EP1412526A2 - Detection amelioree et distinction de l'expression genique differentielle par la ligature et l'amplification de sondes - Google Patents

Detection amelioree et distinction de l'expression genique differentielle par la ligature et l'amplification de sondes

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Publication number
EP1412526A2
EP1412526A2 EP02753074A EP02753074A EP1412526A2 EP 1412526 A2 EP1412526 A2 EP 1412526A2 EP 02753074 A EP02753074 A EP 02753074A EP 02753074 A EP02753074 A EP 02753074A EP 1412526 A2 EP1412526 A2 EP 1412526A2
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European Patent Office
Prior art keywords
target polynucleotide
sensor probes
sensor
pair
polynucleotides
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EP02753074A
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German (de)
English (en)
Inventor
Liang Torrey Mesa Research Institute Inc SHI
Bi-Yu Torrey Mesa Research Institute Inc LI
Xun Torrey Mesa Research Institute Inc WANG
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Syngenta Participations AG
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Syngenta Participations AG
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Publication of EP1412526A2 publication Critical patent/EP1412526A2/fr
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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

Definitions

  • This invention relates to methods for the analysis of nucleic acids and more particularly to the determination of polynucleotide expression and detection of single nucleotide polymorphisms.
  • DNA complement or gene complement is identical in various cells in the body of multi-cellular organisms, there are qualitative and quantitative differences in polynucleotide expression in various cells.
  • a human genome is estimated to contain roughly about 30,000-40,000 genes, however, only a fraction of these genes are expressed in a given cell (International Human Genome Sequence Consortium, Nature, 409:860-921, 2001; Venter et al., Science, 291:1304-1351, 2001).
  • there are quantitative differences among the expressed genes in various cell types Although all cells express certain housekeeping genes, each distinct cell type additionally expresses a unique set of genes. Phenotypic differences between cell types are largely determined by the complement of proteins that are uniquely expressed.
  • RNA profiling RNA profiling
  • differential display RNA profiling
  • hybridization-based methods such as subtractive hybridization (Koyama et al., Proc. Natl Acad. Sci. USA 84: 1609-1613, 1987; Zipfel et al, Mol Cell. Biol 9: 1041-1048, 1989), microarray (U.S. Patent No. 6,150,095), etc.,
  • cDNA tags EST, serial analysis of gene expression (SAGE) (see, e.g. U.S. Patent Nos. 5,695,937 and 5,866,330), and
  • fragment size based often referred to as gel-based methods where a differential display is generated upon electrophoretic separation of DNA fragments on a gel such as a polyacrylamide gel (described in U.S. patent Nos. 5,871,697, 5,459,037, 5,712,126 and PCT publication No. WO 98/51789).
  • Microarray based gene analysis approach enables working with hundreds of thousands of genes simultaneously rather than one or a few genes at a time.
  • Microarray technology has come at an appropriate time, when entire genomes of humans and other organisms are being worked out.
  • Massive sequence information generated as a result of genome sequencing, particularly human genome sequencing has created a demand for technologies that provide high-throughput and speed.
  • Microarrays fill this unique niche.
  • DNA microarrays offer the opportunity to perform fast, comprehensive, moderately quantitative analyses on hundreds of thousands of genes simultaneously.
  • a DNA microarray is composed of an ordered set of DNA molecules of known sequences usually arranged in rectangular configuration in a small space such as 1 cm 2 in a standard microscope slide format. For example, an array of 200 x 200 would contain 40,000 spots with each spot corresponding to a probe of known sequence. Such a microarray can be potentially used to simultaneously monitor the expression of 40,000 genes in a given cell type under various conditions.
  • the probes usually take the form of cDNA, ESTs or oligonucleotides. Most preferred are ESTs and oligonucleotides in the range of 30-200 bases long as they provide an ideal substrate for hybridization.
  • microarrays also known as chips
  • the sample or test material usually consists of RNA that has been amplified by PCR.
  • PCR serves the dual purposes of amplifying the starting material as well as allowing introduction of fluorescent tags.
  • High-density microarrays are built by depositing an extremely minute quantity of DNA solutions at precise location on an array using high precision machines, a number of which are available commercially.
  • An alternative approach pioneered by Packard Instruments, enables deposition of DNA in much the same way that ink jet printer deposits spots on paper.
  • High-density DNA microarrays are commercially available from a number of sources such as Affymetrix, Incyte, Mergen, Genemed Molecular Biochemicals, Sequenom, Genomic Solutions, Clontech, Research Genetics, Operon and Stratagene.
  • labeling for DNA microarray analysis involves fluorescence, which allows multiple independent signals to be read at the same time. This allows simultaneous hybridization of the same chip with two samples labeled with different fluorescent dyes.
  • the calculation of the ratio of fluorescence at each spot allows determination of the relative change in the expression of each gene under two different conditions. For example, comparison between a normal tissue and a corresponding tumor tissue using the approach helps in identifying genes whose expression is significantly altered.
  • the method offers a particularly powerful tool when the gene expression profile of the same cell is to be compared under two or more conditions. High- resolution scanners with capability to monitor fluorescence at various wavelengths are commercially available.
  • polymo ⁇ hisms As greater information on the genome of species is obtained, new markers in the form of genetic variations or polymo ⁇ hisms have been identified for various traits. Numerous types of polymo ⁇ hisms are known to exist. Polymo ⁇ hisms can be created when DNA sequences are either inserted or deleted from the genome, for example, by viral insertion. Another source of sequence variation can be caused by the presence of repeated sequences in the genome variously termed short tandem repeats (STR), variable number tandem repeats (VNTR), short sequences repeats (SSR) or microsatellites. These repeats can be dinucleotide, trinucleotide, tetranucleotide or pentanucleotide repeats. Polymo ⁇ hism results from variation in the number of repeated sequences found at a particular locus.
  • STR short tandem repeats
  • VNTR variable number tandem repeats
  • SSR short sequences repeats
  • microsatellites These repeats can be dinucleotide
  • SNPs single nucleotide polymo ⁇ hisms
  • SNPs are by far the most common source of variation in the genome, as useful genetic markers. SNPs account for approximately 90% of human DNA polymo ⁇ hism (Collins et al., Genome Res., 8:1229-1231, 1998). SNPs are single base pair positions in genomic DNA at which different sequence alternatives (alleles) exist in a population. The term SNP is not limited to single base substitutions, but also includes single base insertions or deletions. In addition, short insertions or deletions of 10 base pairs or less are also often categorized as SNPs because they are often detected with methodologies used to detect single base polymo ⁇ hisms.
  • Nucleotide substitution SNPs are of two types. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine for a pyrimidine or vice versa.
  • the typical frequency at which SNPs are observed is about 1 per 1000 base pairs (Li and Sadler, Genetics, 129:513-523, 1991; Wang et al., Science 280: 1077-1082, 1998; Harding et al., Am. J. Human Genet., 60:772-789, 1997; Taillon-Miller et al., Genome Res., 8:748-754, 1998).
  • the frequency of SNPs varies with the type and location of the change in question.
