WO2004040020A1 - Procedes et composition de detection de cibles - Google Patents

Procedes et composition de detection de cibles Download PDF

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Publication number
WO2004040020A1
WO2004040020A1 PCT/US2002/033801 US0233801W WO2004040020A1 WO 2004040020 A1 WO2004040020 A1 WO 2004040020A1 US 0233801 W US0233801 W US 0233801W WO 2004040020 A1 WO2004040020 A1 WO 2004040020A1
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Prior art keywords
primer
specific portion
nucleic acid
probe
sequence
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PCT/US2002/033801
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English (en)
Inventor
Sabine Short
Michael H. Wenz
Ernest Friedlander
Shirley Johnson
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Applera Corporation
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Application filed by Applera Corporation filed Critical Applera Corporation
Priority to EP02776259A priority Critical patent/EP1558756A4/fr
Priority to PCT/US2002/033801 priority patent/WO2004040020A1/fr
Priority to AU2002342093A priority patent/AU2002342093A1/en
Priority to US10/693,609 priority patent/US20040235005A1/en
Publication of WO2004040020A1 publication Critical patent/WO2004040020A1/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]

Definitions

  • the invention relates to methods and compositions for the detection of targets in a sample.
  • the detection of the presence or absence of (or quantity of) one or more target sequences in a sample containing one or more target sequences is commonly practiced. For example, the detection of cancer and many infectious diseases, such as AIDS and hepatitis, routinely includes screening biological samples for the presence or absence of diagnostic nucleic acid sequences. Also, detecting the presence or absence of nucleic acid sequences is often used in forensic science, paternity testing, genetic counseling, and organ transplantation. [003] An organism's genetic makeup is determined by the genes contained within the genome of that organism. Genes are composed of long strands or deoxyribonucleic acid (DNA) polymers that encode the information needed to make proteins. Properties, capabilities, and traits of an organism often are related to the types and amounts of proteins that are, or are not, being produced by that organism.
  • DNA deoxyribonucleic acid
  • a protein can be produced from a gene as follows. First, the DNA of the gene that encodes a protein, for example, protein "X”, is converted into ribonucleic acid (RNA) by a process known as “transcription.” During transcription, a single-stranded complementary RNA copy of the gene is made. Next, this RNA copy, referred to as protein X messenger RNA (mRNA), is used by the cell's biochemical machinery to make protein X, a process referred to as “translation.” Basically, the cell's protein manufacturing machinery binds to the mRNA, “reads” the RNA code, and “translates” it into the amino acid sequence of protein X. In summary, DNA is transcribed to make mRNA, which is translated to make proteins.
  • mRNA protein X messenger RNA
  • the amount of protein X that is produced by a cell often is largely dependent on the amount of protein X mRNA that is present within the cell.
  • the amount of protein X mRNA within a cell is due, at least in part, to the degree to which gene X is expressed. Whether a particular gene is expressed, and if so, to what level, may have a significant impact on the organism.
  • the method comprises forming a ligation reaction composition comprising the sample, and a ligation probe set for each target nucleic acid sequence.
  • the probe set comprises (a) at least one first probe, comprising a target-specific portion and a 5' primer-specific portion, wherein the 5' primer-specific portion comprises a sequence, and (b) at least one second probe, comprising a target-specific portion and a 3' primer- specific portion, wherein the 3' primer-specific portion comprises a sequence.
  • the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence.
  • the methods further comprise forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5' primer-specific portion, the target-specific portions, and the 3' primer-specific portion.
  • the methods further comprise forming at least one amplification reaction composition comprising: at least a portion of the test composition; a polymerase; a double-stranded-dependent specific label, wherein the double- stranded-dependent label has a first detectable signal value when the double-stranded-dependent label is not exposed to double- stranded nucleic acid; and at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5' primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3' primer-specific portion of the ligation product.
  • the methods further comprise subjecting the at least one amplification reaction composition to at least one amplification reaction.
  • the methods further comprise detecting a second detectable signal value at least one of during and after the at least one amplification reaction, wherein a threshold difference between the first detectable signal value and the second detectable signal value indicates the presence of the target nucleic acid sequence, and wherein no threshold difference between the first detectable signal value and the second detectable signal value indicates the absence of the target nucleic acid sequence.
  • the method comprises forming a ligation reaction composition comprising the sample, and a ligation probe set for each target nucleic acid sequence.
  • the probe set comprises (a) at least one first probe, comprising a target-specific portion and a 5' primer-specific portion, wherein the 5' primer-specific portion comprises a sequence, and (b) at least one second probe, comprising a target-specific portion and a 3' primer- specific portion, wherein the 3' primer-specific portion comprises a sequence.
  • the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence.
  • the methods further comprise forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5' primer-specific portion, the target-specific portions, and the 3' primer-specific portion.
  • the methods further comprise forming at least one amplification reaction composition comprising: at least a portion of the test composition; a polymerase; a double-stranded-dependent specific label; and at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5' primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3' primer-specific portion of the ligation product.
  • the methods further comprise subjecting the at least one amplification reaction composition to at least one amplification reaction. In certain embodiments, the methods further comprise detecting the presence or absence of the target nucleic acid sequence by monitoring a signal at least one of during and after the at least one amplification reaction.
  • the method comprises forming at least one reaction composition comprising: the sample; a ligation probe set for the target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5' primer-specific portion, wherein the 5' primer-specific portion comprises a sequence and (b) at least one second probe, comprising a target-specific portion and a 3' primer-specific portion, wherein the 3' primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence; a polymerase; a double-stranded-dependent label, wherein the double-stranded- dependent label has a first detectable signal value when the double- stranded-dependent label is not exposed to double-stranded nucleic acid; and at least one primer set,
  • the methods further comprise subjecting the reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5' primer-specific portion, the target-specific portions, and the 3' primer-specific portion.
  • the methods further comprise, after the at least one cycle of ligation, subjecting the reaction composition to at least one amplification reaction. In certain embodiments, the methods further comprise detecting a second detectable signal value at least one of during and after the at least one amplification reaction, wherein a threshold difference between the first detectable signal value and the second detectable signal value indicates the presence of the target nucleic acid sequence, and wherein no threshold difference between the first detectable signal value and the second detectable signal value indicates the absence of the target nucleic acid sequence.
  • the method comprises forming at least one reaction composition comprising: the sample; a ligation probe set for the target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5' primer-specific portion, wherein the 5' primer-specific portion comprises a sequence and (b) at least one second probe, comprising a target-specific portion and a 3' primer-specific portion, wherein the 3' primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence; a polymerase; a double-stranded-dependent label; and at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5' primer-specific portion of the ligation product, and (ii) at least one second primer comprising
  • the methods further comprise subjecting the reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5' primer-specific portion, the target-specific portions, and the 3' primer-specific portion.
  • the methods further comprise, after the at least one cycle of ligation, subjecting the reaction composition to at least one amplification reaction. In certain embodiments, the methods further comprise detecting the presence or absence of the target nucleic acid sequence by monitoring a signal at least one of during and after the at least one amplification reaction.
  • kits for detecting at least one target nucleic acid sequence in a sample are provided. In certain embodiments, the kits comprise: ,
  • a ligation probe set for each target nucleic acid sequence comprising (i) at least one first probe, comprising a target-specific portion, a 5' primer- specific portion, wherein the 5' primer-specific portion comprises a sequence, and (ii) at least one second probe, comprising a target-specific portion, a 3' primer-specific portion, wherein the 3' primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target nucleic acid sequence; and
  • the method comprises forming a ligation reaction composition comprising the sample, a ligation probe set for each target nucleic acid sequence, and poly-deoxy-inosinic-deoxy-cytidylic acid.
  • the probe set comprises (a) at least one first probe, comprising a target-specific portion, and (b) at least one second probe, comprising a target- specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence.
  • the methods further comprise forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5' primer-specific portion, the target-specific portions, and the 3' primer-specific portion.
  • the methods further comprise detecting the presence or absence of the ligation product to detect the presence or absence of the at least one target nucleic acid sequence.
  • the method comprises forming a ligation reaction composition comprising the sample, a ligation probe set for each target nucleic acid sequence, and poly-deoxy-inosinic-deoxy-cytidylic acid.
  • the probe set comprises (a) at least one first probe, comprising a target-specific portion, and (b) at least one second probe, comprising a target- specific portion, wherein the probes in each set are suitable fpr ligation together when hybridized adjacent to one another on a complementary target sequence.
  • the methods further comprise forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5' primer-specific portion, the target-specific portions, and the 3' primer-specific portion.
  • the methods further comprise forming at least one amplification reaction composition comprising: at least a portion of the test composition; a polymerase; and at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5' primer-specific portion of the ligation product, and (ii) at least one second primer.
  • the methods further comprise subjecting the at least one amplification reaction composition to at least one amplification reaction. In certain embodiments, the methods further comprise detecting the presence or absence of the target nucleic acid sequence by detecting whether the at least one amplification reaction results in amplification product from ligation product.
  • kits for detecting at least one target nucleic acid sequence in a sample are provided.
  • the kits comprise:
  • At least one first probe comprising a target-specific portion, a 5' primer-specific portion, wherein the 5' primer-specific portion comprises a sequence
  • At least one second probe comprising a target-specific portion, a 3' primer-specific portion, wherein the 3' primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target nucleic acid sequence;
  • compositions for a ligation reaction comprising a ligase and poly-deoxy-inosinic-deoxy-cytidylic acid are provided.
