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WO2009064897A2 - Détection de variations de séquences d'acides nucléiques dans l'acide nucléique circulant dans l'encéphalopathie spongiforme bovine - Google Patents

Détection de variations de séquences d'acides nucléiques dans l'acide nucléique circulant dans l'encéphalopathie spongiforme bovine Download PDF

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WO2009064897A2
WO2009064897A2 PCT/US2008/083420 US2008083420W WO2009064897A2 WO 2009064897 A2 WO2009064897 A2 WO 2009064897A2 US 2008083420 W US2008083420 W US 2008083420W WO 2009064897 A2 WO2009064897 A2 WO 2009064897A2
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seq
nucleic acid
sequence
sequences
bse
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WO2009064897A3 (fr
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Ekkehard Schuetz
Julia Beck
Howard Urnovitz
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Chronix Biomedical Inc
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Chronix Biomedical Inc
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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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

  • BSE Mad cow disease or bovine spongiform encephalopathy
  • BSE tests are post mortem tests performed on brain tissue from animals that have been slaughtered.
  • the majority of these tests detect abnormal proteins known as prions, which are not found in an animal until the disease has progressed into late stage.
  • Early stage spongiform encephalopathies are difficult to detect by prion testing because prion accumulation is most often associated with late-stage disease.
  • Genetic tests for prion gene polymorphisms are currently used to determine the susceptibility of sheep for scrapie (Hunter, et al. Arch Virol 141:809-824, 1996). No such diversity of prion genes is found in BSE.
  • the detection of nucleic acids in cattle sera Schotz et al.
  • CDLI circulating nucleic acids
  • This invention is based on the discovery that single nucleotide variances and polymorphisms in nucleic acid sequences are detected in acellular samples, such as serum or plasma, from animals at risk for transmissible spongiform encephalopathy, e.g., BSE.
  • the invention therefore provides a method of detecting an animal with bovine spongiform encephalopathy (BSE), the method comprising: detection of an individual or multiple single nucleotide polymorphisms (SNPs), also referred to herein as single nucleotide variations (SNVs), in nucleic acids extracted from an acellular sample obtained from the animals.
  • the sample is an acellular fluid such as serum or plasma.
  • the nucleic acid sample can be a DNA sample or RNA sample.
  • BSE specific SNPs are selected from a database of nucleic acid sequences.
  • the database is generated by ultra deep sequencing technology whereby sequences present in BSE animals are compared to sequences present in animals without BSE.
  • SNVs can be detected using methods that are well known in the art.
  • Most assays entail one of several general protocols: hybridization using allele-specific oligonucleotides, primer extension, allele-specific ligation, sequencing, or electrophoretic separation techniques, e.g., singled stranded conformational polymorphism (SSCP) and heteroduplex analysis.
  • Other assays include 5' nuclease assays, template-directed dye terminator incorporation, molecular beacon allele-specific oligonucleotide assays, single-base extension assays, and SNP scoring by real-time pyrophosphate sequences.
  • Analysis of amplified sequences can be performed using various technologies such as microchips, fluorescence polarization assays, and matrix-assisted laser desoprtion ionization (MALDI) mass spectrometry.
  • MALDI matrix-assisted laser desoprtion ionization
  • Two methods that can also be used are assays based on invasive cleavage with Flap nucleases and methodologies employing padlock probes.
  • the presence of SNVs is detected by sequencing.
  • the invention provides a method of selecting technologies to use in an amplification reaction to detect an animal with BSE, the method comprising: identifying nucleic acid sequences that have differences in BSE animals compared to normal; and selecting reagents that detect specific SNP/SNV reactivity.
  • the sequences are . identified in acellular samples, such as serum, hi one embodiment, the invention provides a method for the detection of SNPs/SNVs to detect an animal with BSE, the method comprising: whole genome amplification of circulating nucleic acids, ultra deep sequencing of the amplified products and identification of SNPs/SNVs in the resulting database in BSE animals as compared to normal controls.
  • the invention provides a method of detecting an animal with BSE, the method comprising: extracting nucleic acids from a sample and detecting the statistical presence of a SNP/SNV in the extracted nucleic acids. Detection of a SNP/SNV can be done directly or indirectly, e.g., through amplification of the target nucleic acids and query at a variant position within the target nucleic acid using an oligonucleotide that selectively hybridizes to a reference sequence or known BSE-associated variant; or by direct sequencing..
  • Fig. 1 provides an example of a query repetitive element against a database. Calculations based on sequences Infected and controls compared using Chi-square test.
  • the solid line shows the Chi-square value at each position of this example queried sequence, based on distribution of nucleotide sampling, ie. A, C, G, T or a insertion or deletion, of sequences from animals with BSE as compared to sequences from normal controls.
  • the dotted line depicts the total number of hits in the database for each position on this example queried sequence.
  • Fig. 2 shows exemplary SNV analysis. Whenever the dotted line reaches 1.0 the position has a single nucleotide variation found only in animals with BSE as compared to normal controls.