  • SNPs can affect phenotype. Studies have shown that SNPs can cause major changes in structural folds of mRNA that may affect cell regulation (Shen et al., Proc. Natl Acad. Sci. USA, 96:7871-7876, 1999). When located in a coding region, the presence of a SNP can result in the production of a protein that is non-functional or has decreased function. When present in a non-coding regulatory region, such as a promoter region, the SNP can alter expression of a gene.
  • SNPs SNPs
  • multiplexed allele-specific diagnostic assay MASDA; U.S. Patent No. 5,834,181
  • TaqMan assay U.S. Patent No. 5,962,233
  • molecular beacons U.S. Patent No. 5,925,557
  • MADGE microtiter array diagonal gel electrophoresis
  • PASA PCR amplification of specific alleles
  • Microsphere-based liquid arrays use different coded microspheres to anchor molecular probes and capture target molecules quantitatively. The captured target molecules are then identified, sorted, and quantified by flow cytometry or other readout methods (Bruchez et al., Science, 281 :2013-2016, 1998; Steemers, et al., Nature Biotechnology, 18:91-94; Kettman, et al., Cytometry, 33:234-243, 1998).
  • the Luminex liquid array system uses 100 different types of inexpensive, color-coded microspheres to capture and sort target molecules, with a readout speed of 20,000 microspheres/second (Kettman, et al., Cytometry, 33:234-243, 1998).
  • it provides an inexpensive, multiplexing high-throughput readout platform (Smith et al., Clinical Chemistry, 44:2054-2056, 1998), and has been widely used in detection of protein ligands (Willman, et al., Am. J. Clin. Pathol, 115:764-769, 2001) and DNA polymo ⁇ hism in a high-throughput fashion (Ye, et al., Human Mutation, 17:305-316 2001).
  • ELITE Enzymatic Ligation-based Identification of Transcript Expression
  • the method combines the benefits of the inexpensive T4 DNA ligase in its property to mediate RNA-templated DNA oligonucleotide ligation, the PCR in signal amplification and the liquid array in its multiplexing flexibility and high-throughput readout to achieve sensitive and multiplex detection of polynucleotide expression.
  • a method for determining polynucleotide expression comprising providing a population of test polynucleotides containing one or more target polynucleotides of interest, where at least a portion of the nucleotide sequence of the target polynucleotides is known.
  • a pair of specific sensor probes are provided, where the first probe of the pair has a 3' portion that is complementary to the target polynucleotide and a 5' portion comprising a primer binding site common to all first sensor probes; and the second probe of the pair has a 5' portion complementary to the target polypeptide and a 3' portion comprising a primer binding site common to all second sensor probes.
  • the 5' end of the second probe is phosphorylated.
  • the sensor probes are such that when hybridized to the target polynucleotide, the 3' end of the first probe is immediately adjacent to the 5' end of the second probe, with no gap in between the two probes. If necessary, the target polynucleotides are denatured, then combined with sets of sensor probes under moderately stringent, stringent, or highly stringent conditions and the sensor probes are allowed to hybridize to the target polynucleotides.
  • a ligase reaction is conducted so the hybridized pairs of sensor probes are ligated to form a ligated sensor probe comprising both members of the sensor probe pair.
  • a T4 ligase is used.
  • a single mismatch or more at the 3' end of the first probe or the 5' end of the second probe results in a failure of the ligation reaction.
  • the ligated sensor probes are amplified using any suitable method so that the resulting amplified ligated sensor probes contain a detectable label.
  • amplification is accomplished using the polymerase chain reaction and the common primer binding sites on the sensor probes.
  • the detectable label is inco ⁇ orated into the amplified sensor probes using labeled dNTPs, while in still another embodiment, labeled primers are used.
  • the detector oligonucleotide comprises a detectable label that is unique for that class of detector oligonucleotide and that differs, i.e. is capable of being differentiated, from the detectable label of the sensor probes. At least a portion of the detector oligonucleotide is capable of hybridizing to the ligated and amplified sensor probes.
  • the detector oligonucleotides further comprise a linker which does not hybridize to the ligated sensor probes, but links the detector oligonucleotide to a label, such as a microparticle, for example a fluorescent microbead or microsphere.
  • a label such as a microparticle, for example a fluorescent microbead or microsphere.
  • the fluorescent microbead or microsphere is a Luminex microbead.
  • the labeled amplified ligated sensor probes are combined with detector oligonucleotides and allowed to hybridize under moderately stringent, stringent, or highly stringent conditions. If necessary, the amplified sensor probes are denatured prior to hybridization.
  • the detector oligonucleotide hybridize to the ligated sensor probes at a location that spans the ligation site of the sensor probe pair. The hybridization of the detector oligonucleotides to the corresponding ligated amplified sensor probes is determined using any suitable means.
  • hybridization of the detector oligonucleotide to the amplified ligated sensor probes is determined by detecting the presence of the sensor probe label in association with the detector oligonucleotide label. In one embodiment, the detection is by use of a flow cytometer. If desired, the determination of hybridization can be quantitative.
  • Another aspect provides detection of a single nucleotide polymo ⁇ hism of interest comprising providing a test population of polynucleotides which contains at least one target polynucleotide known, or suspected, to contain a single nucleotide polymo ⁇ hism (SNP).
  • SNP single nucleotide polymo ⁇ hism
  • the first sensor probe of the pair has a 3' end portion that is complementary to the target polynucleotide and a 5' end portion that contains a common primer binding site; and the second sensor probe of the pair has a 5' end portion that is complementary to target polynucleotide and a 3' end portion that contains a common primer binding site.
  • the sensor probes in a pair are such that the 3' end of the first probe hybridizes on the target polynucleotide immediately adjacent to the 5' end of the second probe and that either the 3' terminal nucleotide on the first probe or the 5' terminal nucleotide on the second probe is complementary to an allele of the SNP of interest.
  • a second pair of sensor probes is provided.
  • This second pair of sensor probes has the same characteristics as the first pair and hybridizes to the target polynucleotide at the same location, but either the 3' terminal nucleotide of the first probe in this second pair or the 5' terminal nucleotide of the second probe of this second pair is complementary to an allele of the SNP of interest that is different from the complementary allele for the first pair of sensor probes
  • the target polynucleotides are combined with the sensor probe pairs under moderately stringent, stringent, or highly stringent conditions and the sensor probes are allowed to hybridize to the target polynucleotides. If necessary, the target polynucleotides are denatured prior to hybridization.
  • the hybridized sensor probe pairs are ligated to form a ligated sensor probe under conditions such that if there is a mismatch at the site of the SNP of interest, ligation does not occur. That is, if the SNP allele complementary to either the 3' terminal nucleotide of the first sensor probe, or the 5' terminal of the second sensor probe is not present, ligation does not occur.
  • a T4 ligase is used.
  • the successfully ligated sensor probes are amplified by any suitable means to produce amplified ligated sensor probes such that the amplified sensor probes comprise a detectable label.
  • amplification is achieve by the polymerase chain reaction and the common primer binding sites on the sensor probes.
  • the detectable label is inco ⁇ orated into the amplified ligated sensor probes using labeled dNTPs, while in still another embodiment, labeled primers are used.
  • each class of detector oligonucleotides For each type of sensor probe pair, at least one class of detector oligonucleotides is provided where each class of detector oligonucleotide has a unique detectable label that is different from the detectable label of the amplified sensor probes. At least a portion of the detector oligonucleotide is capable of hybridizing to the ligated and amplified sensor probes.