  • Figure 1 is a schematic showing a ligation probe set according to certain embodiments of the invention.
  • Each probe includes a portion that is complementary to the target
  • Each probe set comprises at least one first probe and at least one second probe that are designed to hybridize with the target with the 3' end of the first probe immediately adjacent to and opposing the 5' end of the second probe.
  • Figure 2 is a schematic showing an exemplary embodiment of certain embodiments comprising ligation and primer extension amplification.
  • FIG. 3 depicts a method for differentiating between two potential alleles in a target locus using certain embodiments of the invention.
  • Fig. 3(A) shows: (i) a target-specific probe set comprising: two first probes (A and B) that have the same target-specific portions except for different pivotal complements (here, T at the 3' end probe A and C at the 3' end probe B) and different primer-specific portions ((P-SPA) and (P-SPB)); and one second probe (Z) comprising a target-specific portion and a primer-specific portion (P- SP2).
  • Fig. 3(B) shows the three probes annealed to the target.
  • the target-specific portion of probe A is fully complementary with the 3' target region including the pivotal nucleotide.
  • the pivotal complement of probe B is not complementary with the 3' target region.
  • the target-specific portion of probe B therefore, contains a base-pair mismatch at the 3' end.
  • the target-specific portion of probe Z is fully complementary to the 5' target region.
  • Fig. 3(C) shows ligation of probes A and Z to form ligation product A-Z. Probes B and Z are not ligated together to form a ligation product due to the mismatched pivotal complement on probe B.
  • Fig. 3(D) shows denaturing the double-stranded molecules to release the A-Z ligation product and unligated probes B and Z.
  • Figure 4 depicts certain embodiments employing flap endonuclease.
  • Figure 5 depicts certain embodiments employing flap endonuclease.
  • Figure 6 depicts certain embodiments employing flap endonuclease.
  • Figure 7 depicts certain embodiments employing flap endonuclease.
  • FIG. 8 is a schematic depicting certain embodiments of the invention.
  • Fig. 8(A) depicts a target sequence and a ligation probe set comprising: two first probes (A and B) that have the same target-specific portions except for different pivotal complements (here, T at the 3' end probe A and G at the 3'end probe B) and different primer-specific portions ((P-SPA) and (P-SPB)); and one second probe (Z) comprising a target-specific portion and a primer- specific portion (P-SP2).
  • Fig. 8(B) depicts the A and Z probes hybridized to the target sequence under annealing conditions.
  • Fig. 8(C) depicts the ligation of the first and second probes in the presence of a ligation agent to form ligation product.
  • Fig. 8(D) depicts denaturing the ligation produc target complex to release a single-stranded ligation product; and performing two separate amplification reactions with either primer set (PA) and (P2) or primer set (PB) and (P2).
  • Figure 9 depicts certain embodiments involving three biallelic loci.
  • Figure 10 depicts certain embodiments involving three biallelic loci.
  • Figure 11 depicts certain embodiments in which one probe of a ligation probe set also serves as a primer.
  • Figure 12 depicts exemplary alternative splicing.
  • Figure 13 depicts certain embodiments involving splice variants.
  • Figure 14 relates to certain embodiments employing ⁇ Ci values. V. Detailed Description of Certain Exemplary Embodiments
  • nucleotide base refers to a substituted or unsubstituted aromatic ring or rings.
  • the aromatic ring or rings contain at least one nitrogen atom.
  • the nucleotide base is capable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an appropriately complementary nucleotide base.
  • nucleotide bases and analogs thereof include, but are not limited to, naturally occurring nucleotide bases adenine, guanine, cytosine, 6 methyl-cytosine, uracil, thymine, and analogs of the naturally occurring nucleotide bases, e.g., 7- deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N6 - ⁇ 2 -isopentenyladenine (6iA), N6 - ⁇ 2 -isopentenyl-2-methylthioadenine (2ms6iA), N2 -dimethylguanine (dmG), 7-methylguanine (7mG), inosine, nebularine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine, 5-
  • Patent Nos. 6,143,877 and 6,127,121 and PCT published application WO 01/38584 disclose ethenoadenine, indoles such as nitroindole and 4-methylindole, and pyrroles such as nitropyrrole.
  • Certain exemplary nucleotide bases can be found, e.g., in Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla., and the references cited therein.
  • nucleotide refers to a compound comprising a nucleotide base linked to the C-1' carbon of a sugar, such as ribose, arabinose, xylose, and pyranose, and sugar analogs thereof.
  • a sugar such as ribose, arabinose, xylose, and pyranose
  • nucleotide also encompasses nucleotide analogs.
  • the sugar may be substituted or unsubstituted.
  • Substituted ribose sugars include, but are not limited to, those riboses in which one or more of the carbon atoms, for example the 2'-carbon atom, is substituted with one or more of the same or different CI, F, -R, -OR, - NR 2 or halogen groups, where each R is independently H, C C ⁇ alkyl or Cs-C ⁇ aryl.
  • Exemplary riboses include, but are not limited to, 2'-(C1 -C6)alkoxyribose, 2'-(C5 -C14)aryloxyribose, 2 , ,3'-didehydroribose, 2'-deoxy-3 , -haloribose, 2'- deoxy-3'-fluororibose, 2'-deoxy-3'-chlororibose, 2'-deoxy-3'-aminoribose, 2'- deoxy-3'-(C1 -C6)alkylribose, 2'-deoxy-3'-(C1 -C6)alkoxyribose and 2'-deoxy-3'- (C5 -C14)aryloxyribose, ribose, 2'-deoxyribose, 2',3'-dideoxyribose, 2'-haloribose, 2'-fluororibose,
  • Modifications at the 2'- or 3'-position of ribose include, but are not limited to, hydrogen, hydroxy, methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo.
  • Nucleotides include, but are not limited to, the natural D optical isomer, as well as the L optical isomer forms (see, e.g., Garbesi (1993) Nucl. Acids Res. 21 :4159-65; Fujimori (1990) J. Amer. Chem. Soc.
  • nucleotide base is purine, e.g. A or G
  • the ribose sugar is attached to the N 9 -position of the nucleotide base.
  • nucleotide base is pyrimidine, e.g. C, T or U
  • the pentose sugar is attached to the N 1 -position of the nucleotide base, except for pseudouridines, in which the pentose sugar is attached to the C5 position of the uracil nucleotide base (see, e.g., Komberg and Baker, (1992) DNA Replication, 2 nd Ed., Freeman, San Francisco, CA).
  • One or more of the pentose carbons of a nucleotide may be substituted with a phosphate ester having the formula:
  • nucleotides are those in which the nucleotide base is a purine, a 7-deazapurine, a pyrimidine, or an analog thereof.
  • Nucleotide 5'-triphosphate refers to a nucleotide with a triphosphate ester group at the 5' position, and is sometimes denoted as "NTP", or "dNTP” and “ddNTP” to particularly point out the structural features of the ribose sugar.
  • the triphosphate ester group may include sulfur substitutions for the various oxygens, e.g. ⁇ -thio-nucleotide 5'- triphosphates.
  • sulfur substitutions for the various oxygens e.g. ⁇ -thio-nucleotide 5'- triphosphates.
  • nucleotide analog refers to embodiments in which the pentose sugar and/or the nucleotide base and/or one or more of the phosphate esters of a nucleotide may be replaced with its respective analog.
  • exemplary pentose sugar analogs are those described above.
  • nucleotide analogs have a nucleotide base analog as described above.
  • exemplary phosphate ester analogs include, but are not limited to, alkylphosphonates, methylphosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroanilidates, phosphoroamidates, boronophosphates, etc., and may include associated counterions.
  • nucleotide analog also included within the definition of "nucleotide analog” are nucleotide analog monomers that can be polymerized into polynucleotide analogs in which the DNA/RNA phosphate ester and/or sugar phosphate ester backbone is replaced with a different type of internucleotide linkage.
  • Exemplary polynucleotide analogs include, but are not limited to, peptide nucleic acids, in which the sugar phosphate backbone of the polynucleotide is replaced by a peptide backbone.
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably and mean single-stranded and double- stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, or internucleotide analogs, and associated counter ions, e.g., H + , NH 4 + , trialkylammonium, Mg 2+ , Na + and the like.
  • DNA 2'-deoxyribonucleotides
  • RNA ribonucleotides linked by internucleotide phosphodiester bond linkages
  • counter ions e.g., H + , NH 4 + , trialkylammonium, Mg 2+ , Na + and the like.
  • a nucleic acid may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof.
  • the nucleotide monomer units may comprise any of the nucleotides described herein, including, but not limited to, naturally occurring nucleotides and nucleotide analogs, nucleic acids typically range in size from a few monomeric units, e.g. 5-40 when they are sometimes referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units.
  • nucleic acid sequence is represented, it will be understood that the nucleotides are in 5' to 3' order from left to right and that "A” denotes deoxyadenosine or an analog thereof, “C” denotes deoxycytidine or an analog thereof, “G” denotes deoxyguanosine or an analog thereof, “T” denotes thymidine or an analog thereof, and “U” denotes uridine or an analog thereof, unless otherwise noted.
  • A denotes deoxyadenosine or an analog thereof
  • C denotes deoxycytidine or an analog thereof
  • G denotes deoxyguanosine or an analog thereof
  • T denotes thymidine or an analog thereof
  • U denotes uridine or an analog thereof, unless otherwise noted.