  • the accompanying solid line is the respective Chi-square value, based on distribution of nucleotide sampling, ie. A, C, G, T or a insertion or deletion, calculated per animal. Throughout this example sequence, more than one position contains a SNV that is present in BSE animals but not in normal controls.
  • a "cohort” refers to birth or feeding cohorts that are defined according to the official EU definition as being raised or born on the same farm within 12 months prior to or after a BSE index case.
  • Animals "with BSE” refer to cattle that are incubating BSE etiologic agents but may or may not show any clinical signs of BSE or PrP res reactivity at the time of sampling.
  • reactivity refers to a change in a characteristic of SNP/SNV detection, in the presence of a nucleic acid sequence that is indicative of BSE.
  • a sample is considered reactive when it exhibits a value of at least 3, preferably 5 standard deviations above a reference standard.
  • a "positive reference” or “positive control” is a sample that is known to contain SNPs/SNVs that are indicative of BSE.
  • a “positive reference” can be from a known cohort animal that was reactive in the assay of the invention.
  • a “positive reference” can be a synthetic construct that shows reactivity in an assay of the invention.
  • a “reference control” is a sample that results in minimal change to the SNP/SNV detection in BSE. Often, such a sample is a known negative, e.g., from healthy animals. For example, in diagnostic applications, such a control is typically derived from a normal animal that is not a cohort with a PrP res animal. A “reference control” is preferably included in an assay, but may be omitted.
  • “Amplifying” refers to a step of submitting a solution to conditions sufficient to allow for amplification of a polynucleotide if all of the components of the reaction are intact.
  • Components of an amplification reaction include, e.g., primers, a polynucleotide template, polymerase, nucleotides, and the like.
  • the term “amplifying” typically refers to an "exponential" increase in target nucleic acid. However, “amplifying” as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid.
  • An "amplification characteristic” refers to any parameter of an amplification reaction. Such reactions typically comprises repeated cycles. An amplification characteristic may be the number of cycles, a melting curve, temperature profile, or band characteristics on a gel or other means of post-amplification detection.
  • the term allele-specific probe does not refer to an allele per se, but to a probe to a variant nucleic acid sequence relative to a reference sequence.
  • an “allele” in the context of this invention refers to a variant nucleic acid sequence in comparison to a references sequence, e.g., the reference sequences set forth in SEQ ID NOs 1-41.
  • a “melting profile” or “melting curve” refers to the melting temperature characteristics of a nucleic acid fragment over a temperature gradient.
  • the melting curve is derived from the first derivative of the melting signal.
  • the melting point of a DNA fragment depends, e.g., on its length, its G/C content, the ionic strength of the buffer and the presence of mismatches (heteroduplexes).
  • mismatches heteroduplexes
  • amplification reaction refers to any in vitro means for multiplying the copies of a target sequence of nucleic acid.
  • methods include but are not limited to polymerase chain reaction (PCR), DNA ligase, (LCR), Q ⁇ RNA replicase, RNA transcription- based (TAS and 3SR) amplification reactions, and nucleic acid sequence based amplification (NASBA).
  • PCR polymerase chain reaction
  • LCR DNA ligase
  • TAS and 3SR RNA transcription- based amplification reactions
  • NASBA nucleic acid sequence based amplification
  • PCR Polymerase chain reaction
  • PCR refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression.
  • PCR is well known to those of skill in the art; see, e.g., U.S. Patents 4,683,195 and 4,683,202; PCR Technology: Principles and Applications for DNA Amplification (Erlich, ed., 1992)and PCR Protocols: A Guide to Methods and Applications, Innis et al, eds, 1990.
  • amplification reaction mixture refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid. These include enzymes, aqueous buffers, salts, amplification primers, target nucleic acid, and nucleoside triphosphates. Depending upon the context, the mixture can be either a complete or incomplete amplification reaction mixture
  • a "primer” refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid and serves as a point of initiation of nucleic acid synthesis.
  • Primers can be of a variety of lengths and are often less than 50 nucleotides in length, for example 12-25 nucleotides, in length.
  • the length and sequences of primers for use in PCR can be designed based on principles known to those of skill in the art, see, e.g., Innis et al., supra.
  • a primer is preferably a single-stranded oligodeoxyribonucleotide.
  • the primer includes a "hybridizing region" exactly or substantially complementary to the target sequence, preferably about 15 to about 35 nucleotides in length.
  • a primer oligonucleotide can either consist entirely of the hybridizing region or can contain additional features which allow for the detection, immobilization, or manipulation of the amplified product, but which do not alter the ability of the primer to serve as a starting reagent for DNA synthesis.
  • a nucleic acid sequence tail can be included at the 5' end of the primer that hybridizes to a capture oligonucleotide.
  • a primer for use in the invention need not exactly correspond to the sequence(s) that it amplifies in a hybridization reaction.
  • the incorporation of mismatches into a probe can be used to adjust duplex stability when the assay format precludes adjusting the hybridization conditions.
  • the effect of a particular introduced mismatch on duplex stability is well known, and the duplex stability can be routinely both estimated and empirically determined, as described above.