  • the detector oligonucleotides further comprise a linker which does not hybridize to the ligated sensor probes, but links the detector oligonucleotide to a label, such as a microsphere, for example a fluorescent microbead or microsphere.
  • the fluorescent microbead or microparticle is a Luminex microbead.
  • the detector oligonucleotides and the amplified, ligated sensor probes are combined and allowed to hybridize under moderately stringent, stringent or highly stringent conditions. If necessary, the amplified ligated sensor probes are denatured prior to hybridization. In one embodiment, the detector oligonucleotide hybridize to the ligated sensor probes at a location that spans the ligation site of the sensor probe pair, that is, spans the location of the SNP of interest.
  • hybridization of the detector oligonucleotides to the corresponding ligated amplified sensor probes is determined using any suitable means. In one embodiment, hybridization of the detector oligonucleotide to the amplified ligated sensor probes is determined by detecting the presence of the sensor probe label in association with the detector oligonucleotide label. In one embodiment, the detection is by use of a flow cytometer. If desired, the determination of hybridization can be quantitative.
  • Another aspect provides a method for determining the physiological or developmental state of a cell or tissue.
  • This aspect comprises obtaining a population of polynucleotides from a test cell or tissue comprising at least one target polynucleotide.
  • the target polynucleotide is isolated from the test cell or tissue using standard methods.
  • the expression of the target polynucleotide or polynucleotides is determined using the methods described herein and the results compared to those obtained using the same method obtained from a reference cell or tissue of a known physiological or developmental state.
  • Still another aspect provides a method for diagnosing a disease condition, disorder, or predisposition of interest.
  • This aspect comprises obtaining a population of polynucleotides from a cell or tissue of a test subject comprising at least one target polynucleotide.
  • the target polynucleotide is isolated from the cell or tissue using standard methods.
  • the expression of the target polynucleotide or polynucleotides is determined using the methods described herein and the results compared to those obtained using the same method obtained from a cell or tissue of a reference subject known to have the disease, condition, disorder, or predisposition of interest.
  • Yet another aspect provides a method for diagnosing a disease, condition, disorder or predisposition associated with a single nucleotide polymo ⁇ hism (SNP).
  • This aspect comprises obtaining a population of polynucleotides from a cell or tissue of a test subject and determining the presence, absence, or frequency of at least one allele of at least one SNP of interest using any of the methods described herein.
  • the presence, absence or frequency of the SNP allele of interest in the test subject is compared to the presence, absence or frequency of the allele in a population of polynucleotides from a cell or tissue obtained from a reference subject known to have said disease, condition, disorder, or predisposition.
  • kits for practicing the methods disclosed herein comprise, at a minimum, at least one pair of sensor probes for at least one target polynucleotide of interest, at least one detector oligonucleotide for each pair of sensor probes provided, and instructions for carrying out any of the methods described herein.
  • the detector oligonucleotides further comprise a microbead or microsphere.
  • the kits may also comprise a ligase, primers, dNTPs, a polymerase and/or buffer solutions.
  • Figure 1 shows a depiction of one embodiment of the present invention.
  • Figure 2 shows the sensitivity of the methods disclosed for polynucleotide expression analysis.
  • Figure 3 shows the dynamic range of the methods disclosed for polynucleotide expression analysis.
  • Figure 4 shows the reproducibility of the methods disclosed for polynucleotide expression analysis.
  • aspects of the present invention provide novel methods for use in the analysis of nucleic acids. These methods are particularly useful for expression analysis, such as gene expression analysis. Additional aspects provide methods for the detection of single nucleotide polymo ⁇ hisms (SNPs). SNPs have a wide variety of uses including diagnosis of genetic diseases and predispositions in plants and animals, including humans, as well as uses to identify valuable phenotypes and in marker assisted selection. In addition to providing multiplex capabilities, the methods provided have the advantages of adaptability, easy of use, and cost effectiveness.
  • SNP single nucleotide polymo ⁇ hism.
  • MFI mean fluorescence intensity
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric ( 2 or more monomers) form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Whether or not specifically stated, polynucleotides and oligonucleotides are considered to have a 5' end and a 3' end. Although nucleotides are usually joined by phosphodiester linkages, the terms also include peptide nucleic acids such as polymeric nucleotides containing neutral amide backbone linkages composed of aminoethyl glycine units (Nielsen et al., Science, 254: 1497, 1991).
  • the terms refer only to the primary structure of the molecule.
  • the terms include double- and single-stranded DNA and RNA as well DNA/RNA hybrids that may be single-stranded, but are more typically double- stranded.
  • the terms also refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all or one or more of the molecules, but more typically involve only a region of some of the molecules.
  • the terms also include known types of modifications, for example, labels, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g. methyl phosphonates, phophotriesters, phosphoamidates, carbamates etc.), those containing pendant moieties, such as, for example, proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc,), those containing alkylators, those with modified linkages (e.g.
  • Polynucleotides include both sense and antisense, or coding and template strands. The terms include naturally occurring and chemically synthesized molecules.
  • detectable label refers to a label which, when attached, provides a means of detection.
  • labels available for this pu ⁇ ose including, without limitation, radioactive labels such as radionuclides, fluorophores or fluorochromes, peptides, enzymes, antigens, antibodies, vitamins or steroids.
  • radioactive nuclides such as 32 P or 35 S, or fluorescent dyes are conventionally used to label PCR primers.
  • Chemiluminescent dyes can also be used for the pu ⁇ ose.
  • the label can be attached directly to the molecule of interest or be attached through a linker.
  • suitable labels include xanthine dyes, rhodamine dyes, naphthylamines, benzoxadiazoles, stilbenes, pyrenes, acridines, Cyanine 3, Cyanine 5, phycoerythrin conjugated streptavidin, Alexa 532, fluorescein, tetramethyl rhodamine, fluorescent nucleotides, digoxigenin, and biotin- deoxyuracil triphosphate.
  • the nucleic acid can be labeled using intercalating dyes such as, for example, YOYO, TOTO, Picogreen, ethidium bromide, and the like.
  • sequence means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.
  • the term "subject” refers to any plant or animal.
  • animal includes human beings.
  • primer or “oligonucleotide primer” or “PCR primer” means an oligonucleotide, either naturally occurring, as in a purified restriction enzyme digest, or produced synthetically, that under the proper conditions, is capable of hybridizing to a template DNA or RNA molecule to initiate primer extension by polymerization, such as by a DNA-dependent DNA polymerase, an RNA-dependent RNA polymerase, or an RNA- dependent DNA polymerase, to produce a DNA or RNA molecule that is complementary to the template molecule.
  • Primers are often between about 5 to about 50, typically between about 10 to about 30 and more typically between about 18 to about 25 nucleotides in length, and do not contain palindromic sequences or sequences resulting in the formation of primer dimers. Often primers are single stranded, however, double stranded primers may be used provided the primer is treated to separate the strands prior to being used for primer extension.
  • a target polynucleotide in some aspects of the invention, can be DNA or RNA. Any of the various types of DNA and RNA can be used, for example, mRNA, cRNA, viral RNA, synthetic RNA, cDNA, genomic DNA, viral DNA, plasmid DNA, synthetic DNA, amplified DNA or any combination thereof.
