-containing probes are selected so as to amplify and detect cDNA copies of RNAs expressed from the allele of interest, e.g. in the presence or absence of the siRNA.
  • reagents for reverse transcribing the RNA are present in the kit.
  • the kit can be sold, for example, in conjunction with the siRNA which is to be validated.
  • the RNAi to be validated can be included as part ofthe kit.
  • a method ofthe invention can be used as a diagnostic assay.
  • One aspect of the invention is a method for detecting an autosomal-dominant disorder (a disorder which results from a mutation in a first allele, which causes reduced expression from that allele compared to the expression of a wild type allele) in a subject, comprising performing real time quantitative PCR, in a single container, of cDNA copies of RNA expressed from each ofthe mutant and the wild type alleles, in the presence of a distinguishable allele-specific probe for each ofthe two alleles, by a method ofthe invention.
  • An example of such a method is illustrated in Example m and Figure 2C.
  • Another aspect of the invention is a method for detecting a recessive disorder ⁇ e.g., a compound heterozygous autosomal recessive disorder) which is characterized by differential expression of more than one RNA from a gene of interest, in a subject, comprising performing real time quantitative PCR, in a single container, of a cDNA of each of the RNAs, in the presence of distinguishable probes specific for each RNA, by a method of the invention.
  • An example of such a method is illustrated in Example III and Figures 2 A and 2B.
  • An X-linked disorder either dominant or recessive, can also be detected by such a method.
  • Another aspect of the invention is a method for detecting a disorder mediated by an alternately spliced RNA from a gene of interest, in a subject, comprising performing real time quantitative PCR, in a single container, of cDNA copies of the alternately spliced RNA and the wild type RNA, in the presence of a probe specific for each of the RNAs, by a method of the invention.
  • This invention relates, e.g. , to a method for quantitating the relative amounts of a plurality of different RNA transcripts from a gene of interest in a subject or cell.
  • a gene of interest includes two or more genes.
  • the RNA transcripts can take any of a variety of forms.
  • a method of the invention can be used to distinguish among two or more known splice variants (including alternative splice variants) transcribed from a gene of interest.
  • PCR primers are designed which extend across unique exons or fused exon borders of the splice variants.
  • the probes used to detect the amplified DNA contain, at their 5' and 3' ends, a fluorophore and a compatible quencher, such that unique, distinguishable fluorophores are present on the probes for each of the splice variants to be quantitated.
  • a fluorophore and a compatible quencher such that unique, distinguishable fluorophores are present on the probes for each of the splice variants to be quantitated.
  • aberrant isoforms are translated from aberrant splice variants.
  • a method of the invention can be used to study the alternative splicing experimentally, and/or to diagnose the presence of a disease condition characterized by alternative splicing, such
  • Another embodiment of the invention is a method to distinguish among transcripts of two or more closely related, but distinguishable, infective organisms, such as microorganisms.
  • a method of the invention can be used as a diagnostic assay to detect expression of multiple variants of HIV or other viruses.
  • Another embodiment of the invention is a method to distinguish among the RNA genomes of closely related, but distinguishable, infective organisms, such as mutant viruses or virus species.
  • a preferred embodiment of the invention is a method to quantitate the relative expression from a plurality of (e.g. two) alleles of a gene of interest in a subject or cell.
  • the method may be used to compare the levels of expression of two alleles (e.g. a wild type and a mutant allele), or it may be used to compare the levels of expression of three or more alleles. The latter may occur, for example, if RNA is expressed from a pseudogene as well as from the full-length allele.
  • two alleles e.g. a wild type and a mutant allele
  • non-limiting disease conditions characterized by differential allelic expression from pseudogenes and/or full length genes include Gaucher disease and congenital adrenal hyperplasia. Expression from three or more alleles can also occur, for example, in a sample from a patient, such as a blood sample, which contains different cells, which, taken together, contain three or more expression products of allelic forms of a gene of interest.
  • Much of the discussion herein is directed to comparing (quantitating) the relative expression from a plurality of alleles. It is to be understood that this discussion also relates to comparing (quantitating) the relative amounts of other types of RNA transcripts expressed from a gene of interest, such as splice variants.
  • RNA transcripts whose expression is quantitated comprise nucleic acid sequence variants which differ in at least one base within a binding site for a specific probe.
  • RNA allelic variants comprise the same nucleic acid sequence variation as the DNAs from which they are transcribed (alleles), wherein T residues in the DNA are comparable to U residues in the RNA. Therefore, a probe that is specific for an allelic RNA of interest is also specific for the DNA from which the allelic RNA is expressed, or for a cDNA copy of the RNA.
  • a probe that is specific for an RNA expressed from a gene of interest (e.g., an allelic expression product) will form a match with one sequence variation but a mismatch with the other(s).