  • Suitable hybridization conditions which depend on the exact size and sequence of the probe, can be selected empirically using the guidance provided herein and well known in the art (see, e.g., the general PCR and molecular biology technique references cited herein).
  • sequence when referring to a nucleic acid refers to a sequence of nucleotides that are contiguous within a second sequence but does not include all of the nucleotides of the second sequence.
  • a "temperature profile” refers to the temperature and lengths of time of the denaturation, annealing and/or extension steps of a PCR reaction.
  • a temperature profile for a PCR reaction typically consists of 10 to 60 repetitions of similar or identical shorter temperature profiles; each of these shorter profiles may typically define a two step or three- step PCR reaction. Selection of a "temperature profile” is based on various considerations known to those of skill in the art, see, e.g., Innis et al., supra.
  • a “template” refers to a double or single stranded polynucleotide sequence that comprises a polynucleotide to be amplified.
  • an "acellular biological fluid” is a biological fluid that substantially lacks cells.
  • such fluids are fluids prepared by removal of cells from a biological fluid that normally contains cells (e.g., whole blood).
  • exemplary processed acellular biological fluids include processed blood (serum and plasma), e.g., from peripheral blood or blood from body cavities or organs; and samples prepared from urine, milk, saliva, sweat, tears, phlegm, cerebrospinal fluid, semen, feces, and the like.
  • serum or plasma is the acellular sample that is analyzed in the assays of the invention.
  • acellular samples that can be used include samples comprising nucleic acids obtained by washing any cell preparation to remove circulating nucleic acids that are associated with the cell surface.
  • samples comprising nucleic acids obtained by washing any cell preparation to remove circulating nucleic acids that are associated with the cell surface.
  • such an acellular sample can be obtained by washing circulating blood cells, such as lymphocytes. The supernatant from the wash can then be analyzed.
  • Nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, or chimeric constructs of polynucleotides chemically linked to reporter molecules, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • biological sample refers to a sample obtained from an organism or from components (e.g., cells) of an organism.
  • the sample may be of any biological tissue or fluid. Frequently the sample will be a "clinical sample” which is a sample derived from a patient, animal or human, with a disease or suspected of having a disease.
  • samples include, but are not limited to, sputum, blood, serum, plasma, body cavity blood or blood products, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, milk, peritoneal fluid, and pleural fluid, or cells therefrom.
  • Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • An "individual” or “patient” as used herein, refers to any animals, often mammals, including, but not limited to humans, nonhuman primates such as chimpanzees and monkeys, horses, cows, deer, sheep, goats, pigs, dogs, minks, elk, cats, lagromorphs, and rodents.
  • a "chronic illness” is a disease, symptom, or syndrome that last for months to years.
  • chronic illnesses in animals include, but are not limited to, cancers and wasting diseases as well as autoimmune diseases, and neurodegenerative diseases such as spongiform encephalopathies and others.
  • Repetitive genomic sequences or “repetitive genomic nucleic acid sequences” (RGNAS) refer to highly repeated DNA elements present in the animal genome. These sequences are usually categorized in sequence families and are broadly classified as tandemly repeated DNA or interspersed repetitive DNA (see, e.g., Jelinek and Schmid, Ann. Rev. Biochem. 51:831-844, 1982; Hardman, Biochem J. 234:1-11, 1986; and Vogt, Hum. Genet. 84:301-306, 1990). Tandemly repeated DNA includes satellite, minisatellite, and microsatellite DNA.
  • Repetitive genomic sequences includes AIu sequences, short interspersed nuclear elements (SINES), long terminal repeats (LTR), LTR and non-LTR transposable elements, LTR and non-LTR retrotransposons, endogenous retroviruses, and long interspersed nuclear elements (LINES) including Ll LINE sequences.
  • Intergenic sequence or "spacer DNA” or “non-coding sequence” refers to those nucleic acid sequences, including non-intronic sequences that do not code for protein sequences.
  • a "rearranged sequence” or “recombined sequence” is a region of the genomic DNA that is rearranged compared to normal, i.e., the rearranged sequence is not contiguous in genomic DNA in healthy animals or in genomic DNA obtained from animals prior to contracting a disease or prior to exposure to a genotoxic agent.
  • a single nucleotide polymorphism is used herein interchangeably with the term "single nucleotide variance" or " single nucleotide variations” (SNV).
  • SNP single nucleotide polymorphism
  • the reference sequence can be derived experimentally from nucleic acids sequenced from the serum of normal individuals and the test sequence may be derived from the serum of an animal with BSE.
  • Such variability can include regions of short nucleotide (1- 5 nucleotide) deletions and insertions.
  • SNPs may occur at any region in the genome or in nucleic acid sequences. In the current invention, the change is in a non-coding region of DNA, including, but not limited to repetitive sequences, and intragenic DNA.
  • Ultra Deep Sequencing (454 Sequencing) is a massively-parallel pyrosequencing system capable of sequencing roughly 100 megabases of raw DNA sequence per 7-hour run using the GSFLX sequencing machine.
  • the system relies on fixing nebulized and adapter- ligated DNA fragments to small DNA-capture beads in a water-in-oil emulsion.