  • Nucleic acids include, but are not limited to, genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, fragmented nucleic acid, nucleic acid obtained from subcellular organelles such as mitochondria or chloroplasts, and nucleic acid obtained from microorganisms or DNA or RNA viruses that may be present on or in a biological sample.
  • Nucleic acids may be composed of a single type of sugar moiety, e.g., as in the case of RNA and DNA, or mixtures of different sugar moieties, e.g., as in the case of RNA/DNA chimeras.
  • nucleic acids are ribopolynucleotides and 2'-deoxyribopolynucleotides according to the structural formulae below:
  • each B is independently the base moiety of a nucleotide, e.g., a purine, a 7-deazapurine, a pyrimidine, or an analog nucleotide
  • each m defines the length of the respective nucleic acid and can range from zero to thousands, tens of thousands, or even more
  • each R is independently selected from the group comprising hydrogen, halogen, -R", --OR", and ⁇ NR"R", where each R" is independently (C1 -C6) alkyl or (C5 -C14) aryl, or two adjacent Rs are taken together to form a bond such that the ribose sugar is 2',3'-didehydroribose; and each R' is independently hydroxyl or
  • ⁇ is zero, one or two.
  • nucleotide bases B are covalently attached to the C1' carbon of the sugar moiety as previously described.
  • nucleic acid may also include nucleic acid analogs, polynucleotide analogs, and oligonucleotide analogs.
  • nucleic acid analog may also include nucleic acid analogs, polynucleotide analogs, and oligonucleotide analogs.
  • nucleic acid analog refers to a nucleic acid that contains at least one nucleotide analog and/or at least one phosphate ester analog and/or at least one pentose sugar analog.
  • nucleic acid analogs include nucleic acids in which the phosphate ester and/or sugar phosphate ester linkages are replaced with other types of linkages, such as N-(2-aminoethyl)-glycine amides and other amides (see, e.g., Nielsen et al., 1991 , Science 254: 1497-1500; WO 92/20702; U.S. Pat. No. 5,719,262; U.S. Pat. No. 5,698,685;); morpholinos (see, e.g., U.S. Pat. No. 5,698,685; U.S. Pat. No. 5,378,841 ; U.S. Pat. No.
  • PNA 2-aminoethylglycine
  • PNA 2-aminoethylglycine
  • PNA 2-aminoethylglycine
  • annealing and “hybridization” are used interchangeably and mean the base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex, triplex, or other higher- ordered structure.
  • the primary interaction is base specific, e.g., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding.
  • base-stacking and hydrophobic interactions may also contribute to duplex stability.
  • An "enzymatically active mutant or variant thereof,” when used in reference to an enzyme such as a polymerase or a ligase, means a protein with appropriate enzymatic activity.
  • an enzymatically active mutant or variant of a DNA polymerase is a protein that is able to catalyze the stepwise addition of appropriate deoxynucleoside triphosphates into a nascent DNA strand in a template-dependent manner.
  • An enzymatically active mutant or variant differs from the "generally-accepted" or consensus sequence for that enzyme by at least one amino acid, including, but not limited to, substitutions of one or more amino acids, addition of one or more amino acids, deletion of one or more amino acids, and alterations to the amino acids themselves. With the change, however, at least some catalytic activity is retained. In certain embodiments, the changes involve conservative amino acid substitutions.
  • Conservative amino acid substitution may involve replacing one amino acid with another that has, e.g., similar hydrophobicity, hydrophilicity, charge, or aromaticity.
  • conservative amino acid substitutions may be made on the basis of similar hydropathic indices.
  • a hydropathic index takes into account the hydrophobicity and charge characteristics of an amino acid, and in certain embodiments, may be used as a guide for selecting conservative amino acid substitutions. The hydropathic index is discussed, e.g., in Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is understood in the art that conservative amino acid substitutions may be made on the basis of any of the aforementioned characteristics.
  • amino acids may include, but are not limited to, glycosylation, methylation, phosphorylation, biotinylation, and any covalent and noncovalent additions to a protein that do not result in a change in amino acid sequence.
  • Amino acid refers to any amino acid, natural or non- natural, that may be incorporated, either enzymatically or synthetically, into a polypeptide or protein.
  • Fragments for example, but without limitation, proteolytic cleavage products, are also encompassed by this term, provided that at least some enzyme catalytic activity is retained.
  • the skilled artisan will readily be able to measure catalytic activity using an appropriate well-known assay.
  • an appropriate assay for polymerase catalytic activity might include, for example, measuring the ability of a variant to incorporate, under appropriate conditions, rNTPs or dNTPs into a nascent polynucleotide strand in a template-dependent manner.
  • an appropriate assay for ligase catalytic activity might include, for example, the ability to ligate adjacently hybridized oligonucleotides comprising appropriate reactive groups.
  • a "target” or “target nucleic acid sequence” comprises a specific nucleic acid sequence that can be distinguished by a probe. Targets may include both naturally occurring and synthetic molecules.
  • Probes comprise oligonucleotides that comprise a specific portion that is designed to hybridize in a sequence-specific manner with a complementary region on a specific nucleic acid sequence, e.g., a target nucleic acid sequence.
  • the specific portion of the probe may be specific for a particular sequence, or alternatively, may be degenerate, e.g., specific for a set of sequences.
  • a "ligation probe set” according to the present invention is a group of two or more probes designed to detect at least one target.
  • a ligation probe set may comprise two nucleic acid probes designed to hybridize to a target such that, when the two probes are hybridized to the target adjacent to one another, they are suitable for ligation together.
  • suitable for ligation refers to at least one first target-specific probe and at least one second target-specific probe, each comprising an appropriately reactive group.
  • exemplary reactive groups include, but are not limited to, a free hydroxyl group on the 3' end of the first probe and a free phosphate group on the 5' end of the second probe.
  • the second probe may be a 5'- adenylated probe, in which the 5'-phosphate of adenosine is attached to the 5' end of the probe (a phosphoanhydride linkage).
  • Exemplary pairs of reactive groups include, but are not limited to: phosphorothioate and tosylate or iodide; esters and hydrazide; RC(O)S " , haloalkyl, or RCH 2 S and ⁇ -haloacyl; thiophosphoryl and bromoacetoamido groups.
  • Exemplary reactive groups include, but are not limited to, S-pivaloyloxymethyl-4-thiothymidine.
  • first and second target-specific probes are hybridized to the target sequence such that the 3' end of the first target-specific probe and the 5' end of the second target-specific probe are immediately adjacent to allow ligation.
  • detectable signal value refers to a value of the signal that is detected from a label.
  • the detectable signal value is the amount or intensity of signal that is detected from a label. Thus, if there is no detectable signal from a label, its detectable signal value is zero (0).
  • the detectable signal value is a characteristic of the signal other than the amount or intensity of the signal, such as the spectra, wavelength, color, or lifetime of the signal.
  • Detectably different signal value means that one or more detectable signal values are distinguishable from one another by at least one detection method.
  • double-stranded-dependent label refers to a label that provides a detectably different signal value when it is exposed to double- stranded nucleic acid than when it is not exposed to double-stranded nucleic acid.
  • threshold difference between detectable signal values refers to a set difference between a first detectable signal value and a second detectable signal value that results when the target nucleic acid sequence that is being sought is present in a sample, but that does not result when the target nucleic acid sequence is absent.
  • the first detectable signal value of a double- stranded-dependent label is the detectable signal value from the label when it is not exposed to double-stranded nucleic acid.
  • the second detectable signal value is detected during and/or after an amplification reaction using a composition that comprises the double-stranded-dependent label.
  • the term "quantitating,” when used in reference to an amplification product, refers to determining the quantity or amount of a particular sequence that is representative of a target nucleic acid sequence in the sample. For example, but without limitation, one may measure the intensity of the signal from a label. The intensity or quantity of the signal is typically related to the amount of amplification product. The amount of amplification product generated correlates with the amount of target nucleic acid sequence present prior to ligation and amplification, and thus, in certain embodiments, may indicate the level of expression for a particular gene.
  • amplification product refers to the product of an amplification reaction including, but not limited to, primer extension, the polymerase chain reaction (PCR), RNA transcription, and the like.
  • exemplary amplification products may comprise at least one of primer extension products, PCR amplicons, RNA transcription products, and the like.
  • Primers refer to oligonucleotides that are designed to hybridize with the primer-specific portion of probes, ligation products, or amplification products in a sequence-specific manner, and serve as primers for amplification reactions.
  • a “universal primer” is capable of hybridizing to the primer-specific portion of more than one species of probe, ligation product, or amplification product, as appropriate.
  • a “universal primer set” comprises a first primer and a second primer that hybridize with a plurality of species of probes, ligation products, or amplification products, as appropriate.
  • a "ligation agent” according to the present invention may comprise any number of enzymatic or chemical (i.e., non-enzymatic) agents that can effect ligation of nucleic acids to one another.
  • sequence encompasses situations where both of the sequences are completely the same or complementary to one another, and situations where only a portion of one of the sequences is the same as, or is complementary to, a portion or the entire other sequence.
  • sequence encompasses, but is not limited to, nucleic acid sequences, polynucleotides, oligonucleotides, probes, primers, primer-specific portions, and target-specific portions.
  • sequence encompasses, but is not limited to, nucleic acid sequences, polynucleotides, oligonucleotides, probes, primers, primer-specific portions, and target-specific portions. Despite the mismatches, the two sequences should selectively hybridize to one another under appropriate conditions.