  • a different allele-specific probe is used for each allele whose expression is to be measured. These probes form a match only with the allele they are specific for, but form mismatches with all the other alleles.
  • RNA transcripts may differ from one another by a single base, two or more non- contiguous bases, a deletion, an insertion, an inversion, combinations thereof, etc. Such differences comprise the defining features of the alleles.
  • a method of the invention can readily distinguish between expression from alleles which differ from one another by only a single base mismatch.
  • allele is not limited to variants of a gene in a diploid DNA genome, but is used in a broader manner.
  • the term allele additionally comprises a variety of other nucleic acid sequences with point mutations, deletions, insertions, etc. of one or more base pairs, such as mitochondrial DNA (mtDNA), messenger RNA (mRNA), viral DNA or RJSTA genomes, or DNA of microorganisms, including viruses. Consequently, the method of the present invention is applicable for the quantification of nucleic acid species in a variety of systems.
  • a method of the invention can be used to detect a variety of RNAs other than mRNA, including, e.g., tRNA, rRNA, microRNAs, etc.
  • RNAs can be "gene products" of a gene of interest.
  • RNA franscripts which are quantitated by a method of the invention can be expressed from a gene of interest in a subject or cell, or they can be expressed in a cell-free system.
  • allelic variants or splice variants from two or more genes is assayed simultaneously.
  • a disease condition such as a cancer
  • expression from the alleles of both of the genes can be analyzed simultaneously in a sample taken from a subject (e.g. a patient).
  • Disease conditions where such an assay can be employed include multigenic cancers (see, e.g., Tayebi et al. (2003) Am. J. Hum. Genet. 72, 519-534; Felix-Lopez et al. (2003) J. Pediatr. Endocrinol. Metab.
  • PCR primers are selected which will amplify a segment of a nucleic acid containing the "defining feature" of an RNA transcript (e.g., the site of an allelic variation, a variant splice site, or the like).
  • Typical amplicons range in size from about 50 base pairs (bp) to about 1200 bp, preferably from about 50 to about 150 bp. (All ranges used herein include the end points of the range.) A skilled worker will recognize how to select an amplicon of an appropriate size.
  • Factors to take into account include the possibility that too long a sequence may include polymorphisms other than particular sequence difference of interest, giving rise to reduced specificity compared to a shorter sequence; and that relatively short sequences can be amplified and analyzed more rapidly than relatively long sequences.
  • Methods for designing PCR primers and for carrying out PCR reactions are conventional and can be optimized readily by one of skill in the art. See, e.g. , Innis et al., editors, PCR Protocols (Academic Press, New York, 1990); McPherson et al, editors, PCR: A Practical Approach, Volumes 1 and 2 (IRL Press, Oxford, 1991, 1995); Barany (1991) PCR Methods and Applications I, 5-16; Diffenbach et al., editors, PCR Primers, A Laboratory Manual (Cold Spring Harbor Press); etc.
  • a preferred DNA polymerase is the Taq polymerase isolated from Thermus aquaticus. hi addition to the wild type enzyme, many variants of Taq are available, including Hot-start Taq, Hi fidelity Taq, Platinum Taq, and many others. Furthermore, many Taq-related enzymes which exhibit the desired properties can be used, including Pfu (e.g., Pfu Ultra, Pfu Turbo, Hot start Pfu) and Herculase. Suitable polymerases are available from commercial sources, such as Stratagene (La Jolla, CA), Sigma- Aldrich (St.
  • the amplification need not be carried out by a thermostable DNA polymerase (PCR).
  • PCR thermostable DNA polymerase
  • Other DNA polymerases may be used, which operate at lower temperatures, provided that the polymerase is strand-displacing and is associated with a 5'->3' exonuclease activity, and that the amplification can be measured in real time.
  • ligase based amplification schemes such as ligase chain reaction (LCR); Q-beta replicase-based amplification schemes; and strand displacement amplification (SDA) schemes.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • types of strand displacing DNA polymerases which can be used in methods of the invention are: M2 DNA polymerase; VENTTM DNA polymerase; Klenow fragment of DNA polymerase I; T5 DNA polymerase; modified T7 DNA polymerase; SequenaseTM; and T4 DNA polymerase holoenzyme.
  • PCR reactions of the invention are carried out in a single reaction mixture (reaction buffer), in a single reaction chamber (e.g. , container, well of a plate, etc.), and/or in a single thermocycling protocol.
  • reaction buffer e.g. , container, well of a plate, etc.
  • thermocycling protocol e.g., thermocycling protocol
  • Any of a variety of reaction chambers can be used, in any of a variety of formats, provided that the material is compatible with the detection of fluorescence.
  • Suitable "optical" plasticware is available from several distributors, including Applied Biosystems (Foster City, CA), Axygen, and Bio Rad.