  • the DNA fixed to these beads is then amplified by PCR.
  • each DNA-bound bead is placed into a ⁇ 44 ⁇ m well on a PicoTiterPlate, a fiber optic chip. A mix of enzymes such as polymerase, ATP sulfurylase, and luciferase are also packed into the well.
  • the PicoTiterPlate is then placed into the GS20 for sequencing.
  • Contig refers to sequences that are computationally assembled from several overlapping physically contiguous sequences into one contiguous sequence. Such a contig is usually, but not necessarily longer than the initial sequences.
  • “Whole genome amplification” is a technique in which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis.
  • CNAs refers to DNA or RNA that is found in acellular fluids.
  • stringent hybridization conditions will be those in which the salt concentration is about 0.2XSSC at pH 7 and the temperature is at least about 6O 0 C.
  • a nucleic acid of the invention or fragment thereof can be identified in standard filter hybridizations using the nucleic acids disclosed here under stringent conditions, which for purposes of this disclosure, include at least one wash (usually 2) in 0.2X SSC at a temperature of at least about 60°C, usually about 65°C, sometimes 70°C for 20 minutes, or equivalent conditions.
  • an annealing temperature of about 5°C below Tm is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 72°C, e.g., 40 0 C, 42°C, 45°C, 52°C, 55 0 C, 57 0 C, or 62°C, depending on primer length and nucleotide composition.
  • High stringency PCR amplification, a temperature at, or slightly (up to 5°C) above, primer Tm is typical, although high stringency annealing temperatures can range from about 50 0 C to about 72°C, and are often 72°C, depending on the primer and buffer conditions (Ahsen et al, Clin Chem. 47:1956-61, 2001).
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C-95°C for 30 sec-2 min., an annealing phase lasting 30 sec-10 min., and an extension phase of about 72°C for 1 -15 min.
  • BSE is clinically characterized by increasing perturbation of central nervous function in the affected animal, ultimately leading to severe symptoms, e.g., an inability to stand, forcing the sacrifice of the animal.
  • the bovine form does not appear to be associated with a mutation in the prion gene, but may be caused by a post-translational misfolding of the prion protein, which leads to aggregation in the central nervous system.
  • the diagnosis is based on the fact that misfolded prion protein has enhanced resistance to protease K digestion. As disease-specific prion accumulation in the plasma or blood of animals has not been identified, the diagnostic target has been the brain stem.
  • the invention provides a method for diagnosing an increased risk for BSE by amplification and analysis of circulating nucleic acids (CNA) from test animals.
  • CNA circulating nucleic acids
  • Nucleic acid molecules detected in the methods of the invention may be free, single or double stranded, molecules or complexed with protein or lipids or both.
  • the detected nucleic acids can be DNA or RNA molecules.
  • RNA molecules need not be transcribed from a gene, but can be transcribed from any sequence in the chromosomal DNA.
  • Exemplary RNAs include miRNA, intergenic RNA, small nuclear RNA (snRNA), mRNA, tRNA, rRNA, and interference RNA (iRNA).
  • the nucleic acid molecules may comprise sequences transcribed from repetitive genomic sequences or intergenic or non-coding DNA in the genome of the individual from which the sample is derived.
  • the detected nucleic acid molecules may also be the products of rearrangement of germline sequences and/or sequences introduced into the genome, e.g., exogenous viral sequences.
  • a polynucleotide detected using this method may be a particular polynucleotide or may be a population of polynucleotides that are present in the sample.
  • the polynucleotide in a particular sample need not have that sequence, i.e., the sequence of the polynucleotide in the sample may be altered in comparison to the known sequence.
  • Such alterations can include mutations, e.g., insertions, deletions, substitutions, and various other rearrangements.
  • the resulting amplified products may be as result of the amplification reaction and not reflect the original pool of polynucleotides.
  • the test samples are typically biological samples that comprise target nucleic acids.
  • a target nucleic acid can be from any source, but is typically from a biological sample that comprises small quantities of nucleic acid, e.g., nucleic acid samples obtained from samples that are not readily quantified by standard PCR methodology.
  • the test sample is a nucleic acid, e.g., RNA or DNA that is isolated from serum or plasma. SNP/SNV Detection Reactions
  • Detection techniques for evaluating nucleic acids for the presence of a SNP or SNV involve procedures well known in the field of molecular genetics. Further, many of the methods involve amplification of nucleic acids. Ample guidance for performing such technicques is provided in the art. Exemplary references include manuals such as PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N. Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds.
  • microarrays can be utilized for genomewide SNP detection assays (Genomewide SNP assay reveals mutations underlying Parkinson disease. Simon-Sanchez J, Scholz S, Del Mar Matarin M, Fung HC, Hernandez D, Gibbs JR, Britton A, Hardy J, Singleton A. Hum Mutat. 2007 Nov 9)
  • Suitable amplification methods include ligase chain reaction ⁇ see, e.g., Wu & Wallace, Genomics 4:560-569, 1988); strand displacement assay ⁇ see, e.g., Walker et al, Proc. Natl. Acad. ScL USA 89:392-396, 1992; U.S. Pat. No. 5,455,166); and several transcription-based amplification systems, including the methods described in U.S. Pat. Nos. 5,437,990; 5,409,818; and 5,399,491; the transcription amplification system (TAS) (Kwoh et al, Proc. Natl.