  • the term “selectively hybridize” means that, for particular identical sequences, a substantial portion of the particular identical sequences hybridize to a given desired sequence or sequences, and a substantial portion of the particular identical sequences do not hybridize to other undesired sequences.
  • a “substantial portion of the particular identical sequences” in each instance refers to a portion of the total number of the particular identical sequences, and it does not refer to a portion of an individual particular identical sequence.
  • "a substantial portion of the particular identical sequences” means at least 90% of the particular identical sequences.
  • "a substantial portion of the particular identical sequences” means at least 95% of the particular identical sequences.
  • the number of mismatches that may be present may vary in view of the complexity of the composition. Thus, in certain embodiments, fewer mismatches may be tolerated in a composition comprising DNA from an entire genome than a composition in which fewer DNA sequences are present. For example, in certain embodiments, with a given number of mismatches, a probe may more likely hybridize to undesired sequences in a composition with the entire genomic DNA than in a composition with fewer DNA sequences, when the same hybridization conditions are employed for both compositions. Thus, that given number of mismatches may be appropriate for the composition with fewer DNA sequences, but fewer mismatches may be more optimal for the composition with the entire genomic DNA.
  • sequences are complementary if they have no more than 20% mismatched nucleotides. In certain embodiments, sequences are complementary if they have no more than 15% mismatched nucleotides. In certain embodiments, sequences are complementary if they have no more than 10% mismatched nucleotides. In certain embodiments, sequences are complementary if they have no more than 5% mismatched nucleotides. [087] In this application, a statement that one sequence hybridizes or binds to another sequence encompasses situations where the entirety of both of the sequences hybridize or bind to one another, and situations where only a portion of one or both of the sequences hybridizes or binds to the entire other sequence or to a portion of the other sequence.
  • sequence encompasses, but is not limited to, nucleic acid sequences, polynucleotides, oligonucleotides, probes, primers, primer-specific portions, and target-specific portions.
  • the term "to a measurably lesser extent” encompasses situations in which the event in question is reduced at least 10 fold. In certain embodiments, the term “to a measurably lesser extent” encompasses situations in which the event in question is reduced at least 100 fold.
  • a statement that a component may be, is, or has been "substantially removed” means that at least 90% of the component may be, is, or has been removed. In certain embodiments, a statement that a component may be, is, or has been "substantially removed” means that at least 95% of the component may be, is, or has been removed.
  • target nucleic acid sequences may include RNA and DNA.
  • RNA target sequences include, but are not limited to, mRNA, rRNA, tRNA, viral RNA, and variants of RNA, such as splicing variants.
  • DNA target sequences include, but are not limited to, genomic DNA, plasmid DNA, phage DNA, nucleolar DNA, mitochondrial DNA, and chloroplast DNA.
  • target nucleic acid sequences include, but are not limited to, cDNA, yeast artificial chromosomes (YAC's), bacterial artificial chromosomes (BAC's), other extrachromosomal DNA, and nucleic acid analogs.
  • Exemplary nucleic acid analogs include, but are not limited to, LNAs, PNAs, PPG's, and other nucleic acid analogs.
  • PPG is pyrrazolopyrimidine dG, which is discussed, e.g., in Sedelnikova et al., Antisense Nucleic Acid Drug Dev 2000, 0(6):443-452 (Dec 2000).
  • a variety of methods are available for obtaining a target nucleic acid sequence for use with the compositions and methods of the present invention.
  • certain isolation techniques include, but are not limited to, (1) organic extraction followed by ethanol precipitation, e.g., using a phenol/chloroform organic reagent (e.g., Ausubel et al., eds., Current Protocols in Molecular Biology Volume 1, Chapter 2, Section I, John Wiley & Sons, New York (1993)), in certain embodiments, using an automated DNA extractor, e.g., the Model 341 DNA Extractor available from Applied Biosystems (Foster City, CA); (2) stationary phase adsorption methods (e.g., Boom et al., U.S.
  • the above isolation methods may be preceded by an enzyme digestion step to help eliminate unwanted protein from the sample, e.g., digestion with proteinase K, or other like proteases. See, e.g., U.S. Patent Application Serial No. 09/724,613.
  • a target nucleic acid sequence may be derived from any living, or once living, organism, including but not limited to prokaryote, eukaryote, plant, animal, and virus.
  • the target nucleic acid sequence may originate from a nucleus of a cell, e.g., genomic DNA, or may be extranuclear nucleic acid, e.g., plasmid, mitrochondrial nucleic acid, various RNAs, and the like.
  • the sequence from the organism is RNA, it may be reverse-transcribed into a cDNA target nucleic acid sequence.
  • the target nucleic acid sequence may be present in a double-stranded or single stranded form.
  • Exemplary target nucleic acid sequences include, but are not limited to, amplification products, ligation products, transcription products, reverse transcription products, primer extension products, methylated DNA, and cleavage products.
  • Exemplary amplification products include, but are not limited to, PCR and isothermal products.
  • nucleic acids in a sample may be subjected to a cleavage procedure. In certain embodiments, such cleavage products may be targets.
  • Different target nucleic acid sequences may be different portions of a single contiguous nucleic acid or may be on different nucleic acids. Different portions of a single contiguous nucleic acid may or may not overlap.
  • a target nucleic acid sequence comprises an upstream or 5' region, a downstream or 3' region, and a "pivotal nucleotide" located in the upstream region or the downstream region (see, e.g., Figure 1).
  • the pivotal nucleotide may be the nucleotide being detected by the probe set and may represent, for example, without limitation, a single polymorphic nucleotide in a multiallelic target locus. In certain embodiments, more than one pivotal nucleotide is present. In certain embodiments, one or more pivotal nucleotides is located in the upstream region, and one or more pivotal nucleotide is located in the downstream region.
  • more than one pivotal nucleotide is located in the upstream region or the downstream region.
  • a ligation probe set comprises two or more probes that comprise a target-specific portion that is designed to hybridize in a sequence-specific manner with a complementary region on a specific target nucleic acid sequence (see, e.g., probes 2 and 3 in Fig. 2).
  • a probe of a ligation probe set may further comprise a primer-specific portion.
  • any of the probe's components may overlap any other probe component(s).
  • the target-specific portion may overlap the primer-specific portion.
  • sequence-specific portions of probes are of sufficient length to permit specific annealing to complementary sequences in primers and targets as appropriate.
  • the length of the primer-specific portions are any number of nucleotides from 6 to 35.
  • the length of the target-specific portions are any number of nucleotides from 6 to 35.
  • a ligation probe set comprises at least one first probe and at least one second probe that adjacently hybridize to the same target nucleic acid sequence.
  • a ligation probe set is designed so that the target-specific portion of the first probe will hybridize with the downstream target region (see, e.g., probe 2 in Fig. 2) and the target-specific portion of the second probe will hybridize with the upstream target region (see, e.g., probe 3 in Fig. 2).
  • the sequence-specific portions of the probes are of sufficient length to permit specific annealing with complementary sequences in targets and primers, as appropriate.
  • adjacently hybridized probes may be ligated together to form a ligation product, provided that they comprise appropriate reactive groups, for example, without limitation, a free 3'-hydroxyl and 5'-phosphate group.
  • some ligation probe sets may comprise more than one first probe or more than one second probe to allow sequence discrimination between target sequences that differ by one or more nucleotides (see, e.g., Figure 3).
  • a ligation probe set is designed so that the target-specific portion of the first probe will hybridize with the downstream target region (see, e.g., the first probe in Fig. 1) and the target-specific portion of the second probe will hybridize with the upstream target region (see, e.g., the second probe in Fig. 1).
  • a nucleotide base complementary to the pivotal nucleotide, the "pivotal complement” or “pivotal complement nucleotide” is present on the proximal end of the second probe of the target-specific probe set (see, e.g., 5' end (PC) of the second probe in Fig. 1 ).
  • the first probe may comprise the pivotal complement rather than the second probe (see, e.g., Fig. 3).
  • the pivotal nucleotide(s) may be located anywhere in the target sequence and that likewise, the pivotal complement(s) may be located anywhere within the target-specific portion of the probe(s).
  • the pivotal complement may be located at the 3' end of a probe, at the 5' end of a probe, or anywhere between the 3' end and the 5' end of a probe.
  • the hybridized first and second probes may be ligated together to form a ligation product (see, e.g., Figure 3(B)-(C)).
  • Figure 3 (B)-(C) a mismatched base at the pivotal nucleotide, however, interferes with ligation, even if both probes are otherwise fully hybridized to their respective target regions.
  • other mechanisms may be employed to avoid ligation of probes that do not include the correct complementary nucleotide at the pivotal complement.
  • conditions may be employed such that a probe of a ligation probe set will hybridize to the target sequence to a measurably lesser extent if there is a mismatch at the pivotal nucleotide.
  • non-hybridized probes will not be ligated to the other probe in the probe set.
  • the first probes and second probes in a ligation probe set are designed with similar melting temperatures (T m ).
  • T m melting temperatures
  • the T m for the probe(s) comprising the pivotal complement(s) of the target pivotal nucleotide sought will be approximately 4-15° C lower than the other probe(s) that do not contain the pivotal complement in the probe set.
  • the probe comprising the pivotal complement(s) will also be designed with a T m near the ligation temperature. Thus, a probe with a mismatched nucleotide will more readily dissociate from the target at the ligation temperature.