  • Preferable containers can be closed to form a leak-proof seal, in order to reduce or prevent cross-contamination of samples.
  • Suitable formats for performing the PCR reactions include computer-controlled thermal cyclers with a fluorescence detectors, e.g., ABI7300, ABI7500, ABI7900 (Applied Biosystems, Foster City, CA), Biorad iCycler (Biorad, Hercules, CA), Mx3000P (Stratagene, La Jolla, CA), Lightcycler (Roche, Indianapolis, IN), and Smart Cycler (Cepheid, Sunnyvale, CA).
  • the sequences of interest are amplified in the presence of distinguishable probes specific for each of the RNA transcripts whose expression is to be measured.
  • the probes have a fluorophore, sometimes referred to herein as a "reporter,” at one end but do not fluoresce when free in solution because they have a "quencher” at the other end that absorbs fluorescence from the reporter.
  • the polymerase e.g. Taq
  • the polymerase encounters a probe specifically base-paired with its target and unwinds it.
  • the polymerase cleaves the partially unwound probe and liberates the reporter fluorophore from the quencher, thereby increasing net fluorescence.
  • the presence of two probes, each labeled with a different fluor allows one to detect both variants (e.g. alleles) in a single tube. Because only perfect double stranded nucleic acids are cleaved by the exonuclease, the method can readily distinguish between RNA transcripts that do or do not bind to a given probe.
  • variant-specific probe or allele-specific probe refer to a probe which binds specifically to a predetermined sequence of a variant or allele.
  • a probe which binds "specifically” to a sequence binds selectively to that species (e.g., hybridizes to it, or duplexes with it) in comparison to its binding to other sequences, e.g. when the target sequence is present in a preparation comprising other alleles expressed from the gene of interest, splice variants expressed from the gene of interest, or other, unrelated sequences.
  • the phrases "specific for” or “specific to” a polynucleotide have a functional meaning that the polynucleotide can be used to identify the presence of one or more target genes in a sample and distinguish them from non-target genes. It is specific in the sense that it can be used to detect polynucleotides above background noise ("non-specific binding").
  • a specific sequence is a defined order of nucleotides which occurs in the polynucleotide, e.g., in the nucleotide sequence of an allelic variant or splice variant of interest, and which is characteristic of that sequence, and substantially no non-target sequences.
  • the probe can be of any size which is necessary to confer specificity, e.g. about 10, 15, 20, 25 or more nucleotides. In the present case, the probe binds specifically to the variant for which it is designed under the ' conditions of PCR amplification used in a method of the invention.
  • the probes used in methods of the invention are designed so that they cannot be extended in the 3 ' direction by the polymerase in the reaction.
  • the quenchers bound to the 3 ' end of a probe may prevent it from being extended.
  • the probe and primers generally bind at different temperatures. In general, the probe binds in the range of about 65-94 ° C, while the primers bind from about 55-65 ° C, depending on their sequence. See, e.g., the discussion in the Applied Biosystems manual (KJ. Livak, ABI Prism 7700 Sequence Detection System, User Bulletin no. 2, PE Applied Biosystems, 1977).
  • a minor groove binding protein may be attached to a probe; this assists in more efficient binding of the probe and will help to bring the annealing temperature of the probe within PCR conditions. Furthermore, by using a minor groove binder, probe sequences can be shortened.
  • the labeling moieties at the 5' and 3' termini of the specific ⁇ e.g., allele- specific fluorophore-quencher reporter oligonucleotide probes a wide variety of conventional fluorophores can be used. A skilled worker will recognize sets of spectrally resolvable fluorophores which emit at different frequencies and thus can be readily distinguished.
  • a plurality of fluorescent probes can be used. For example, three or more nucleic variants can be quantitated simultaneously. When quantitating three or more variants, the conditions can be optimized to reduce potential problems with specificity and cross hybridization, using conventional procedures.
  • Suitable fluorescer-quencher dye sets will be evident to the skilled worker. Some examples are described, e.g., in Holland et al. (1991) Proc. Natl. Acad. Sci. 88, 7276-7280; WO 95/21266; Lee et al. (1993) Nucleic Acids Research 21, 3761-3766; Livak et al. (1995), supra; U.S. Pat. No. 4,855,225 (Fung e? ⁇ J); U.S. Pat. No. 5,188,934 (Menchen ef ⁇ /.); PCT/US9O/O5565 (Bergot et al. ⁇ and others.
  • Suitable fluorophores include rhodamine dyes and fluorescein dyes, including, e.g., fluorescein; 6-carboxyfluorescein (FAMTM), 2',4',5',7',-tetracliloro-4,7-dichlorofluorescein(HEXTM), 2',7'-dimethoxy-4',5 '-6-carboxyrhodamine (JOETM), N',N',N',N'-tetramethyl-6-carboxyrhodamine (TAMRATM) and 6-carboxy-X-rhodamine (ROXTM).