  • the target polynucleotides can be obtained from any source containing nucleic acids. Sources typically include cells and tissues from prokaryotes and eukaryotes such as bacteria, yeast, fungi, plants and animals. The target polynucleotides can also be obtained from viruses.
  • tissue By tissue is meant a plurality of cells that in their native state are organized to perform one or more specific functions.
  • Non-limiting examples of tissues include muscle tissue, cardiac tissue, nervous tissue, leaf tissue, stem tissue, root tissue, etc.
  • Cells from which target polynucleotides are obtained can be haploid, diploid, or polyploid.
  • hybridization refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing. Techniques and conditions for hybridization are well known to practitioners in the field. It is also well known in the field that when double stranded nucleic acid molecules are used in hybridization, the double stranded molecules are made typically single stranded, generally by heat or chemical denaturation, prior to hybridization. As used herein, hybridization includes such denaturation, if required, whether or not such denaturation is specifically mentioned.
  • One aspect provides a method for determining expression of a polynucleotide, for example, gene expression.
  • the determination can be, if desired, quantitative.
  • a population of test polynucleotides for example DNA or RNA
  • the population of polynucleotides can be obtained from any source.
  • the polynucleotides can be obtained from plant or animal cells or tissues.
  • the polynucleotides are isolated or purified. Methods for the isolation or purification of polynucleotides are well known in the art and can be found in standard texts such as Sambrook et al., Molecular Cloning, 2 nd ed., Cold Spring Harbor Laboratory Press, 1989.
  • the population of polynucleotides contains one or more target polynucleotides of interest where the target polynucleotides comprise a known nucleic acid sequence.
  • the population of polynucleotides contains at least 20 different target polynucleotides, in another embodiment at least 50 different target polynucleotides, and in still another embodiment at least 100 different polynucleotides.
  • the sequence of all polynucleotides in the population need not be known.
  • the complete sequence of the target polynucleotides need not be known, but rather only a portion of the sequence of the target polynucleotide need be known.
  • each target polynucleotide For each target polynucleotide, at least one pair of specific sensor probes is provided.
  • the specific sensor probes are chosen so that under the conditions used, the sensor probes will preferably hybridize to only one target molecule in the population. It will be apparent that, if desired, more that one pair of sensor probes can be used. That is, that for a particular target polynucleotide, different sensor probe pairs that hybridize at different locations on the target polynucleotide or hybridize under different conditions can be used.
  • Each pair of sensor probes are selected so that a 3' portion of one member of the pair is complementary to the target polynucleotide while the other member of the pair has a 5' portion that is complementary to the target polynucleotide.
  • each sensor probe is also chosen so that when hybridized to the target polynucleotide, the 3' end of one member of the pair is immediately adjacent to the 5' end of the other member of the pair. By immediately adjacent is meant that there is no gap between the 3' end of one member of the pair and the 5' end of the other member of the pair. In one embodiment each sensor probe contains three portions.
  • the sensor probes are selected so that when hybridized to the target polynucleotide, the first portion of each member of a pair will be immediately adjacent to each other.
  • Common primer binding sites are portions of the sensor probes which have little or no homology to known nucleotide sequences from the organism from which the target polynucleotides are obtained.
  • the common primer regions may or may not be homologous to the detector oligonucleotides.
  • the common primer binding sites are common to all sets of sensor probes. The use of common primer binding sites allows the use of the same primers for amplification of all sensor probes.
  • the portion of the sensor probe that is complementary to the target polynucleotide is about 15 to about 30 nucleotides in length, in another embodiment about 18 to about 24 nucleotides in length, while in still another embodiment about 20 nucleotides in length.
  • the common primer binding site of the sensor probes is about 12 to about 24 nucleotides long, in another embodiment about 15 to about 20 nucleotides long and in still another embodiment about 18 nucleotides long.
  • the population of polynucleotides containing the target polynucleotides of interest are then combined with the pairs of specific sensor probes under conditions that allow for hybridization of the sensor probes to the target polynucleotides. If necessary, prior to hybridization the target polynucleotides are denatured using any means known in the art, for example heat or chemical denaturation.
  • the hybridization conditions can be moderately stringent, stringent or highly stringent. In one embodiment, hybridization is carried out under stringent conditions.
  • the T m (melting temperature) of a nucleic acid hybrid is the temperature at which 50% of the bases are base-paired.
  • the T m melting temperature
  • the T m reflects a time-independent equilibrium that depends on the concentration of oligonucleotide.
  • the T m corresponds to a situation in which the strands are held together in structure possibly containing alternating duplex and denatured regions.
  • the T m reflects an intramolecular equilibrium that is independent of time and polynucleotide concentration.
  • T m is dependent on the composition of the polynucleotide (e.g. length, type of duplex, base composition, and extent of precise base pairing) and the composition of the solvent (e.g. salt concentration and the presence of denaturants such formamide).
  • concentration e.g. salt concentration and the presence of denaturants such formamide.
  • T m 81.5°C - 16.6(log 10 [Na + ]) + 0.41(% G + C) - 0.63(% formamide) - 600/L)
  • stringent hybridization conditions include 5X SSPE, 50% formamide at 42°C or 5X SSPE at 68°C.
  • Stringent wash conditions are often determined empirically in preliminary experiments, but usually involve a combination of salt and temperature that is approximately 12-25°C below the T m .
  • One example of highly stringent wash conditions is IX SSC at 60°C.
  • An example of very highly stringency wash conditions is 0.1X SSPE, 0.1% SDS at 42 °C (Meinkoth and Wahl, Anal. Biochem., 138:267-284, 1984).
  • An example of extremely stringent wash conditions is 0.1X SSPE, 0.1%> SDS at 50-65°C.
  • various combinations of factors can result in conditions of substantially equivalent stringency. Such equivalent conditions are within the scope of the present inventive discovery.
  • pairs of sensor probes that have hybridized to their target polynucleotide immediately adjacent to each other are ligated together to form a ligated sensor probe.
  • Conditions for ligation used as such that the presence of a single mismatch between the adjacent 3' and 5' ends of a pair of sensor probes will prevent ligation from occurring.
  • Any suitable ligase known in the art can be used.
  • a T4 ligase is used. It has long been recognized in the art that T4 DNA ligase can mediate ligation of DNA oligonucleotides hybridized to RNA (Kleppe, et al., Proc. Natl. Acad. Sci. USA, 67:68-73, 1970) and that this ligation permits discrimination of single nucleotide mismatches between the target and the probe (Nilsson, et al., Nature Biotech. 18:791-793, 2000).
  • the ligated sensor probes are amplified to produce amplified sensor probes.
  • Any known method of amplification can be used including, but not limited to, the polymerase chain reaction (PCR) (U.S. Patent Nos. 4,965,188; 4,800,159; 4,683,202; 4,683,195), ligase chain reaction (Wu and Wallace, Genomics, 4:560-569, 1989; Landegren et al., Science, 241 :1077- 1080, 1988), transcription amplification (Kwoh et al. Proc. Natl. Acad. Sci. USA, 86:1173- 1177, 1989), self-sustained sequenced replication (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 1874-1878, 1990) and nucleic acid based sequence amplification (NASBA).