  • ligation probe sets do not comprise a pivotal complement at the terminus of the first or the second probe (e.g., at the 3' end or the 5' end of the first or second probe). Rather, the pivotal complement is located somewhere between the 5' end and the 3' end of the first or second probe.
  • probes with target-specific portions that are fully complementary with their respective target regions will hybridize under high stringency conditions. Probes with one or more mismatched bases in the target-specific portion, by contrast, will hybridize to their respective target region to a measurably lesser extent. Both the first probe and the second probe must be hybridized to the target for a ligation product to be generated.
  • highly related sequences that differ by as little as a single nucleotide can be distinguished.
  • ligation of probes with a pivotal complement that is not complementary to the pivotal nucleotide may occur, but such ligation occurs to a measurably lesser extent than ligation of probes with a pivotal complement that is complementary to the pivotal nucleotide.
  • a locus may have one of three or four possible different nucleotides.
  • one may employ three or four different first or second ligation probes that each have a different pivotal complement.
  • each of the different probes also has a different primer-specific portion.
  • double-stranded-dependent labels include, but are not limited to, intercalating agents, including, but not limited to, SYBR Green 1, Ethidium Bromide, Acridine Orange, and Hoechst 33258 (all available from Molecular Probes Inc., Eugene, Oregon); TOTAB, TOED1 and TOED2 (Benson et al., Nucleic Acid Research, 21(24):5727-5735 (1993)); TOTO and YOYO (Benson et al., Analytical Biochemistry, 231 :247-255 (1995).
  • intercalating agents including, but not limited to, SYBR Green 1, Ethidium Bromide, Acridine Orange, and Hoechst 33258 (all available from Molecular Probes Inc., Eugene, Oregon); TOTAB, TOED1 and TOED2 (Benson et al., Nucleic Acid Research, 21(24):5727-5735 (1993)); TOTO and YOYO (Benson e
  • Exemplary double-stranded-dependent labels include, but are not limited to, certain minor groove binder dyes, including, but not limited to, 4',6-diamino-2-phenylindole (Molecular Probes Inc., Eugene, Oregon). Certain of the above-noted double-stranded-dependent labels and others are discussed, e.g., in Handbook of Fluorescent Probes and Research Chemicals, Sixth Edition, by Richard Haugland, Molecular Probes, Inc., Eugene, Oregon (1996) (See, e.g., pages 149 to 151. Certain exemplary double- stranded-dependent labels are described, for example, in U.S. Patent Nos. 5,994,056 and 6,171 ,785.
  • a threshold difference between first and second detectable signal values to detect the presence or absence of a target nucleic acid in a sample.
  • the difference between the first and second detectable signal values is the same as or greater than the threshold difference, i.e., there is a threshold difference, one concludes that the target nucleic acid is present. If the difference between the first and second detectable signal values is less than the threshold difference, i.e., there is no threshold difference, one concludes that the target nucleic acid is absent.
  • a double-stranded-dependent label that is not exposed to double-stranded nucleic acid may have a first detectable signal value of zero.
  • the detectable signal value may increase linearly during and/or after an amplification reaction.
  • the second detectable signal value is linearly increased from the first detectable signal value.
  • the detectable signal value when an amplification reaction is carried out with a composition that includes a ligation product and appropriate primers for amplifying the ligation product, the detectable signal value may increase exponentially during and/or after an amplification reaction. (In other words, the second detectable signal value is exponentially increased from the first detectable signal value.)
  • one employs threshold time values (T t ) to determine whether a particular target nucleic acid sequence is present.
  • the threshold time value is the minimum time of an amplification reaction that results in a set detectable signal value of a label.
  • the time that results in a set intensity value 1 may be X seconds. The threshold time value for such a reaction is thus X.
  • the time threshold value when an amplification reaction is carried out with a composition that includes a ligation product and appropriate primers for amplifying the ligation product, the time threshold value may be Y seconds.
  • the time threshold value for such a reaction is Y.
  • the standard deviation is 1
  • the standard deviation is 1
  • one may seek to detect the presence or absence of two different alleles at a particular locus.
  • one may use threshold time values to determine if a sample is homozygous for one or the other allele or if the sample is heterozygous containing both alleles.
  • one may use two different primer sets in separate amplification reactions for detecting two different alleles.
  • one primer set includes primers PA and PZ and another primer set includes primers PB and PZ for detecting alleles A and B, respectively.
  • one may determine the ⁇ T t as follows: ⁇ T t T t (amplification with primers PB and PZ) minus T t (amplification with primers PA and PZ). In certain embodiments, one can then set various ⁇ T t values to determine whether the sample is heterozygous or is homozygous for one of the two alleles.
  • T t is in seconds
  • the sample is homozygous for allele A if the ⁇ T t is greater than or equal to 270; homozygous for allele B if the ⁇ T t is less than or equal to -120; heterozygous if ⁇ T t is greater than or equal to -60 and less than or equal to 210; and make no call if ⁇ T t is greater than -120 and less than -60 or greater than 210 and less than 270.
  • one may set the ranges of ⁇ T t values at other levels as appropriate for determining the presence or absence of various alleles.
  • one employs threshold cycle (C t ) values to determine whether a particular target nucleic acid sequence is present.
  • the C value is the minimum number of cycles in an amplification reaction that result in a set detectable signal value of a label.
  • the number of cycles that result in a set intensity value 1 may be 40.
  • the C t value for such a reaction is thus 40.
  • the C t value when an amplification reaction is carried out with a composition that includes a ligation product and appropriate primers for amplifying the ligation product, the C t value may be 30. Thus, the value for such a reaction is 30.
  • one may use the difference between such C t values ( ⁇ C t ) (here 40 minus 30 0) to assess whether the target nucleic acid sequence is present. For example, in certain embodiments, one may conclude that a ⁇ C t of somewhere above or equal to a set value slightly above 0 indicates the presence of the target nucleic acid sequence, and value below that threshold indicates the absence of the target nucleic acid sequence. In certain embodiments, one may use the standard deviation of the C t value for the amplification reaction without any ligation product to set the appropriate ⁇ C t to signify presence of target nucleic acid sequence.
  • the standard deviation is 1
  • one primer set includes primers PA and PZ and another primer set includes primers PB and PZ for detecting alleles A and B, respectively.
  • one may determine the ⁇ as follows: ⁇ C t C t (amplification with primers PB and PZ) minus C t (amplification with primers PA and PZ). [0122] In certain embodiments, one can then set various ⁇ C t values to determine whether the sample is heterozygous or is homozygous for one of the two alleles.
  • T t and/or C t values may be used with various methods employing double-stranded-dependent labels as discussed herein.
  • T t and/or C t values in any of a variety of methods employing ligation and amplification reactions. Exemplary methods include, but are not limited to, those discussed in U.S. Patent No. 6,027,889, PCT Published Patent Application No. WO 01/92579, and U.S. Patent Application Nos. 09/584,905, 10/011,993, and 60/412,225.
  • a ligation probe set that can be used in a FEN-OLA technique
  • FEN flap endonuclease
  • OLA oligonucleotide ligation
  • a first probe of a ligation probe set comprises a target-specific portion that is designed to hybridize to the target nucleic acid sequence.
  • a second probe of the ligation probe set comprises a flap portion, a target-specific portion, and a FEN cleavage position nucleotide between the flap portion and the target-specific portion.
  • the target- specific portion of the second probe is designed to hybridize to the target nucleic acid sequence such the end of the target-specific portion nearest the flap portion is adjacent to the hybridized target-specific portion of the first probe.
  • the flap portion is designed such that a substantial portion of the flap portions do not hybridize to the target nucleic acid sequence.
  • a "substantial portion of the flap portions do not hybridize” refers to a portion of the total number of flap portions, and it does not refer to a portion of an individual flap portion.
  • "a substantial portion of flap portions that do not hybridize” means that at least 90% of the flap portions do not hybridize. In certain embodiments, at least 95% of the flap portions do not hybridize.
  • FEN will cleave the second probe between the cleavage position nucleotide and the target-specific portion, if the proper target nucleic acid sequence is present. Specifically, such cleavage occurs if the target-specific portions of the first and second probes hybridize to the target nucleic acid sequence, and the FEN cleavage position nucleotide is complementary to the nucleotide of the target nucleic acid sequence that is directly adjacent to the portion of the target nucleic sequence that hybridizes to the target specific portion of the second probe.
  • Figure 4 shows certain nonlimiting examples that help to illustrate certain ligation probe sets that may be used in FEN-OLA techniques according to certain embodiments.
  • the second probe may then be ligated to the adjacent hybridized first probe of a ligation probe set. If the flap is not cleaved, the second probe will not be ligated to the adjacent hybridized first probe.
  • FIG 5 Certain nonlimiting examples of probes used in a FEN-OLA technique are depicted in Figure 5.
  • a probe set comprising: two first probes, differing in their primer-specific portions and their pivotal complements (see, e.g., probes A and B in Fig. 5(A)); and two second probes that comprise different FEN cleavage position nucleotides that correspond to the pivotal complements of the two first probes (see, e.g., probes Y and Z in Fig. 5(A)).
  • FEN will cleave the flap of a second probe only if the second probe comprises a FEN cleavage position nucleotide that is complementary to the pivotal nucleotide of target nucleic acid sequence (see, e.g., Fig. 5(B)).