  • fluorescein fluorescein
  • FAMTM 6-carboxyfluorescein
  • HEXTM 2',4',5',7',-tetracliloro-4,7-dichlorofluorescein(HEXTM)
  • HEXTM 2',7'-dimethoxy-4',5 '-6
  • dyes which can be used include TETTM; VICTM; Texas Red®, Cy3TM, Cy5TM, SYBR ⁇ GreenI, NEDTM, CAL Fluor Orange 560, BHQ-I, and others.
  • Suitable pairings include, e.g., FAMTM/R0XTM; FAMTM/SYBR® Green I; VIC@/JOETM; NEDTM/TAMRATM/ROX HEXTM FAMTM/SYBR® Green I; V1C®/JOETM; NEDTM/ TAMRATM/ Cy3TM; ROXTM/Texas Red®; Cy5TM dyes; and CAL Fluor Orange 560/ BHQ- 1.
  • These and other suitable dyes are available commercially, e.g. from ⁇ nvitrogen (Carlsbad, CA), Applied Biosystems (Foster City, CA), Biosearch Technologies (Novato, CA), and others.
  • RNA is generated from a DNA clone of each RNA transcript to be quantitated ⁇ e.g.
  • allelic or splice variants preferably in vitro; the RNA is converted to cDNA; and the cDNAs are mixed in a series of ratios and amplified by real time PCR in the presence of probes that are specific for the RNA transcripts, as described above.
  • cloned DNAs comprising the defining (distinguishing) features of each allele are optionally amplified ⁇ e.g. by PCR) and are mixed in a series of ratios and amplified by real time PCR in the presence of specific probes as described above.
  • an "isolated" DNA refers to a DNA molecule ⁇ e.g. a polynucleotide or oligonucleotide) which is removed from its original environment (e.g. , the natural environment if it is naturally occurring), and is isolated or separated from at least one other component with which it is naturally associated.
  • a naturally-occurring polynucleotide present in its natural living host is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a composition, and still be isolated in that such composition is not part of its natural environment.
  • An isolated DNA representing an RNA transcript of interest e.g.
  • RNA transcript a full-length gene or cDNA, or a fragment thereof which comprises a defining feature of the RNA transcript
  • a full-length gene or cDNA, or a fragment thereof which comprises a defining feature of the RNA transcript can be prepared by PCR-amplifying and/or cloning the sequence, synthesizing it chemically, or other methods which will be evident to a skilled worker.
  • An "isolated" sequence, as used herein, is to be distinguished from that sequence when it is present in genomic DNA.
  • a standardization curve prepared as above may not be reliable.
  • Samples for analysis can be obtained from any suitable source.
  • cells or tissues from eukaryotes e.g., plants or animals, such as mammals, including humans
  • tumor samples e.g., tumor samples, biopsy samples or other tissues, or bodily fluids, such as blood or blood fractions, urine, seminal fluid, etc.
  • RNA e.g. expressed RNA
  • Methods of isolating RNA and other molecular biology methods used in the invention can be carried out using conventional procedures. See, e.g., discussions in Sambrook, et al.
  • RNA transcripts can be used to quantitate the relative amounts of RNA expressed from a variety of templates.
  • a method of the invention can be used to measure nucleic acids, or expression from nucleic acids, which include cloned fragments or subclones thereof, chemically synthesized nucleic acids, genomic nucleic acid samples, cDNAs, nucleic acid molecules obtained from nucleic acid libraries, etc.
  • Methods of the invention can be readily adapted to a high throughput format, using automated ⁇ e.g. robotic) systems, which allow many measurements to be carried out simultaneously. Furthermore, the methods can be miniaturized ⁇ e.g. , carried out in reaction buffers of about 25 ⁇ l, 1 ⁇ l, 0.1 ⁇ l, or less).
  • reaction buffers of about 25 ⁇ l, 1 ⁇ l, 0.1 ⁇ l, or less.
  • a method of the invention can be substituted for any method in which the relative amounts of a plurality of RNA transcripts expressed from a gene of interest is quantitatively measured, m one embodiment, the relative expression of an imprinted gene and a non-imprinted correlate can be monitored. See, e.g., the imprinted genes discussed in Lo et al. (2003) Genome Research 13, 1855-1862.
  • Methods of the invention can be used to monitor the relative amounts of a plurality of RNA transcripts from a gene of interest ⁇ e.g. the relative expression of variant alleles) which are associated with a disease condition ⁇ e.g. a pathological disorder). Such measurements can be used in experimental studies of the disease condition, in methods of prognosis, or in diagnostic methods, such as methods to detect the presence of the condition, to follow the progression of the condition or its response to a therapeutic treatment; etc.