  • TAS transcription amplification system
  • oligonucleotide primers and/or probes can be prepared by any suitable method, usually chemical synthesis. Oligonucleotides can be synthesized using commercially available reagents and instruments. Alternatively, they can be purchased through commercial sources. Methods of synthesizing oligonucleotides are well known in the art ⁇ see, e.g, Narang et al, Meth. Enzymol. 68:90-99, 1979; Brown et al, Meth. Enzymol. 68:109-151, 1979; Beaucage et al, Tetrahedron Lett.
  • modified phosphodiester linkages e.g., phosphorothioate, methylphosphonates, phosphoamidate, or boranophosphate
  • linkages other than a phosphorous acid derivative may be used to prevent cleavage at a selected site
  • 2 '-amino modified sugars tends to favor displacement over digestion of the oligonucleotide when hybridized to a nucleic acid that is also the template for synthesis of a new nucleic acid strand.
  • This technique also commonly referred to as allele specific oligonucleotide hybridization (ASO) (e.g., Stoneking et al., Am. J. Hum. Genet. 48:70-382, 1991; Saiki et al., Nature 324, 163-166, 1986; EP 235,726; and WO 89/11548), relies on distinguishing between two DNA molecules differing at a polymorphic position, typically by one nucleotide, by hybridizing an oligonucleotide probe that is specific for one of the variants to an amplified product obtained from amplifying the nucleic acid sample.
  • This method typically employs short oligonucleotides, e.g., 15-20 bases in length.
  • probes are designed to differentially hybridize to one variant versus another.
  • probes are designed to hybridize to the version of the nucleic acid sequence that is present in normal cows. Principles and guidance for designing such probe is available in the art, e.g., in the references cited herein.
  • Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.
  • Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15-base oligonucleotide at the 7 position; in a 16-based oligonucleotide at either the 8 or 9 position) of the probe, but this design is not required.
  • the amount and/or presence of an allele is determined by measuring the amount of allele-specific oligonucleotide that is hybridized to the sample.
  • the oligonucleotide is labeled with a label such as a fluorescent label. After stringent hybridization and washing conditions, fluorescence intensity is measured for each SNP oligonucleotide.
  • the nucleotide present at the polymorphic site is identified by hybridization under sequence-specific hybridization conditions with an oligonucleotide probe exactly complementary to one of the polymorphic alleles in a region encompassing the polymorphic site.
  • the probe hybridizing sequence and sequence-specific hybridization conditions are selected such that a single mismatch at the polymorphic site destabilizes the hybridization duplex sufficiently so that it is effectively not formed.
  • sequence-specific hybridization conditions stable duplexes will form only between the probe and the exactly complementary allelic sequence.
  • oligonucleotides from about 10 to about 35 nucleotides in length, preferably from about 15 to about 35 nucleotides in length, which are exactly complementary to an allele sequence in a region which encompasses the polymorphic site are within the scope of the invention.
  • the nucleotide present at the polymorphic site is identified by hybridization under sufficiently stringent hybridization conditions with an oligonucleotide substantially complementary to one of the SNP/SNV alleles in a region encompassing the polymorphic site, and exactly complementary to the allele at the polymorphic site. Because mismatches which occur at non-polymorphic sites are mismatches with both allele sequences, the difference in the number of mismatches in a duplex formed with the target allele sequence and in a duplex formed with the corresponding non-target allele sequence is the same as when an oligonucleotide exactly complementary to the target allele sequence is used.
  • the hybridization conditions are relaxed sufficiently to allow the formation of stable duplexes with the target sequence, while maintaining sufficient stringency to preclude the formation of stable duplexes with non-target sequences. Under such sufficiently stringent hybridization conditions, stable duplexes will form only between the probe and the target allele.
  • oligonucleotides from about 10 to about 35 nucleotides in length, preferably from about 15 to about 35 nucleotides in length, which are substantially complementary to an allele sequence in a region which encompasses the polymorphic site, and are exactly complementary to the allele sequence at the polymorphic site, are within the scope of the invention.
  • oligonucleotides may be desirable in assay formats in which optimization of hybridization conditions is limited.
  • probes for each target are immobilized on a single solid support.
  • Hybridizations are carried out simultaneously by contacting the solid support with a solution containing target DNA.
  • the hybridization conditions cannot be separately optimized for each probe.
  • the incorporation of mismatches into a probe can be used to adjust duplex stability when the assay format precludes adjusting the hybridization conditions.
  • duplex stability can be routinely both estimated and empirically determined, as described above.
  • Suitable hybridization conditions which depend on the exact size and sequence of the probe, can be selected empirically using the guidance provided herein and well known in the art.
  • the use of oligonucleotide probes to detect single base pair differences in sequence is described in, for example, Conner et al., 1983, Proc. Natl. Acad. Sci. USA 80:278-282, and U.S. Pat. Nos. 5,468,613 and 5,604,099, each incorporated herein by reference.