  • the first and second probes of the probe set are ligated together if the pivotal complement of the first probe is complementary to the pivotal nucleotide of the target nucleic acid sequence (see, e.g., Fig. 5(C)). If there is a mismatch at the pivotal nucleotide, no ligation occurs.
  • cleavage of probes with a FEN cleavage position nucleotide that is not complementary to the pivotal nucleotide may occur, but such cleavage occurs to a measurably lesser extent than cleavage of probes with a FEN cleavage position nucleotide that is complementary to the pivotal nucleotide.
  • ligation of probes with a pivotal complement that is not complementary to the pivotal nucleotide may occur, but such ligation occurs to a measurably lesser extent than ligation of probes with a pivotal complement that is complementary to the pivotal nucleotide.
  • probes used in a FEN-OLA technique are also depicted in Figure 6.
  • Figure 6 one employs a probe set comprising two first probes, which comprise different primer-specific portions and different pivotal complements and the pivotal complement of each first probe is at the penultimate nucleotide position at the 3' end of the first probes (see, e.g., probes A and B in Fig. 6(A)).
  • the probe set further comprises a second probe that comprises a FEN cleavage position nucleotide that is the same as the nucleotide at the 3' end of the two first probes (see, e.g., probe Z in Fig. 6(A)).
  • FEN will cleave the flap of a second probe only if the second probe comprises a FEN cleavage position nucleotide that is complementary to the nucleotide immediately 5' of the pivotal nucleotide of target nucleic acid sequence (see, e.g., Fig. 6(B)).
  • the first and second probes of the probe set are ligated together if: (1) the pivotal complement of the first probe is complementary to the pivotal nucleotide of the target nucleic acid sequence; and (2) the nucleotide at the 3' end of the first probe is complementary to the nucleotide immediately 5' of the pivotal nucleotide of target nucleic acid sequence (see, e.g., Fig. 6(C)). If there is a mismatch at the pivotal nucleotide, no ligation occurs.
  • cleavage of probes with a FEN cleavage position nucleotide that is not complementary to the nucleotide immediately 5' of the pivotal nucleotide may occur, but such cleavage occurs to a measurably lesser extent than cleavage of probes with a FEN cleavage position nucleotide that is complementary to the nucleotide immediately 5' of the pivotal nucleotide.
  • ligation of probes with a pivotal complement that is not complementary to the pivotal nucleotide may occur, but such ligation occurs to a measurably lesser extent than ligation of probes with a pivotal complement that is complementary to the pivotal nucleotide.
  • ligation of first probes with a nucleotide at the 3' end that is not complementary to the nucleotide immediately 5' of the pivotal nucleotide may occur, but such ligation occurs to a measurably lesser extent than ligation of first probes with a nucleotide at the 3' end that is complementary to the nucleotide immediately 5' of the pivotal nucleotide.
  • probes used in a FEN-OLA technique are also depicted in Figure 7.
  • Figure 7 one employs a probe set comprising two second probes, which comprise the same FEN cleavage position nucleotide and comprise different primer-specific portions and different pivotal complements (the pivotal complement of each second probe is immediately 3' to the FEN cleavage position nucleotide) (see, e.g., probes A and B in Fig. 7(A)).
  • the probe set further comprises a first probe that comprises a nucleotide at the 3' end that is the same as the FEN cleavage position nucleotide (see, e.g., probe Z in Fig. 7(A)).
  • FEN will cleave the flap of a second probe only if the second probe comprises a FEN cleavage position nucleotide that is complementary to the nucleotide immediately 3' of the pivotal nucleotide of target nucleic acid sequence (see, e.g., Fig. 7(B)).
  • the first and second probes of the probe set are ligated together if: (1 ) the pivotal complement of the second probe is complementary to the pivotal nucleotide of the target nucleic acid sequence; and (2) the nucleotide at the 3' end of the first probe is complementary to the nucleotide immediately 3' of the pivotal nucleotide of target nucleic acid sequence (see, e.g., Fig. 7(C)). If there is a mismatch at the pivotal nucleotide, no ligation occurs.
  • cleavage of probes with a FEN cleavage position nucleotide that is not complementary to the nucleotide immediately 3' of the pivotal nucleotide may occur, but such cleavage occurs to a measurably lesser extent than cleavage of probes with a FEN cleavage position nucleotide that is complementary to the nucleotide immediately 3' of the pivotal nucleotide.
  • ligation of probes with a pivotal complement that is not complementary to the pivotal nucleotide may occur, but such ligation occurs to a measurably lesser extent than ligation of probes with a pivotal complement that is complementary to the pivotal nucleotide.
  • ligation of first probes with a nucleotide at the 3' end that is not complementary to the nucleotide immediately 3' of the pivotal nucleotide may occur, but such ligation occurs to a measurably lesser extent than ligation of first probes with a nucleotide at the 3' end that is complementary to the nucleotide immediately 3' of the pivotal nucleotide.
  • a group of probes may all be used for a specific six nucleotide portion of a particular target nucleic acid sequence.
  • each of the probes in the group may comprise the same six nucleotide sequence portion that is complementary to the particular target nucleic acid sequence.
  • the probes in the group further comprise additional adjacent degenerate portions that randomly have the four different nucleotides at each of the positions of the degenerate portion so that both the specific six nucleotide portion and the degenerate portion of at least one of the probes in the group will hybridize to any nucleic acid that includes the specific six nucleotide portion.
  • each probe of a group of probes may include the same six nucleotide sequence portion that is complementary to the particular target nucleic acid sequence.
  • Each of the probes of the group may further comprise a four nucleotide degenerate portion.
  • the probes in the series may have all of the possible combinations for a four nucleotide sequence.
  • one of the probes in the group will have a random four nucleotide sequence that will also hybridize to the target. Accordingly, the length of the portion of at least one probe in the group that hybridizes to the target increases to ten nucleotides rather than six nucleotides.
  • Exemplary, but nonlimiting, universal nucleotides are discussed, e.g., in Berger et al. Angew. Chem. Int. Ed. Engl. (2000) 39: 2940- 42; and Smith et al. Nucleosides & Nucleotides (1998) 17: 541-554.
  • An exemplary, but nonlimiting, universal nucleotide is 8-aza-7-deazaadenine, which is discussed, e.g., in Sella and Debelak, Nucl. Acids Res., 28:3224-3232 (2000).
  • a primer set comprises at least one primer capable of hybridizing with the primer-specific portion of at least one probe of a ligation probe set.
  • a primer set comprises at least one first primer and at least one second primer, wherein the at least one first primer specifically hybridizes with one probe of a ligation probe set (or a complement of such a probe) and the at least one second primer of the primer set specifically hybridizes with a second probe of the same ligation probe set (or a complement of such a probe).
  • the first and second primers of a primer set have different hybridization temperatures, to permit temperature-based asymmetric PCR reactions.
  • a ligation probe set typically comprises a plurality of first probes and a plurality of second probes.
  • sequence-specific primers and probes are well known to persons of ordinary skill in the art. Detailed descriptions of primer design that provide for sequence-specific annealing can be found, among other places, in Diffenbach and Dveksler, PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, 1995, and Kwok et al. (Nucl. Acid Res. 18:999-1005, 1990).
  • sequence-specific portions of the primers are of sufficient length to permit specific annealing to complementary sequences in ligation products and amplification products, as appropriate.
  • a primer set of the present invention comprises at least one second primer.
  • the second primer in that primer set is designed to hybridize with a 3' primer-specific portion of a ligation or amplification product in a sequence-specific manner (see, e.g., Figure 2C).
  • the primer set further comprises at least one first primer.
  • the first primer of a primer set is designed to hybridize with the complement of the 5' primer-specific portion of that same ligation product or amplification product in a sequence-specific manner.
  • a universal primer or primer set may be employed according to certain embodiments.
  • a universal primer or a universal primer set hybridizes with two or more of the probes, ligation products, or amplification products in a reaction, as appropriate.
  • PCR amplification reaction
  • qualitative or quantitative results may be obtained for a broad range of template concentrations.
  • Certain exemplary minor groove binders and certain exemplary methods of attaching minor groove binders to oligonucleotides are discussed, e.g., in U.S. Patent Nos. 5,801,155 and 6,084,102.
  • Certain exemplary minor groove binders are those available from Epoch Biosciences, Bothell, Washington.
  • a minor groove binder may be attached to at least one of the following: at least one probe of a ligation probe set and at least one primer of a primer set.
  • a minor groove binder is attached to a ligation probe that includes a 3' primer-specific portion.
  • the presence of the minor groove binder facilitates use of a short primer that hybridizes to the 3' primer-specific portion in an amplification reaction.
  • the short primer, or segment of the primer that hybridizes to the primer-specific portion or its complement may have a length of anywhere between 8 and 15 nucleotides.
  • a minor groove binder is attached to at least one of a forward primer and a reverse primer to be used in an amplification reaction.
  • a primer with a minor groove binder attached to it may be a short primer.
  • the short primer, or segment of the primer that hybridizes to the primer-specific portion or its complement may have a length of anywhere between 8 and 15 nucleotides.
  • both the forward and reverse primers may have minor groove binders attached to them.
  • a minor groove binder is attached to the 3' end of the second probe, and a minor groove binder is attached to a primer that hybridizes to the complement of the 5' primer-specific portion of the first probe.
  • the presence of the minor groove binders facilitates use of short forward and reverse primers in an amplification reaction.
  • the short primer, or segment of the primer that hybridizes to the primer-specific portion or its complement may have a length of anywhere between 8 and 15 nucleotides.