  • the conditions which can be detected by a method of the invention are autosomal- dominant disorders (disorders which result from a mutation in one allele, which causes reduced expression from that allele compared to the expression of a wild type allele).
  • Typical (non-limiting) examples of human autosomal-dominant disorders include incontinentia pigmenti (Berlin et al. (2002) J. Am. Acad. Dermatol. 47, 169-187), Hutchinson-Gilford progeria syndrome (Scaffidi et al. (2005) Nat. Med. U, 440-445), neurofibromatosis (Eisenbarth et al. (2000) Am. J. Hum. Genet.
  • myotonic dystrophy (Korade-Mirnics et al. (1999) Hum. MoI. Genet. 8, 1017-1023), sialuria, Machado- Joseph disease/spinocerebellar ataxia, frontotemporal dementia (Miller et al. (2003) Proc. Natl. Acad. ScL U.S.A. 100, 7195-7200), amyotrophic lateral sclerosis (ALS) (Maxwell etal. (2004) Proc. Natl. Acad. ScL U.S.A. 101, 3178-3183), slow channel congenital myasthenic syndrome (Abdelgany et al. (2003) Hum. MoI. Genet.
  • a cancer suppressor gene such as p53
  • p53 maybe down-regulated, or an oncogene maybe upregulated
  • autosomal recessive disorders ⁇ e.g., compound heterozygous autosomal recessive disorders
  • Such differential expression can result from a variety of mechanisms, including e.g. a nonsense, missense or splice site mutation in a first allele which results in RNA decay or alternate splicing and thus altered expression compared to expression of the comparable wild type allele.
  • RNA expression Other mechanisms that can be responsible for up or down regulation of RNA expression include insertions, deletions, frame shift mutations, etc, within coding or non-coding sequences of a gene, or mechanisms such as genetic imprinting, X-inactivation, etc.
  • Typical (non-limiting) examples of such autosomal recessive disorders include diabetes (Pugliese et al. (2002) Diabetes Metab. Res. Rev. 18, 13-25), cystic fibrosis, homocystenuria, hereditary inclusion body myopathy, Hermansky-Pudlak syndrome, cystinosis, Zellweger syndrome, beta-thalessemia, alkaptonuria, individual genetic variations on drug disposition, efficacy and safety (Lamba et al. (2002) Adv.
  • a method of the invention can be used to characterize the progression of, or the staging of, a disease condition of interest. It has been suggested that differences in allelic expression can modify the phenotype of a number of diseases. For example, expression of the Decorin gene 179 allelic variant is associated with a slower progression of renal disease in patients with type I diabetes (DeCosmo et al. (2002), Nephron 92, 72-76); differences in allelic expression of the CLCNl gene influence the myotonia congenital phenotype (Duna et al. (2004) Eur. J. Hum. Genet.
  • Heriditary Inclusion Body Myopathy HBM; MIM 600737
  • sialuria MM 600737
  • GNE coding for the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase (GNE)/ 7V-acetylmannosamine kinase (MNK).
  • GNE/MNK catalyzes the first two committed, rate-limiting steps in the biosynthesis of sialic acid, and is feedback-inhibited at its allosteric site by the end product, CMP-sialic acid.
  • Sialuria exhibits autosomal dominant inheritance; patients are heterozygous for a missense mutation in the allosteric site of GNE (codons 263-266), leading to a loss of feedback-inhibition. Consequently, cytoplasmic accumulation and urinary excretion of large quantities of free sialic acid occur.
  • HIBM is inherited in an autosomal recessive fashion; patients harbor two recessive GNE mutations (mostly missense) outside of the allosteric site. HIBM mutations lead to decreased GNE/MNK enzyme activity and, in some patients, decreased sialylation of muscle glycoproteins.
  • sialuria and HIBM have a variable phenotypic expression and disease progression, not only among patients with the same GNE mutations but also among affected patients within the same family. Although the mutations tested in the Examples herein did not reveal changes in allelic expression, these experiments provide proof of principle for the use of the rapid, convenient methods of the invention.
  • an undesirable (detrimental) allele e.g. aberrant expression, such as over-expression, of one allele (often a mutant) of a gene
  • the gene may be inhibited partially, or it may be substantially inhibited, e.g. silenced or knocked out.
  • Methods which can be used for such specific inhibition of expression include, e.g., the use of antisense molecules, ribozymes or the like.
  • a method of great current interest is the inhibition of the expression of an undesirable (detrimental) allele by RNA silencing, using an allele-specific siRNA.
  • RNAi silencing For example, Machado-Joseph disease/spinocerebellar ataxia, frontotemporal dementia, ALS, slow channel congenital myasthenic syndrome, and spinobulbar muscular atrophy have been reported to be responsive to RNAi silencing.