  • amplification is accomplished by PCR.
  • the common primer binding sites located on the sensor probes can be utilized. Because the primer sites are common across pairs of sensor probes directed to different target molecules, a single primer or set of primers can be used. This limits the bias in amplification that can result due to differences in the efficiency of binding of different primers under the same binding conditions. The lessening or elimination of this bias allows for the amplification to be quantitative.
  • the presence of two primer binding sites on ligated sensor probes allows for preferential amplification of ligated sensor probes. Thus, ligated sensor probes are amplified exponentially, while non-ligated sensor probes are not amplified or are amplified only linearly.
  • the amplified sensor probes comprise a detectable label or marker.
  • the detectable label or marker is inco ⁇ orated into the amplified sensor probes during amplification.
  • the label can be inco ⁇ orated by using primers, one or both of which contain a label, labeled nucleoside triphosphates (NTPs), or a combination of labeled primers and NTPs.
  • NTPs labeled nucleoside triphosphates
  • the labels are biotin-deoxyuracil triphosphate and phycoerythrin conjugated streptavidin.
  • the present invention also provides for each different pair of sensor probes at least one class of detector oligonucleotide that is capable of identifying sensor probes for a particular target polynucleotide.
  • the ability of the detector oligonucleotide to identify a particular group of sensor probes is due to the presence of a detectable label or maker associated with a particular class of detector oligonucleotide.
  • the detectable label may be coupled directly to the detector oligonucleotide or by means of a linker molecule.
  • the detectable label is different and so unique for each class of detector oligonucleotide used.
  • detector oligonucleotides each class comprising a different label
  • the detectable labels or markers used with detector oligonucleotides should be different from the detectable label or marker used with the amplified sensor probes.
  • different is meant that the labels are capable of being differentiated from each other, for example on the basis of their emission spectra.
  • detector oligonucleotides are about 18 to about 30 nucleotides in length. In another embodiment, detector oligonucleotides are about 24 nucleotides long.
  • the detector oligonucleotide further comprises a microbead or microsphere.
  • the microbead is attached to the detector oligonucleotide by means of a linker, for example a 5' amino-unilinker (Oligo Etc., Wilsonville, OR).
  • a linker for example a 5' amino-unilinker (Oligo Etc., Wilsonville, OR).
  • the identity of the detector oligonucleotide can be accomplished using microbeads of different sizes, shapes and/or colors (labels).
  • the microbeads can range in size from about 0.1 micrometers to about 1000 micrometers, generally about 1 to about 100 micrometers, typically about 2 to about 50 micrometers, more typically about 3 to about 25 micrometers, usually about 6 to about 12 micrometers.
  • the microbeads can be made of any suitable material including, but not limited to, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polycarbonate, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze, polysulfone, or combinations thereof.
  • suitable material including, but not limited to, brominated polystyrene, poly
  • polymer materials such as carbohydrate, e.g., carboxymethyl cellulose, hydroxyethyl cellulose, agar, gel, proteinaceous polymer, polypeptide, eukaryotic and prokaryotic cells, viruses, lipid, metal, resin, latex, rubber, silicone, e.g., polydimethyldiphenyl siloxane, glass, ceramic, charcoal, kaolinite, bentonite, and the like can also be used.
  • commercially available Luminex microbeads Luminex Co ⁇ ., Austin, TX are used.
  • Luminex microbeads are extensively discussed in U.S. Patent No. 6,268,222 and PCT publications WO 99/37814 and WO 01/13120. Briefly, the microbeads are microparticles that inco ⁇ orate polymeric nanoparticles stained with one or more fluorescent dyes. All of the nanoparticles in a given population are dyed with the same concentration of a dye, and by inco ⁇ orating a known quantity of these nanoparticles into the microsphere, along with known quantities of other nanoparticles stained with different dyes, a multifluorescent microsphere results. By varying the quantity and ratio of different populations of nanoparticles, it is possible to establish and distinguish a large number of discrete populations of microspheres with unique emission spectra.
  • the fluorescent dyes used are of the general class known as cyanine dyes, with emission wavelengths between 550 nm and 900 nm. These dyes may contain methine groups; the number of methine groups influences the spectral properties of the dye.
  • the monomethine dyes that are pyridines typically have a blue to blue-green fluorescence emission, while quinolines have a green to yellow-green fluorescence emission.
  • the trimethine dye analogs are substantially shifted toward red wavelengths, and the pentamethine dyes are shifted even further, often exhibiting infrared fluorescence emission.
  • any dye compatible with the composition of the beads can be used.
  • the dyes When a number of different microbeads are used in practicing the methods described herein, it is preferable, but not required, that the dyes have the same or overlapping excitation spectra, but possess distinguishable emission spectra. Multiple classes or populations of particles can be produced from just two dyes. The ratio of nanoparticle populations with red/orange dyes is altered by an adequate increment in proportion so that the obtained ratio does not optically overlap with the former ratio. In this way a large number of differently fluorescing microbead classes are produced.
  • Detector oligonucleotides may be coupled to the microbead by any suitable means known in the art. The exact method of coupling will vary with the composition of the microbead and the type of linker present, if any.
  • detector oligonucleotides are coupled to microbeads by the well known carbodiimide coupling procedure. Multiple detector oligonucleotides may be coupled to a single microbead. Microbeads of the same class or group, that is having the same label or fluorescent signature, will have detector oligonucleotides specific for the same target polynucleotide attached to them.
  • the association of the detector oligonucleotide to the target polynucleotide is accomplished using the sensor probes.
  • more than one group of sensor probe pairs may be directed to a single target polynucleotide.
  • the sequence of detector oligonucleotides attached to a single class of microbeads may be different, although all sequences will be associated with the same target polynucleotide.
  • the detector oligonucleotides are such that at least a portion of the detector oligonucleotide is complementary to a portion of the amplified sensor probe that is, in turn, complementary to the target polynucleotide to allow hybridization of the detector oligonucleotide to the amplified ligated sensor probe.
  • hybridization is typically performed under moderately stringent, stringent or highly stringent conditions. In one embodiment, hybridization of detector oligonucleotides to amplified sensor probes is performed under stringent conditions.
  • the portion of the amplified sensor probes that hybridizes with the sensor oligonucleotide comprises the ligation site where the members of the sensor probe pair were ligated prior to amplification.
  • conditions can be modified to favor hybridization of detector oligonucleotides to those sensor probe pairs that have been successfully ligated.
  • the exact conditions used will vary with the composition of the sensor probes and detector oligonucleotides used. Optimization of hybridization conditions is routine in the art and can be accomplished by the skilled artisan using the guidance provided herein and standard reference texts in molecular biology.
  • Identification, and if desired quantification, of detector oligonucleotides that have hybridized to ligated and amplified sensor probes is accomplished by means of the detectable labels. Successful hybridization will result in a molecule that comprises both the label associated with the amplified sensor probe and the label associated with the detector molecule.
  • the presence of each target polynucleotide in the original population of polynucleotides can be determined based on the label associated with the detector oligonucleotide while the amount of each target can be determined by quantifying the amount of sensor probe label associated with each class of detector oligonucleotide.