  • Exemplary, but nonlimiting, non-natural nucleotides are discussed, e.g., in Wu et al. J. Am. Chem. Soc. (2000) 122: 7621-32; Berger et al. Nuc. Acids Res. (2000) 28: 2911-14, Ogawa et al. J. Am. Chem. Soc. (2000) 122: 3274-87
  • ligase is an enzymatic ligation agent that, under appropriate conditions, forms phosphodiester bonds between the 3'-OH and the ⁇ '-phosphate of adjacent nucleotides in DNA or RNA molecules, or hybrids.
  • exemplary ligases include, but are not limited to, Tth K294R ligase and Tsp AK16D ligase. See, e.g., Luo et al., Nucleic Acids Res., 24(14):3071 -3078 (1996); Tong et al., Nucleic Acids Res., 27(3):788-794 (1999); and Published PCT Application No. WO 00/26381.
  • Temperature sensitive ligases include, but are not limited to, T4 DNA ligase, T7 DNA ligase, and E. coli ligase.
  • thermostable ligases include, but are not limited to, Taq ligase, Tth ligase, Tsc ligase, and Pfu ligase.
  • Certain thermostable ligases may be obtained from thermophilic or hyperthermophilic organisms, including but not limited to, prokaryotic, eukaryotic, or archael organisms.
  • Certain RNA ligases may be employed in certain embodiments.
  • the ligase is a RNA dependent DNA ligase, which may be employed with RNA template and DNA ligation probes.
  • An exemplary, but nonlimiting example, of a ligase with such RNA dependent DNA ligase activity is T4 DNA ligase.
  • the ligation agent is an "activating" or reducing agent.
  • Chemical ligation agents include, without limitation, activating, condensing, and reducing agents, such as carbodiimide, cyanogen bromide (BrCN), N-cyanoimidazole, imidazole, 1-methylimidazole/carbodiimide/ cystamine, dithiothreitol (DTT) and ultraviolet light.
  • activating condensing
  • reducing agents such as carbodiimide, cyanogen bromide (BrCN), N-cyanoimidazole, imidazole, 1-methylimidazole/carbodiimide/ cystamine, dithiothreitol (DTT) and ultraviolet light.
  • BrCN cyanogen bromide
  • N-cyanoimidazole imidazole
  • 1-methylimidazole/carbodiimide/ cystamine 1-methylimidazole/carbodiimide/ cystamine
  • DTT dithiothreitol
  • UV light ultraviolet light
  • At least one polymerase is included. In certain embodiments, at least one thermostable polymerase is included.
  • thermostable polymerases include, but are not limited to, Taq polymerase, Pfx polymerase, Pfu polymerase, Vent® polymerase, Deep VentTM polymerase, Pwo polymerase, Tth polymerase, UITma polymerase and enzymatically active mutants and variants thereof. Descriptions of these polymerases may be found, among other places, at the world wide web URL: the-scientist.com/yr1998/jan/profile 1_980105. html; at the world wide web URL: the-scientist.com/yr2001/jan/profile _010903. html; at the world wide web URL: the-scientist.com/yr2001/sep/profile2 _010903. html; at the article The Engineer 12(1 ):17 (Jan. 5, 1998); and at the article The Engineer 15(17):1 (Sep. 3, 2001).
  • a genomic DNA sample may comprise both the target sequence and its complement.
  • ligation probes may be designed to specifically hybridize to an appropriate sequence, either the target sequence and/or its complement.
  • Ligation according to the present invention comprises any enzymatic or chemical process wherein an internucleotide linkage is formed between the opposing ends of nucleic acid sequences that are adjacently hybridized to a template. Additionally, the opposing ends of the annealed nucleic acid sequences should be suitable for ligation (suitability for ligation is a function of the ligation method employed).
  • the internucleotide linkage may include, but is not limited to, phosphodiester bond formation.
  • Such bond formation may include, without limitation, those created enzymatically by a DNA or RNA ligase, such as bacteriophage T4 DNA ligase, T4 RNA ligase, T7 DNA ligase, Thermus thermophilus (Tth) ligase, Thermus aquaticus (Taq) ligase, or Pyrococcus furiosus (Pfu) ligase.
  • a DNA or RNA ligase such as bacteriophage T4 DNA ligase, T4 RNA ligase, T7 DNA ligase, Thermus thermophilus (Tth) ligase, Thermus aquaticus (Taq) ligase, or Pyrococcus furiosus (Pfu) ligase.
  • internucleotide linkages include, without limitation, covalent bond formation between appropriate reactive groups such as between an ⁇ -haloacyl group and a phosphothioate group to form a thiophosphorylacetylamino group; and between a phosphorothioate and a tosylate or iodide group to form a 5'-phosphorothioester or pyrophosphate linkages.
  • chemical ligation may, under appropriate conditions, occur spontaneously such as by autoligation.
  • activating or reducing agents may be used.
  • activating agents and reducing agents include, without limitation, carbodiimide, cyanogen bromide (BrCN), imidazole, 1- methylimidazole/carbodiimide/cystamine, N-cyanoimidazole, dithiothreitol (DTT) and ultraviolet light.
  • Non-enzymatic ligation may utilize specific reactive groups on the respective 3' and 5' ends of the aligned probes.
  • ligation generally comprises at least one cycle of ligation, for example, the sequential procedures of: hybridizing the target- specific portions of a first probe and a second probe, that are suitable for ligation, to their respective complementary regions on a target nucleic acid sequence; ligating the 3' end of the first probe with the 5' end of the second probe to form a ligation product; and denaturing the nucleic acid duplex to separate the ligation product from the target nucleic acid sequence.
  • the cycle may or may not be repeated.
  • thermocycling the ligation reaction may be employed to linearly increase the amount of ligation product.
  • gap-filling ligation including, without limitation, gap-filling OLA and LCR, bridging oligonucleotide ligation, FEN-LCR, and correction ligation.
  • Descriptions of these techniques can be found, among other places, in U.S. Patent Number 5,185,243, published European Patent Applications EP 320308 and EP 439182, published PCT Patent Application WO 90/01069, published PCT Patent Application WO 02/02823, and U.S. Patent Application Serial No. 09/898,323.
  • Poly [d(l-C)] is an organic radical that is produced by the ligation reaction.
  • Poly [d(l-C)] in a ligation reaction with various methods employing ligation probes discussed herein.
  • Poly [d(l-C)] with different types of ligation methods.
  • Poly [d(l-C)] in any of a variety of methods employing ligation reactions. Exemplary methods include, but are not limited to, those discussed in U.S. Patent No. 6,027,889, PCT Published Patent Application No. WO 01/92579, and U.S. Patent Application Nos. 09/584,905; 10/011 ,993; and 60/412,225.
  • such double-stranded nucleic acid also will not include a sequence that is the same as or is similar to the sequences of the primer-specific portions of the ligation probes.
  • such double-stranded nucleic acid also will not include a sequence that is the same as or is similar to the sequences of the target-specific portions of the ligation probes.
  • the double-stranded nucleic acid assists in reducing the amount of ligation that may occur between ligation probes when the sought target nucleic acid sequence is not present.
  • one uses any number between 15 to 80 ng/microliter of unrelated double-stranded nucleic acid in a ligation reaction. In certain embodiments, one uses 30 ng/microliter of unrelated double-stranded nucleic acid in a ligation reaction.
  • One may use unrelated double-stranded nucleic acid in a ligation reaction with various methods employing ligation probes discussed herein. In certain embodiments, one may use unrelated double-stranded nucleic acid with different types of ligation methods. For example, one may use unrelated double- stranded nucleic acid in any of a variety of methods employing ligation reactions.
  • Exemplary methods include, but are not limited to, those discussed in U.S. Patent No. 6,027,889, PCT Published Patent Application No. WO 01/92579, and U.S. Patent Application Nos. 09/584,905; 10/011 ,993; and 60/412,225.
  • Exemplary, but nonlimiting ligation reaction conditions may be as follows.
  • the ligation reaction temperature may range anywhere from about 45° C to 55° C for anywhere from two to 10 minutes.
  • any number from 2 to 100 cycles of ligation are performed.
  • 60 cycles of ligation are performed.
  • allele specific ligation probes (a probe of a probe set that is specific to a particular allele at a given locus) are in a concentration anywhere from 2 to 100 nM.
  • allele specific ligation probes are in a concentration of 50 nM.
  • allele specific ligation probes are in a concentration anywhere from 1 to 7 nM.
  • the locus specific ligation probes are in a concentration anywhere from 2 to 200 nM. In certain embodiments, locus specific ligation probes are in a concentration of 100 nM. In certain embodiments, fragmented genomic DNA is in a concentration anywhere from 5 ng/ ⁇ l to 200 ng/ ⁇ l in the ligation reaction. In certain embodiments, fragmented genomic DNA is in a concentration of 130 ng/ ⁇ l in the ligation reaction. In certain embodiments, the pH for the ligation reaction is anywhere from 7 to 8. In certain embodiments, the Mg++ concentration is anywhere from 2 to 22 nM.
  • the ligase concentration is anywhere from 0.04 to 0.16 u/ ⁇ l. In certain embodiments, the ligase concentration is anywhere from 0.02 to 0.12 u/ ⁇ l. In certain embodiments, the K+ concentration is anywhere from 0 to 70 mM. In certain embodiments, the K+ concentration is anywhere from 0 to 20 mM. In certain embodiments, the Poly [d(l-C)] concentration is anywhere from 0 to 30 ng/ ⁇ l. In certain embodiments, the Poly [d(l-C)] concentration is anywhere from 0 to 20 ng/ ⁇ l. In certain embodiments, the NAD+ concentration is anywhere from 0.25 to 2.25 mM.