  • the proportional change in stability between a perfectly matched and a single-base mismatched hybridization duplex depends on the length of the hybridized oligonucleotides. Duplexes formed with shorter probe sequences are destabilized proportionally more by the presence of a mismatch. In practice, oligonucleotides between about 15 and about 35 nucleotides in length are preferred for sequence-specific detection. Furthermore, because the ends of a hybridized oligonucleotide undergo continuous random dissociation and re- annealing due to thermal energy, a mismatch at either end destabilizes the hybridization duplex less than a mismatch occurring internally. Preferably, for discrimination of a single base pair change in target sequence, the probe sequence is selected which hybridizes to the target sequence such that the polymorphic site occurs in the interior region of the probe.
  • Suitable assay formats for detecting hybrids formed between probes and target nucleic acid sequences in a sample include the immobilized target (dot-blot) format and immobilized probe (reverse dot-blot or line-blot) assay formats.
  • Dot blot and reverse dot blot assay formats are described in U.S. Pat. Nos. 5,310,893; 5,451,512; 5,468,613; and 5,604,099; each incorporated herein by reference.
  • amplified target DNA is immobilized on a solid support, such as a nylon membrane.
  • a solid support such as a nylon membrane.
  • the membrane-target complex is incubated with labeled probe under suitable hybridization conditions, unhybridized probe is removed by washing under suitably stringent conditions, and the membrane is monitored for the presence of bound probe.
  • a preferred dot-blot detection assay is described in the examples.
  • the probes are immobilized on a solid support, such as a nylon membrane or a microtiter plate.
  • the target DNA is labeled, typically during amplification by the incorporation of labeled primers.
  • One or both of the primers can be labeled.
  • the membrane-probe complex is incubated with the labeled amplified target DNA under suitable hybridization conditions, unhybridized target DNA is removed by washing under suitably stringent conditions, and the membrane is monitored for the presence of bound target DNA.
  • a preferred reverse line-blot detection assay is described in the examples.
  • An allele-specific probe that is specific for one of the polymorphism variants is often used in conjunction with the allele-specific probe for the other polymorphism variant.
  • the probes are immobilized on a solid support and the target sequence in an individual is analyzed using both probes simultaneously. Examples of nucleic acid arrays are described by WO 95/11995. The same array or a different array can be used for analysis of characterized polymorphisms.
  • Polymorphisms are also commonly detected using allele-specific amplification or primer extension methods. These reactions typically involve use of primers that are designed to specifically target a polymorphism via a mismatch at the 3' end of a primer. The presence of a mismatch effects the ability of a polymerase to extend a primer when the polymerase lacks error-correcting activity. The presence of the particular allele can be determined by the ability of the primer to initiate extension. If the 3' terminus is mismatched, the extension is impeded. Thus, for example, if a primer matches the "C" allele nucleotide at the 3' end, the primer will be efficiently extended.
  • the primer is used in conjunction with a second primer in an amplification reaction.
  • the second primer hybridizes at a site unrelated to the polymorphic position.
  • Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present.
  • Allele-specific amplification- or extension- based methods are described in, for example, WO 93/22456; U.S. Pat. Nos. 5,137,806; 5,595,890; 5,639,611; and U.S. Pat. No. 4,851,331.
  • identification of the alleles requires only detection of the presence or absence of amplified target sequences.
  • Methods for the detection of amplified target sequences are well known in the art. For example, gel electrophoresis and probe hybridization assays described are often used to detect the presence of nucleic acids.
  • the amplified nucleic acid is detected by monitoring the increase in the total amount of double-stranded DNA in the reaction mixture, is described, e.g., in U.S. Pat. No. 5,994,056; and European Patent Publication Nos. 487,218 and 512,334.
  • the detection of double-stranded target DNA relies on the increased fluorescence various DNA-binding dyes, e.g., SYBR Green, exhibit when bound to double- stranded DNA.
  • allele-specific amplification methods can be performed in reaction that employ multiple allele-specific primers to target particular alleles.
  • Primers for such multiplex applications are generally labeled with distinguishable labels or are selected such that the amplification products produced from the alleles are distinguishable by size.
  • both alleles in a single sample can be identified using a single amplification by gel analysis of the amplification product.
  • an allele-specific oligonucleotide primer may be exactly complementary to one of the polymorphic alleles in the hybridizing region or may have some mismatches at positions other than the 3' terminus of the oligonucleotide, which mismatches occur at non-polymorphic sites in both allele sequences.
  • Identification of the presence of a polymorphism can also be performed using a "TaqMan®” or "5'-nuclease assay", as described in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al, 1988, Proc. Natl. Acad. Sd. USA 88:7276-7280.
  • TaqMan® assay labeled detection probes that hybridize within the amplified region are added during the amplification reaction. The probes are modified so as to prevent the probes from acting as primers for DNA synthesis.
  • the amplification is performed using a DNA polymerase having 5' to 3' exonuclease activity.