  • the determination of the labels present and, if desired, their amount can be accomplished using a single measuring device and be accomplished essentially simultaneously or they can be accomplished using different measuring devices and accomplished in series.
  • detector oligonucleotides can be identified and sorted on the basis of their unique labels. Following sorting, the amount of sensor probe label associated with each class of detector oligonucleotide can be determined, thus allowing the identification and quantification of each target polynucleotide present in the original population of polynucleotides.
  • a device such as a flow cytometer can be used to individually analyze each detector oligonucleotide and identify it based on its unique marker. The detector oligonucleotide is then analyzed to determined the presence of the amplified sensor probe label. If the sensor probe label is present, an event is recorded for the target polynucleotide associated with that detector molecule.
  • detector oligonucleotides and sensor probe labels are differentiated on the basis of differences in emission spectra.
  • any detection system can be used to detect the difference in spectral characteristics between the two labels, including a solid state detector, photomultiplier tube, photographic film, or eye, any of which may be used in conjunction with additional instrumentation such as a spectrometer, luminometer microscope, plate reader, fluorescent scanner, flow cytometer, or any combination thereof, to complete the detection system.
  • the labels preferably have emission wavelengths of perceptibly different colors to enhance visual discrimination.
  • filters and diffraction gratings allow the respective emission maxima to be independently detected.
  • detector oligonucleotides comprise fluorescent microbeads
  • detector oligonucleotides are identified using a flow cytometer, for example a fluorescence-activated cell sorter, wherein the different classes of beads in a mixture can be physically separated from each other based on the fluorochrome identity, size and/or shape of each class of bead; and the presence of the target polynucleotide qualitatively or quantitatively determined based on the presence of the detectable label for each sorted pool containing beads of a particular class.
  • a flow cytometer for example a fluorescence-activated cell sorter
  • Flow cytometers with multiple excitation lasers and detectors are preferred.
  • the Luminex 100 is used.
  • the exact setting necessary for optimum detection will vary with factors such as the instrument used, the labels used, and the particles used. Optimization of settings and conditions for the use of a flow cytometer for practicing the methods disclosed herein can be accomplished by the skilled technician without undue experimentation.
  • General guidance on the use of flow cytometers can be found in texts such as Shapiro, Practical Flow Cytometry, 3 rd ed., Wiley-Liss, 1995 and Jaroszeski et al., Flow Cytometry Protocols, Humana Press, 1998.
  • the methods described herein can also be used to detect single nucleotide polymo ⁇ hisms (SNP).
  • SNP single nucleotide polymo ⁇ hisms
  • the detection of SNPs can be accomplished with only minor modifications to the methods previously described.
  • a population of polynucleotides is obtained wherein the population contains or is suspected to contain one or more target polynucleotides containing at least one SNP.
  • the pairs of sensor probes are provided wherein a first member of each pair has a 3' portion that is complementary to the target polynucleotide and the second member has a 5' portion that is complementary to the target polynucleotide.
  • the sensor probes in each pair are designed so that the 3' end of the first probe is immediately adjacent to the 5' end of the second probe when hybridized to the target polynucleotide.
  • the terminal nucleotide on either the 3' end of the first sensor probe or the 5' end of the second sensor probe is complementary to one of the alleles of the SNP of interest.
  • different pairs of sensor probes can be used to detect the presence of alternate alleles of the same SNP.
  • one set of sensor probes have a terminal nucleotide complementary to one allele of the SNP of interest, while the additional sets of sensor probes have terminal nucleotides complementary to the alternate allele or alleles for the SNP of interest.
  • the sensor probe pairs are ligated together to form ligated sensor probes under conditions such that if there is a single mismatch at either the 3' of the first probe or the 5' end of the second probe, ligation will not occur.
  • the ligated sensor probes are then amplified as described herein such that the amplified sensor probe comprise a detectable label.
  • a detector oligonucleotide having the characteristics previously described is provided. If necessary, the amplified ligated sensor probes are denatured and hybridized to the detector oligonucleotides under moderately stringent, stringent or highly stringent conditions.
  • the hybridization of the detector oligonucleotide to the amplified ligated sensor probes is then determined using any of the methods described herein.
  • the presence, absence or frequency of an allele of the SNP of interest is then determined based on the identity of the detector oligonucleotide.
  • the present method allows determination of if a subject is homozygous or heterozygous for a particular allele of the SNP of interest. For example, when sensor probes are provided for only one of two possible alleles, if the allele is not present, there will be no signal above background. If the subject is homozygous for the allele then the maximum signal will be obtained, while if the subject is heterozygous, a signal approximately half that observed with a homozygous individual will be obtained. In another example where sensor probes to each of two alternative alleles are provided, a heterozygous subject will exhibit signals of approximately equal magnitude for both alleles while a homozygous individual will exhibit signals above background for only one of the two alleles.
  • a population of polynucleotides is obtained from a cell or tissue of a test subject.
  • the presence or absence of an allele of at least one SNP of interest in said population of polynucleotides is determined using the methods discussed herein.
  • the presence, absence, or frequency of the SNP allele or alleles of interest in the polynucleotides from the test subject is compared to the presence, absence, or frequency of the allele or alleles or interest in polynucleotides obtained from a reference subject known to have said disease, condition, disorder or predisposition.
  • Expression profiling involves the determination of changes in the expression of polynucleotides, e.g. genes, under different conditions and physiological states. Because changes in polynucleotide expression can be related to pathological conditions, the methods described herein are useful for diagnosing a disease, condition, disorder, or predisposition associated with a change in expression patterns.
  • information on the expression of one or more target polynucleotides is obtained from an test subject using the methods described herein and compared to the expression pattern for a subject known to have the disease, condition, disorder, or predisposition of interest.
  • data representing the expression pattern of a subject with a known disease, condition, disorder or predisposition is stored on a computer readable medium so that the expression pattern from the test subject can be compared to the stored expression pattern.
  • predisposition refers to the likelihood that an individual subject will develop a particular disease, condition, or disorder. For example, a subject with an increased predisposition will be more likely than average to develop a disease, condition, or disorder, while a subject with a decreased predisposition will be less likely than average to develop a disease, condition, or disorder.
  • the disease, condition, disorder, or predisposition may be genetic or may be due to a microorganism.
  • the methods disclosed herein can be used to determine the developmental or physiological state of a cell or tissue. In this aspect, polynucleotide expression from a test cell or tissue is compared to the expression pattern from a cell of known physiological or developmental state.
  • data representing the expression pattern of a cell or tissue of a known developmental or physiological state is stored on a computer readable medium so that the expression pattern from the test cell or tissue type can be compared to the stored expression pattern.
  • kits for practice of the novel methods disclosed herein One embodiment provides a kit comprising, at least one pair of sensor probes for at least one target polynucleotide, at least one detector oligonucleotide for each pair of sensor probes, and instructions for carrying out the novel methods described herein for the determination of polynucleotide expression. Another embodiment provides a kit comprising, at least one pair of sensor probes for at least one target polynucleotide, at least one detector oligonucleotide for each pair of sensor probes, and instructions for carrying out the novel methods described herein for the detection of single nucleotide polymo ⁇ hisms.