  • methods for confirming in vitro that such inhibition has occurred rely on crude, cumbersome techniques such as visual inspection of the treated cells under a microscope to observe whether morphological changes have taken place.
  • the inventive methods can be used, as an alternative, improved, procedure to determine the level of expression from the allele before and after specific inhibition of expression ⁇ e.g. siRNA treatment), thereby validating the efficacy of the inhibitory agent (e.g. an siRNA).
  • the ability of an siRNA to inhibit the expression of the targeted gene compared to a control can be tested, to validate the specificity of the inhibitory agent (e.g., siRNA).
  • Example IV illustrates allele-specific silencing of an allele (the dominant disease allele in siluria) with an allele-specific siRNA; and shows that, using a method of the invention, one can validate that an allele-specific siRNA of interest is specific and effective.
  • PCR primers and/or fluor- containing probes for one or more RNA transcripts of interest can be packaged individually or in various combinations.
  • PCR primers and probes suitable for the detection of two or more RNA transcripts expressed from a gene of interest are packaged together in a single container (such as an Eppendorf tube).
  • Components for reverse transcribing an RNA to be measured e.g. a reverse transcriptase and, optionally, suitable reaction components
  • components for generating a standard curve maybe included (e.g., a set of isolated DNA molecules, each of which represents one of the RNA transcripts of interest).
  • kits may supply reagents in pre-measured amounts so as to simplify the performance of the subj ect methods.
  • kits of the invention comprise instructions for performing the method.
  • Other optional elements of a kit of the invention include suitable buffers, packaging materials, a thermostable strand-displacing DNA polymerase suitable for use in fluorescer-quencher probe assays, i.e., having 5'-3' exonuclease activity, such as Taq DNA polymerase, etc.
  • the kits of the invention may further comprise additional reagents that are necessary for performing the subject methods.
  • Such reagents include, but are not limited to dNTP mixtures, buffers, etc.
  • the reagents of the kit may be in containers in which they are stable, e.g., in lyophilized form or as stabilized liquids.
  • DNAs used in methods of the invention can have one or more modified nucleotides, provided that the functionality of the DNA is not destroyed.
  • they may contain one or more modifications to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T as well as different purine or pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl.
  • a modified base includes but is not limited to 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils
  • 5-methylcytosine can increase the stability of dv ⁇ lex formation.
  • Base modifications often can be combined with for example a sugar modification, such as 2'-O-methoxyethyl, to achieve unique properties such as increased duplex stability.
  • a sugar modification such as 2'-O-methoxyethyl
  • There are numerous United States patents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, which detail and describe a range of base modifications.
  • Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxyribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; O-, S-, orN-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl maybe substituted or unsubstituted Cl to ClO, alkyl or C2 to ClO alkenyl and alkynyl.
  • 2' sugar modifications also include but are not limited to ⁇ 0[(CH 2 )n0]m CH 3 , -O(CH 2 )nOCH 3 , -O(CH 2 )nNH 2 , ⁇ O(CH 2 )nCH 3 , -O(CH 2 )n-ONH 2 , and - O(CH 2 )nON[(CH 2 )nCH 3 )] 2 , where n and m are from 1 to about 10.
  • modifications at the 2' position include but are not limited to: Cl to ClO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 , CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH 2 and S.
  • Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Nucleotide analogs can also be modified at the phosphate moiety.
  • Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'- alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl- phosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • these phosphate or modified phosphate linkages between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included. Numerous United States patents teach how to make and use nucleotides containing modified phosphates and include but are not limited to, U.S. Pat. Nos.
  • nucleotide analogs need only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.
  • Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes include molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes include molecules that will recognize and hybridize to complementary nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamateback- bones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • PNA aminoethylglycine
  • DNA molecules of the invention can be made up of different types of nucleotides or the same type of nucleotides.
  • one or more of the nucleotides in a primer can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; about 10% to about 50% of the nucleotides can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; about 50% or more of the nucleotides can be ribonucleotides, 2'-O-methyl ribonucleotides, or a mixture of ribonucleotides and 2'-O-methyl ribonucleotides; or all of the nucleotides are ribonucleotides, 2'
  • the nucleotides can be comprised of bases (that is, the base portion of the nucleotide) and can comprise different types of bases.
  • bases can be universal bases, such as 3-nitropyrrole or 5-nitroindole; about 10% to about 50% of the bases can be universal bases; about 50% or more of the bases can be universal bases; or all of the bases can be universal bases.
  • HEBM patient Hl was a 27-year-old male of English/lrish/Scottish descent who had progressive weakness beginning at 20 years of age and recently required crutches to ambulate. He was compound heterozygous for two missense GNE mutations; c.735C>T (Arg246Trp) and c.403G>T (Glyl35Val).