  • any probe which hybridizes to the target nucleic acid downstream from the primer being extended is degraded by the 5' to 3' exonuclease activity of the DNA polymerase.
  • the synthesis of a new target strand also results in the degradation of a probe, and the accumulation of degradation product provides a measure of the synthesis of target sequences.
  • the hybridization probe can be an allele-specific probe that discriminates between the SNP alleles.
  • the method can be performed using an allele-specific primer and a labeled probe that binds to amplified product.
  • any method suitable for detecting degradation product can be used in a 5' nuclease assay.
  • the detection probe is labeled with two fluorescent dyes, one of which is capable of quenching the fluorescence of the other dye.
  • the dyes are attached to the probe, preferably one attached to the 5' terminus and the other is attached to an internal site, such that quenching occurs when the probe is in an unhybridized state and such that cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase occurs in between the two dyes.
  • Amplification results in cleavage of the probe between the dyes with a concomitant elimination of quenching and an increase in the fluorescence observable from the initially quenched dye.
  • the accumulation of degradation product is monitored by measuring the increase in reaction fluorescence.
  • SNPs/SNVs can also be detected by direct sequencing. Methods include e.g., dideoxy sequencing-based methods and other methods such as Maxam and Gilbert sequence (see, e.g., Sambrook and Russell, supra).
  • Other detection methods include PyrosequencingTM of oligonucleotide-length products. Such methods often employ amplification techniques such as PCR. For example, in pyrosequencing, a sequencing primer is hybridized to a single stranded, PCR-amplified, DNA template; and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5' phosphosulfate (APS) and luciferin. The first of four deoxynucleotide triphosphates (dNTP) is added to the reaction.
  • dNTP deoxynucleotide triphosphates
  • DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.
  • PPi pyrophosphate
  • ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5 ' phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP.
  • the light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogramTM. Each light signal is proportional to the number of nucleotides incorporated.
  • Apyrase a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added.
  • Another similar method for characterizing SNPs/SNVs does not require use of a complete PCR, but typically uses only the extension of a primer by a single, fluorescence- labeled dideoxyribonucleic acid molecule (ddNTP) that is complementary to the nucleotide to be investigated.
  • ddNTP dideoxyribonucleic acid molecule
  • the nucleotide at the polymorphic site can be identified via detection of a primer that has been extended by one base and is fluorescently labeled (e.g., Kobayashi et al, MoI. Cell. Probes, 9:175-182, 1995).
  • Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution (see, e.g., Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, W. H. Freeman and Co, New York, 1992, Chapter 7).
  • Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described, e.g, in Orita et ah, Proc. Nat. Acad. ScL 86, 2766-2770 (1989).
  • Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products.
  • Single- stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence difference between alleles of target
  • Oligonucleotides can be labeled by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • Useful labels include fluorescent dyes, radioactive labels, e.g., 32 P, electron-dense reagents, enzyme, such as peroxidase or alkaline phsophatase, biotin, or haptens and proteins for which antisera or monoclonal antibodies are available. Labeling techniques are well known in the art (see, e.g., Current Protocols in Molecular Biology, supra; Sambrook & Russell, supra).
  • a BSE animal is detected by detecting the presence of any one of the 420 polymorphisms set forth in Table 1, or detecting a combination of those polymorphisms. Analysis is generally performed by querying more than one of the 420 variant positions. Thus, anywhere from 1 to all of the 420 variant positions set forth in Table 1 can be analyzed to detect BSE. Generally at least 10, 20, 30, 40, 50 60, 70, or 100 or more positions are analyzed.
  • Table 1 and Table 2 provide a description of SNPs found only in BSE animals. Table 1 provides details of SNPs found only in BSE animals. Column A contains the sequence identification tags for each reference query sequence as described in Table 2. Column B describes the position of the SNP in the sequence referred to in Column A. Column C is the consensus sequence derived from the database of sequences of normal and BSE animals and designates the position (shown in a capital letter) at which a diagnostic SNP can be found in sequences from BSE animals only. Column E is the Sequence ID number for those sequences found in Column F.
  • Column F is the consensus sequence derived from the database of sequences of BSE animals and designates the actual polymorphism (shown in a capital letter) found in sequences from BSE animals only: a "-" designates a deletion, a "N” designates an insertion.
  • Column G is the Sequence ID number for those sequences found in Column H.
  • Column H is an alternative consensus sequence to that found in Column F and derived from the database of sequences of BSE animals and designates the actual polymorphism (shown in a capital letter) found in sequences from BSE animals only: a "-" designates a deletion, a "N” designates an insertion.
  • the position of the polymorphism is determined with reference to one of reference sequences SEQ ID NOs 1-41. Thus, the number of the position indicates where that position occurs in the context of the reference sequence (Le., one of SEQ ID NOs: 1-41). An insertion occurs after the designated position,
  • Table 2 provides a summary table of queried sequences containing diagnostic SNPs found only in BSE animals.
  • Column A contains the sequence identification tags for each query sequence.
  • Column B contains the repetitive element nomenclature that has the highest homology with the query sequence when applicable.
  • Column C is the length of the query sequence.