  • the detector oligonucleotides can further comprise microbeads, ligases, primers, dNTPs, polymerase, and/or buffer solutions.
  • the expression of the Arabidopsis putative plastocyanin gene was analyzed. This gene is found to be expressed at low levels in the dark, and can be induced by light through the phytochrome A signaling pathway. In the phyA null mutant, the light induction is blocked (Tepperman, et al., Proc. Natl. Acad. Sci. USA, 98:9437-9442, 2001).
  • Wild-type and phyA null mutant seedlings of Arabidopsis thaliana ecotype RLD were grown on agar plates in the dark condition as described in Tepperman et al., 2001, supra. After 5 days of germination, seedlings are exposed to continuous far-red light (FRc), and whole seedling samples are collected at 7 time points: 0, 1, 3, 6, 12, 18, and 24 hours after the light treatment. An 8 th sample was also collected from seedlings maintained in darkness for a further 24 hours (6-day dark controls). RNA samples were prepared as described in Tepperman et al., 2001, supra.
  • A. Detector Oligonucleotides Twenty-four-mer detector oligonucleotide sequences were selected for the target mRNA sequences. Since cDNA synthesis is not required in the present method, the detector oligonucleotide sequences can be chosen from any region of mRNA. The Tm of the detector oligonucleotides ranged from 65°C to 75°C. The Tm of each half 12 bases ranged from 32°C to 40°C. The detector oligonucleotides were in the sense direction as the RNA templates, and modified with an amino-Unlinker at 5 ' end for coupling to carboxylated microspheres. The oligonucleotides were synthesized by Operon Technologies (Alameda, CA), and HPLC purified.
  • Sensor Probes were 38 nucleotides in length. For each pair of sensor probes, a 20 base sequence on the 3' end of a first probe and a 20 base sequence on the 5' end of the second probe and continuous in sequence and complementary to a continuous 40 nucleotide stretch in the target mRNA. Sensor probes were designed so that after ligation, the center 24 bases would hybridize with the detector oligonucleotides. The final 18 bases on the 5' end of the first probe and the last 18 bases on the 3' end of the second probe were common for sensor probes and served as common binding sites for PCR primers. The first probe of each pair was cartridge purified, while the second probe was 5' phosphorylated and PAGE purified. C. Primers. The sequence of the primer 1 was the same as the 5' end portion of the first sensor probe and the sequence of the primer 2 was complementary to the 3' end of the second sensor primer. The primers were cartridge purified prior to use.
  • Carboxylated fluorochrome microbeads (Luminex, Austin TX) were coupled to 5' amino-Unilinker modified detector oligonucleotides by a one-sep carbodiimide couple procedure following the manufacturer's instructions.
  • the conjugated microspheres were stored in 0.1 M MES buffer (2-(N-mo ⁇ holino) ethane sulfonic acid, Sigma Chemical Co, St. Louis, MO) in the dark at a concentration of 5x10 4 beads/ul.
  • the ligation reaction contained 5-500 ng of total target RNA, and 1 ul of 0.1 mM mixture of each member of a pair of sensor probes in a final volume of 4 ul.
  • All sensor probes were pre-mixed to yield a concentration of 0.1 mM for each sensor probe.
  • One ul of the 0.1 mM pre-mix was then mixed with 500 ng of target RNA to give a final volume of 4 ul.
  • the mixture was heated to 65°C for 10 minutes, and then slowly cooled to room temperature prior to addition of 2 ml of DNA ligase reaction mixture (1.2 ml 5x buffer, 0.3 ml nuclease-free, and 0.5 ml of T4 DNA ligase, lU/ml, Invitrogen, Carlsbad, CA).
  • the reaction was incubated at 37°C for 4 hours, after which the reaction was terminated by heating to 70°C for 10 minutes and the ligation product was stored at 4°C.
  • Ligated sensor probes were amplified using two universal primers.
  • Primer 1 had the same sequence as the 5' common primer binding region located on the first sensor probe of each probe pair and was biotinylated on the 5' end.
  • the sequence of primer 1 was 5' Biotin- gctgctagtgtccgatgt 3' (SEQ ID NO: 1).
  • Primer 2 was complementary to the 3' common primer binding portion of the second sensor probe of each set and had a sequence of 5' gatctcctagagtcgtga 3' (SEQ ID NO: 2).
  • a 20 ul reaction contained 6 ul of ligation product, 1.5 mM MgCl 2 , 200 mM of each dNTP, 05 mM of each PCR primer, 1 U Taq DNA polymerase (Invitrogen) and lx PCR buffer (Invitrogen).
  • the PCR reaction was carried out in a thermocycler with a hold of 94°C for 3 minutes, followed by 25 cycles of 94°C for 30 seconds, 56°C for 30 seconds and 72°C for 30 seconds, followed by a hold at 72°C for 5 minutes.
  • Detector oligonucleotides conjugated to fluorescent microbeads were diluted with 1.5X TMAC buffer (3M TMAC, 0.1% SDS, 50mM Tris-HCl, pH 8.0, 4 mM EDTA, pH 8.0) at a ratio of 1 :330 v/v and preheated to 55°C.
  • 1.5X TMAC buffer 3M TMAC, 0.1% SDS, 50mM Tris-HCl, pH 8.0, 4 mM EDTA, pH 8.0
  • different color-coded (different emission spectra) microbeads, each with a different detector oligonucleotide were mixed equally in 1.5X TMAC buffer at a ratio of 1 :330 (v/v) and pre-heated to 55°C.
  • PCR products Five ul of PCR products were mixed with 12 ul of nuclease-free water and denatured at 95°C for 10 minutes. Next, 33 ul of 1.5X TMAC buffer diluted microbead conjugated detector oligonucleotides were added to the denatured PCR products and mixed. The reaction contained approximately 5000 of each class of detector oligonucleotides. The PCR products were then hybridized with the detector oligonucleotides for 1 hour at 55°C. Following hybridization, the mixture was centrifuged and the supernatant removed.
  • the pellet was resuspended in 80 ul of strepavidin conjugated R-phycoerythrin solution (PE, Molecular Probes, Eugene OR) at 55°C and incubated in the dark for 10 minutes. Strepavidin conjugated R-phycoerythrin was pre-diluted to 2 ug/ml with IX TMAC buffer prior to addition to the pellet. Fifty ul of PE stained microbead conjugated detector oligonucleotides from each reaction was analyzed using the Luminex 100 system (Luminex, Austin TX) and 100 events counted for each class of detector oligonucleotide. The fluorescence intensity associated with each class of microbead was recorded. The mean fluorescence intensity (MFI) from 100 beads was calculated as a measurement of the relative amount target RNA in the sample.
  • PE strepavidin conjugated R-phycoerythrin solution
  • Strepavidin conjugated R-phycoerythrin was pre-diluted to 2 ug
  • oligos I of genes 12078,12739 and 13234 were paired with oligos II of genes 13234,12078 and 12739,respectively.
  • the first experiment addressed the specificity of the interaction between target RNA and representative sensor probes.
  • the sensor probe pairs were used to detect the expression of Arabidopsis plastocyanin gene in a variety of conditions shown in Table 1 A.
  • plastocyanin gene transcripts were only detected from light-induced Arabidopsis wild-type seedlings total RNA.