  • HIBM patient H2 was a 37-year old female of non- Jewish descent. She had onset of leg weakness in her early twenties and became wheelchair-bound in her mid thirties.
  • the human GNE coding sequence (GenBank accession number NM_005476) was amplified from cDNA of normal human fibroblasts and cloned, using EcoRI and JJr ⁇ /restriction sites, into the pETl 7b vector (EMD Biosciences, San Diego, CA), which contained an N-terminal T7-tag and a T7 promotor sequence.
  • This GNE-pETl 7b plasmid was used to create each of the patient-specific GNE target mutations by site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) . All constructs were verified by sequencing before experimental use.
  • PCR amplifications of the GNE coding sequence were performed using pure Taq Ready-To-Go PCR beads (Amersham, Piscataway, NJ). Amplifications were performed in a 25 ⁇ l reaction volume containing 25 ng GNE-pETl 7b plasmid DNA (normal or mutated) and 0.4 ⁇ M of each primer flanking the T7-promotor (forward, 5 ' -GACTC ACTATAGGG- 3') (SEQ ID NO:21) and the GNE termination codon (reverse, 5'-GCTAGTTATTGCTCAGCGG- 3') (SEQ ID NO:22).
  • RNA samples were treated with RNase-free DNase (rDNAse; Ambion, Austin, TX), and purified using RNeasy mini columns (Qiagen, Valencia, CA).
  • RNA, genomic DNA and cDNA were measured with a GeneQuant Pro spectrophotometer (Amersham, Piscataway, NJ).
  • PCR-generated fluorescent VIC and FAM signals can be measured either at their endpoint (after the last PCR cycle, performed for Figure 1) or in 'real time', where the increase in fluorescence is followed on a per-cycle basis (performed for Figure 2).
  • a total reaction mixture of 25 ⁇ l contained 50 nM primers and 50 nM allele-specific VIC/FAM probes, 25 ng of genomic DNA or 50 ng of cDNA, and Taqman Universal PCR Master Mix (Applied Biosystems, Foster City, CA). All real-time PCR reactions and subsequent analyses were performed on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA) . The pre-run thermal cycling conditions were 10 min at 95 0 C to activate the Taq DNA-polymerase, followed by 40 cycles of 95 0 C for 15 s and 6O 0 C annealing/extension for 1 min. Each experiment was performed in duplicate. Within each experiment, reactions were ran in duplicate.
  • a standard curve was constructed by mixing two allelic cDNA samples to allelic ratios of 8, 4, 2, 1, 0.5, 0.25 and 0.125 in a total concentration of 50 ng/ ⁇ l. In addition, 10 ng/ ⁇ l bacterial cDNA was added to each standard curve reaction mixture.
  • the Taqman real-time assays for each of the five GNE target mutations were validated using genomic DNA from normal controls and from HIBM (Hl, Figures IA and IB; H2, Figures 1 C and ID) and sialuria (S 1 , Figure IE) patients.
  • the endpoint readings of each fluorescent probe (Rn, normalized reporter signal) after the final PCR cycle were determined and visualized in a scatter plot.
  • Figure 1 shows that the patients ' samples cluster in the heterozygous area of each plot (3 independent experiments), while the normal control samples cluster in the homozygous area for the normal allele, demonstrating accurate allele-specificity of each assay.
  • Non-template control assays (ntc) show very minimal fluorescence. Since none of the patients was homozygous for the tested GNE mutations, no results are shown in the homozygous mutated allele area on each plot (indicated by dotted circles).
  • Example III Allele-specific RNA expression To determine whether any of the patients' GNE mutations resulted in different allelic niRNA expression levels, standard curves were prepared for each assay. Allelic cDNAs were prepared from cell-free transcribed RNA for each target GNE mutation and mixed in various ratios (normal allele:mutated allele ranging from 8 - 0.125) to a total cDNA concentration of 50 ng/ ⁇ l. Since each standard curve mixture consisted of 'pure' cDNA produced from in vitro transcribed RNA, we added bacterial cDNA (non-specific cDNA) to reflect the situation in the patient's samples as closely as possible. BLAST analysis (see the web site at ncbi.nih.gov/BLAST/) showed that our primer-probe assays were not located in an area homologous to an expressed bacterial gene.
  • Ct is the PCR cycle number at which the fluorescence generated by cleavage of a probe reaches a fixed threshold above baseline. At a given threshold, a higher Ct value indicates a lower starting copy number. The Ct was manually assigned or determined by the auto-Ct option using the SDS 2.1 software of Applied Biosystems.
  • Figure 2 shows the regression curves of observed 2 ⁇ Ct plotted against mixed allelic ratios for each GNE target mutation. For each of the assays, the experimental curves (solid lines) closely resembled the expected curves (dotted lines) (r 2 >0.98), indicating that the probes showed minimal cross-reactivity.