  • Column D contains the percentage of the query length that has homology (>70%) to the reference repetitive element in Column B.
  • Column E contains, where appropriate, the BLAST reference of the query sequence when searching against the cow genome.
  • Column F contains the percentage of the query length, where appropriate, that has homology (>70%) to the BLAST reference in Column E.
  • Column G contains the highest number of individual sequences in the database, derived from the ultra deep sequencing of both BSE and normal animals, at positions that contains SNPs found only in BSE animals.
  • Column H contains the total number of significant SNPs in the queried sequence found only in BSE animals.
  • Column I contains the maximum number of BSE animals, out of a total of 15 BSE animals but not any normal control animals, that can be detected when using a single SNP from Column H.
  • Column J contains the maximum number of BSE animals, out of a total of 15 BSE animals but not any normal control animals, that can be detected when using a combination of SNPs from Column H.
  • Column K refers to the Sequence Number of oligos (as detailed in Table 1) that are located within the query sequence of Column A and containing the SNP position referred to in Column H.
  • a polymorphic position described herein (i.e., a query position) can be evaluated using sequencing or any number of methods employing oligonucleotides that are competent to discriminate between the residue(s) present in the reference sequence and the indicated polymorphism present only in BSE animals.
  • Such oligonucleotides can bind selectively to the normal sequence or in some embodiments, are designed to bind selectively to the variant sequence known to be associated with BSE.
  • Exemplary oligonucleotides that discriminate between the reference sequence and BSE-associated variant sequence are provided in Table 1.
  • a BSE animal is detected by sequence analysis of one or more polymorphic positions.
  • EXAMPLES Example 1. Detection of polymorphisms to detect animals with BSE
  • This example describes detection of SNP/SNVs associated with BSE.
  • Samples were obtained from an experimental study whereby cows were inoculated orally with BSE- infectious or control brain material.
  • Fleckvieh/Brown Swiss cattle were fed 100 g of either PrP res -positive brain stem macerate or normal brain material (controls). Serum samples were taken 40 months post-inoculation (15 infected, 6 control non-infected and 12 randomly selected normal animals).
  • Serum collection Special care was taken in collection, processing and storage of serum samples. Blood from the tail vein or artery was collected into 18 mL plastic tubes equipped with a coagulation accelerator and kept at room temperature for 30 min to ensure proper coagulation. Until further processing, the tubes were stored at 2 - 8 °C for not longer than 24 hours. Centrifugation was done at 2 - 8 °C, 1000 x g for 15 min. The serum supernatant was transferred into 1.5 mL microcentrifuge cups in 0.5 mL aliquots and frozen immediately at -20°C or -80 0 C until use
  • Ultra deep sequencing of products from steps above was performed using a Roche/454 genome sequencer (GS20/GSFLX) with system reagents according to the manufacturers instructions.
  • Blast analysis A total of 117 contigs were compared against a database containing 808,634 sequences (total letters: 86,785,049) using Blast. Database sequences segregate into 410984 sequences from 15 animals artificially infected with BSE and 397650 sequences from 18 un-infected controls.
  • the ultra deep sequencing approach had generated 41 sequences (Jean is this a table, figure, attachment?) in which SNP/SNVs were found only in animals with BSE and not in normal controls.
  • the sequences were mostly derived from repetitive genomic sequences wherein most of the prevalent sequences had homology to bovine Ll LINE or SINE repetitive elements. Two sequences showed were neither repetitive nor coding sequences homology.
  • Table 1 shows that a total of 421 SNPs could be identified from the 41 sequences
  • Seq. ID: 11 1231 aggaaatartcaCycaagtccaaga Seq. ID: 223 aggaaatartca-ccaagtccaaga Seq. ID: 596 aggaaahagtya-ccaagtccaara
  • Seq. ID: 27 640 aagcagggtgacAatatacagcctt Seq. ID: 394 aagcagggtgacTatatacagcctt Seq. ID: 675 aarcagggtgacTatatacagcctt
  • Seq. ID: 27 1340 acagttcttctgTgtattcttgcca Seq. ID: 353 acagttcttctg-gtattcttgcca Seq. ID: 693 ayagttcttctg-gtattcttgcca
  • Seq. ID: 34 576 aamarggtmvyrAarattrkaangw Seq. ID: 422 aamaaggtcvyrGarattakaaagw Seq. ID: 713 aavaargkvryrGarawkataragw
  • TTTTGT 1 1 1 I CATGTGTTTGTT-AGYTNKGTGBTWDHNADTHAAATTCAACAYCCATTTATGATAAAAACTCTCCAGAAA

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Abstract

La présente invention concerne des procédés et des compositions permettant la détection d'une encéphalopathie spongiforme bovine transmissible (par exemple, ESB), basée sur la présence de polymorphismes associés à l'ESB dans des échantillons d'acides nucléiques dérivés d'échantillons acellulaires.
PCT/US2008/083420 2007-11-14 2008-11-13 Détection de variations de séquences d'acides nucléiques dans l'acide nucléique circulant dans l'encéphalopathie spongiforme bovine Ceased WO2009064897A2 (fr)

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