  • RNA template was omitted, or target RNA was absent in the RNA samples, as in the yeast tRNA sample or mouse lung total RNA sample, the expression signal was similar to the background of water control.
  • the second experiment was designed to examine the specificity of the ligation.
  • Expression of three Arabidopsis genes, a putative plastocyanin, a glycolate oxidase, and a protein with unknown function was detected using the varied representative sensor probes in a light-grown Arabidopsis seedling total RNA samples.
  • Each gene was represented by sensor probe pairs with perfect match; with one base mismatch at the ligation site; or with mis-paired sensor probes, i.e. one member of the sensor probe pair is homologous not to the target, but different gene.
  • the expression signals of all three genes studied were detected from the light-grown Arabidopsis seedling total RNA samples.
  • Single-base mismatches in the sensor probes at the ligation site lowered the detected signal to background levels. Similar results were obtained when mispaired sensor probes were used.
  • the third experiment examined the importance of the T4 DNA ligase in the ligation reaction. Reactions were conducted with or without T4 DNA ligase. In contrast to the positive control experiment, no signal was detected in the reaction without T4 DNA ligase, clearly demonstrating that the T4 DNA ligase is necessary in the reaction.
  • the fourth experiment was designed to demonstrate the specificity of hybridization between PCR amplified ligated sensor probes and detector oligonucleotides.
  • three different microsphere conjugated detector oligonucleotides were mixed prior to hybridization and detection. Only the complimentary sensor probes were picked up by the corresponding detector oligonucleotides, while the hybridization signals of non-matching sensor probes were similar to background
  • T4 DNA ligase can specifically catalyze the ligation of the sensor probes designed to specifically hybridize to the target.
  • the complexity of the non-target RNA in the reaction did not affect the specificity of the ligation, as demonstrated by the results from Arabidopsis and mouse lung samples.
  • the ligation reaction did not tolerant the non-specific sensor probes, such as single-base mismatched sensor probe pairs and mis-paired sensor probes, even if they are adjacently located by non-specific binding to the target RNA.
  • the ligation also required specific target polynucleotides to be presented in the reaction. Without RNA or without target mRNA, the oligonucleotide pair was not ligated by T4 DNA ligase.
  • Table IB Mean Fluorescence Intensity (MFI) for Various Treatments
  • a spike-in experiment was conducted to determine the sensitivity of the present methods.
  • a 40-base oligonucleotide putative plastocyanin gene sequence (Fig. 2), was added at a serial dilution of 5, 0.5, 0.05, 0.005, 0.0005, 0.00005 and 0 fmole.
  • the sensor probe pair (probes 1 and 2), PCR primers and detector oligonucleotides were identical to the previous plastocyanin experiment and are listed in Table 1 A.
  • each cell contains 10 mRNA molecules and there are total 10 7 RNA molecules per cell (assuming that mRNA is 1% of total RNA and the molecular weight of different RNA species is same), 1 of 3x10 7 equates to less than one copy of mRNA target/cell. If calculated by weight, 0.00005 fmole is equivalent to 16.5 pg for a 1 kb long mRNA molecule. Thus the method can detect as low as 16.5 pg mRNA target in a 500 ng total RNA sample. If a target RNA has more copies in a cell, the required total RNA amount will be reduced.
  • the previous data showed the expression level of putative plastocyanin gene in dark-grown wild type Arabidopsis seedling is just above the background, the present method detected its expression in 500 ng of total RNA, while in light-grown Arabidopsis, 5 ng of RNA was enough for detection.
  • Dynamic range is contributed by both PCR amplification of the DNA oligonucleotide sensors and dynamic range of the label associated with amplified ligated sensor probes. In order to achieve the linear amplification, PCR reactions are generally limited to 25-30 cycles. So the detection dynamic range observed is mainly determined by the dynamic range of the sensor probe readout. Based on the above sensitivity study, the signal intensity of method disclosed herein have a linear response to the spike-in template amount between the range of 0.0005 fmole and 0.05 fmole. The dynamic range in response to different RNA concentration was determined using total RNA from Arabidopsis light-grown wild-type seedlings in a serial concentration from 5 ng-2 ug as templates.
  • the putative plastocyanin gene transcript was selected as the target. With a 30-cycle PCR reaction, the plastocyanin gene transcript could be detected from as low as 5 ng of total RNA and the signal intensity qualitatively increased as the amount of starting total RNA increased (Fig. 3). The linear increase of signal intensity was observed within 5 ng-1 ug of total RNA. The signal intensity change was correlated with the absolute RNA concentration, while the change ratio of signal intensity declined when the total RNA amount was over lug. The saturation of signal intensity was mainly limited by the detection dynamic range of liquid array. No signal was detected from as much as 500 ng of the control mouse lung total RNA sample. An increase in PCR cycles beyond 35 did not significantly increase the signal, as the PCR amplification often reached the plateau at 35 cycles (data not shown). The increase in signal intensity was from the increase of the amount of PCR amplified DNA sensor probes.
  • the reproducibility was characterized by conducting 3 replicate experiments using the same RNA sample in serial dilutions.
  • sensor production by ligation reaction, sensor amplification by PCR, and sensor hybridization and detection were conducted independently with 5-500 ng total RNA in each assay.
  • CV coefficient of variance
  • the CVs among three replicates in different dilution concentrations ranged from 2 % to 21 % (Fig. 4), and accounted for the variation of sensor preparation and the variation of hybridization beads preparation.
  • all reactions in one experiment will share the same master mix of reagents and microspheres, thus the overall variation among samples will be reduced compared to this reproducibility assay. Since the variation among replicates are generally less than 20%, the signal intensity with greater than 2-fold differences between samples can be safely inte ⁇ reted as an expression difference.
  • RNA samples of wild-type and phyA -null mutant used in the previous study to profile the expression changes in a time course of light treatments were used in this study for direct comparison.
  • a total of 12 pairs of sensor probes were mixed with each total RNA sample to represent the 12 target mRNA transcripts.
  • 12 kinds of color-coded beads with respective detector oligonucleotides were mixed in the hybridization for simultaneous detection.
  • the polyubiquitin gene showed a consistent expression over the time course in both wild-type and mutant, suggesting that it is neither phyA-regulated, nor light sensitive.
  • Five photosynthesis related genes including putative plastocyanin, magnesium chelatase subunit, chloroplast FtsH protease, and 2 representative sequences of photosystem II type I chlorophyll a/b binding protein, were quickly induced in the wild-type seedlings in the first two hours after transfer to FRc, and maintained at the high expression level over 24 hour time period, but were completely repressed in phyA null mutant.
  • the transcription level of PhyA suppressor spal increased quickly in the early stage, and decreased its expression to the basal level later.

Abstract

L'invention a pour objet de nouveaux procédés prévus pour être utilisés dans l'analyse d'acides nucléiques. Ces applications comprennent l'analyse de l'expression des polynucléotides, et la détection de polymorphismes de nucléotides uniques. L invention traite aussi de procédés de diagnostic et de kits pour mettre en oeuvre ces procédés.
EP02753074A 2001-06-29 2002-06-28 Detection amelioree et distinction de l'expression genique differentielle par la ligature et l'amplification de sondes Withdrawn EP1412526A2 (fr)

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