  • This ratio means that the C allele and the T-allele at position 735 of GNE mRNA in this patient are expressed in similar amounts.
  • Allelic ratios of approximately 1 were also obtained for the other tested GNE mutations, i.e., 0.8 for the Hl c.403G>T allele, 0.83 for H2 c.646T>C (Fig. 2B), and 1.1 for the Sl c.787G>T allele (Fig 2C), indicating that the GNE gene is not susceptible to allelic mutation-specific alterations in expression, at least for the tested mutations.
  • the cDNA obtained from wild type controls yielded 2 ⁇ Ct readings outside the standard curve readings for each assay.
  • the regression curve diverged slightly from the expected curve (as in Fig. 2C), which can be due to reduced quality of the control cDNA and/or to cross reactivity of the allele-specific probes.
  • the observed and expected standard curves deviated too much from each other, probably due to extensive cross-hybridization of the probes, and accurate readings could not be made. In this case, further optimization of the primer/probe set would be needed for reliable measurements of allelic expression.
  • Sialuria ('French type' sialuria; MM 269921) is an autosomal dominant disorder characterized by mild coarse facies and slight motor delay, and profound cytoplasmic accumulation and urinary excretion of large quantities (>1 gram/day) of free sialic acid. Additional sporadic clinical features are hepatosplenomegaly, delayed skeletal development, microcytic anemia, and mild intellectual impairment.
  • the molecular defect of sialuria is a failure to regulate sialic acid synthesis, due to impaired allosteric feedback inhibition of UDP-GIcNAc 2-epimerase by CMP-sialic acid ( Figure 3A).
  • the loss of feedback inhibition due to a mutation in one allele is enough to constitutively produce cytoplasmic free sialic acid.
  • AU described sialuria patients are heterozygous for a missense mutation in one of two amino acids, arginine at position 263 (R263L) or arginine at position 266 (R266Q;

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Abstract

La présente invention concerne, par exemple, un procédé permettant de quantifier les quantités relatives d'une pluralité de produits de transcription d'ARN différents, exprimés par un gène auquel on s'intéresse (tels que des produits d'expression de deux allèles d'un gène auquel on s'intéresse), chez un sujet ou dans une cellule par exemple. Ledit procédé comprend la réalisation, en temps réel et dans un récipient unique, d'une PCR quantitative portant sur les ADNc des produits de transcription en présence de sondes spécifiques de chacun des différents produits de transcription, la sonde de chaque produit de transcription étant marquée avec un fluorophore distinguable. La quantité d'expression de chaque produit parmi la pluralité de produits de transcription peut être quantifiée par une comparaison à une courbe étalon tracée à l'aide d'une PCR quantitative en temps réel portant sur des mélanges, dans des rapports différents, d'ADN isolés représentant chaque produit parmi la pluralité de produits de transcription. L'invention concerne également des procédés de validation de l'efficacité et de la spécificité des petits ARN interférents (siRNA) spécifiques d'allèles, ainsi que des procédés de diagnostic et des kits permettant de mettre en pratique les procédés de l'invention.
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WO2012136712A1 (fr) * 2011-04-08 2012-10-11 Deutsches Krebsforschungszentrum Moyens et procédés de quantification d'acides nucléiques
US8512591B2 (en) 2007-10-12 2013-08-20 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US8628681B2 (en) 2007-10-12 2014-01-14 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US8911641B2 (en) 2010-05-20 2014-12-16 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
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US8512591B2 (en) 2007-10-12 2013-08-20 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US8628681B2 (en) 2007-10-12 2014-01-14 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US8926856B2 (en) 2010-02-16 2015-01-06 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US9175202B2 (en) 2010-02-16 2015-11-03 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US8911641B2 (en) 2010-05-20 2014-12-16 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US10266736B2 (en) 2010-06-25 2019-04-23 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US10844260B2 (en) 2010-06-25 2020-11-24 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
US11760911B2 (en) 2010-06-25 2023-09-19 Mexichem Amanco Holding S.A. De C.V. Heat transfer compositions
WO2012136712A1 (fr) * 2011-04-08 2012-10-11 Deutsches Krebsforschungszentrum Moyens et procédés de quantification d'acides nucléiques
US10330364B2 (en) 2014-06-26 2019-06-25 Hudson Technologies, Inc. System and method for retrofitting a refrigeration system from HCFC to HFC refrigerant
CN116949156A (zh) * 2023-09-19 2023-10-27 美迪西普亚医药科技(上海)有限公司 一种基于核酸变体的通用型检测人t细胞的分析方法
CN116949156B (zh) * 2023-09-19 2023-12-08 美迪西普亚医药科技(上海)有限公司 一种基于核酸变体的通用型检测人t细胞的分析方法

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