  • the test composition may be used directly in the subsequent amplification reaction.
  • the test composition prior to the amplification reaction, the test composition may be subjected to a purification technique that results in a test composition that includes less than all of the components that may have been present after the at least one cycle of ligation. For example, in certain embodiments, one may purify the ligation product.
  • Purifying the ligation product comprises any process that removes at least some unligated probes, target nucleic acid sequences, enzymes, and/or accessory agents from the ligation reaction composition following at least one cycle of ligation.
  • Such processes include, but are not limited to, molecular weight/size exclusion processes, e.g., gel filtration chromatography or dialysis, sequence-specific hybridization-based pullout methods, affinity capture techniques, precipitation, adsorption, or other nucleic acid purification techniques.
  • purifying the ligation product prior to amplification reduces the quantity of primers needed to amplify the ligation product, thus reducing the cost of detecting a target sequence.
  • purifying the ligation product prior to amplification may decrease possible side reactions during amplification and may reduce competition from unligated probes during hybridization.
  • Hybridization-based pullout comprises a process wherein a nucleotide sequence complementary to at least a portion of one probe (or its complement), for example, the primer-specific portion, is bound or immobilized to a solid or particulate pullout support (see, e.g., U.S. Patent No. 6,124,092).
  • a composition comprising ligation product, target sequences, and unligated probes is exposed to the pullout support.
  • the ligation product under appropriate conditions, hybridizes with the support-bound sequences.
  • the unbound components of the composition are removed, substantially purifying the ligation products from those ligation reaction composition components that do not contain sequences complementary to the sequence on the pullout support.
  • additional cycles of HBP using different complementary sequences on the pullout support may remove all or substantially all of the unligated probes, further purifying the ligation product.
  • a ligation reaction one exposes the composition to a support that binds to the binding moiety.
  • the unbound components of the composition are removed, substantially purifying the ligation products from those ligation reaction composition components that do not include the binding moiety, including the unligated probes without a binding moiety.
  • the unligated first and second probes will be substantially removed from the ligation product.
  • the binding moiety is biotin, which binds to streptavidin on the support.
  • binding moieties e.g., a first binding moiety and a second binding moiety
  • one may substantially remove unligated ligation probes using certain exonucleases that act specifically on single stranded nucleic acid.
  • one may employ a ligation probe set or sets that include a protective group on one end such that, when the ligation probes are ligated to one another, both ends of the ligation product will be protected from exonuclease digestion.
  • unligated probes are not protected on one end such that unligated probes are digested by exonuclease.
  • the 5' end of the first probe includes a protective group
  • the 3' end of the second probe includes a protective group.
  • Amplification according to the present invention encompasses a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially.
  • Exemplary amplification techniques include, but are not limited to, PCR or any other method employing a primer extension step, and transcription or any other method of generating at least one RNA transcription product.
  • Other nonlimiting examples of amplification are ligase detection reaction (LDR), and ligase chain reaction (LCR).
  • Other nonlimiting examples of amplification are whole-genome amplification reactions. Amplification methods may comprise thermal-cycling or may be performed isothermally.
  • the term "amplification product" includes products from any number of cycles of amplification reactions, primer extension reactions, and RNA transcription reactions, unless otherwise apparent from the context.
  • amplification methods comprise at least one cycle of amplification, for example, but not limited to, the sequential procedures of: hybridizing primers to primer-specific portions of the ligation product or amplification products from any number of cycles of an amplification reaction; synthesizing a strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands.
  • the cycle may or may not be repeated.
  • amplification methods comprise at least one cycle of amplification, for example, but not limited to, the sequential procedures of: interaction of a polymerase with a promoter; synthesizing a strand of nucleotides in a template- dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands.
  • the cycle may or may not be repeated.
  • Descriptions of certain amplification techniques can be found, among other places, in H. Ehrlich et al., Science, 252:1643-50 (1991), M. Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, NY (1990), R. Favis et al., Nature Biotechnology 18:561-64 (2000), and H.F. Rabenau et al., Infection 28:97-102 (2000); Sambrook and Russell, Ausbel et al.
  • Primer extension according to the present invention is an amplification process comprising elongating a primer that is annealed to a template in the 5' to 3' direction using a template-dependent polymerase.
  • a template dependent polymerase incorporates nucleotides complementary to the template strand starting at the 3'-end of an annealed primer, to generate a complementary strand.
  • Transcription is an amplification process comprising an RNA polymerase interacting with a promoter on a single- or double-stranded template and generating a RNA polymer in a 5' to 3' direction.
  • the transcription reaction composition further comprises transcription factors.
  • RNA polymerases including but not limited to T3, T7, and SP6 polymerases, according to certain embodiments, can interact with double- stranded promoters. Detailed descriptions of transcription according to certain embodiments can be found, among other places in Sambrook et al., Sambrook and Russell, and Ausbel et al.
  • Certain embodiments of amplification may employ multiplex amplification, in which multiple target sequences are simultaneously amplified (see, e.g., H. Geada et al., Forensic Sci. Int. 108:31-37 (2000) and D.G. Wang et al., Science 280:1077-82 (1998)).
  • PCR may be optimized by altering times and temperatures for annealing, polymerization, and denaturing, as well as changing the buffers, salts, and other reagents in the reaction composition. Optimization may also be affected by the design of the amplification primers used. For example, the length of the primers, as well as the G-C:A-T ratio may alter the efficiency of primer annealing, thus altering the amplification reaction. See James G. Wetmur, "Nucleic Acid Hybrids, Formation and Structure," in Molecular Biology and Biotechnology, pp.605-8, (Robert A. Meyers ed., 1995).
  • dUTP and uracil-N- glycosidase Discussion of use of dUTP and UNG may be found, for example, in Kwok et al., "Avoiding false positives with PCR," Nature, 339:237- 238 (1989); and Longo et al. "Use of uracil DNA glycosylase to control carry-over contaimination in polymerase chain reactions," Gene, 93:125-128 (1990).
  • a double-stranded-dependent label is included in the amplification reaction.
  • the double-stranded-dependent label indicates the presence or absence (or amount) of a specific nucleic acid sequence in the reaction.
  • the amount of double-stranded-dependent label that gives a signal typically relates to the amount of nucleic acid produced in the amplification reaction.
  • the amount of signal is related to the amount of product created in the amplification reaction.
  • Devices have been developed that can perform a thermal cycling reaction with compositions containing a fluorescent indicator, emit a light beam of a specified wavelength, read the intensity of the fluorescent dye, and display the intensity of fluorescence after each cycle.
  • Devices comprising a thermal cycler, light beam emitter, and a fluorescent signal detector, have been described, e.g., in U.S. Patent Nos. 5,928,907; 6,015,674; and 6,174,670, and include, but are not limited to the ABI Prism® 7700 Sequence Detection System (Applied Biosystems, Foster City, California) and the ABI GeneAmp® 5700 Sequence Detection System (Applied Biosystems, Foster City, California).
  • each of these functions may be performed by separate devices.
  • the reaction may not take place in a thermal cycler, but could include a light beam emitted at a specific wavelength, detection of the fluorescent signal, and calculation and display of the amount of amplification product.
  • thermal cycling and fluorescence detecting devices can be used for precise quantification of target nucleic acid sequences in samples.
  • fluorescent signals can be detected and displayed during and/or after one or more thermal cycles, thus permitting monitoring of amplification products as the reactions occur in "real time.”
  • one can use the amount of amplification product and number of amplification cycles to calculate how much of the target nucleic acid sequence was in the sample prior to amplification.
  • One skilled in the art can easily determine, for any given sample type, primer sequence, and reaction condition, how many cycles are sufficient to determine the presence of a given target polynucleotide.
  • the amplification products can be scored as positive or negative as soon as a given number of cycles is complete.
  • the results may be transmitted electronically directly to a database and tabulated.
  • large numbers of samples may be processed and analyzed with less time and labor required.
  • the present invention is directed to methods, reagents, and kits for detecting the presence or absence of (or quantitating) target nucleic acid sequences in a sample, using ligation and amplification reactions.
  • a ligation product is formed that includes at least one particular primer-specific portion.
  • Double- stranded-dependent labels are employed that provide a different detectable signal value depending upon whether a double-stranded nucleic acid is present or absent.
  • one or more nucleic acid species are subjected to ligation and amplification reactions, either directly or via an intermediate, such as a cDNA target generated from an mRNA by reverse transcription or a whole-genome amplification reaction.
  • the initial nucleic acid comprises mRNA and a reverse transcription reaction may be performed to generate at least one cDNA, followed by at least one ligation reaction and at least one amplification reaction.
  • DNA ligation probes hybridize to target RNA, and an RNA dependent DNA ligase is employed in a ligation reaction, followed by an amplification reaction. The ligation products and amplification products may be detected (or quantitated) using labeled probes.
  • a ligation probe set comprising at least one first probe and at least one second probe, is combined with the sample to form a ligation reaction composition.
  • the ligation composition may further comprise a ligation agent.
  • the first and second probes in each ligation probe set are suitable for ligation together and are designed to hybridize to adjacent sequences that are present in the target nucleic acid sequence. When the target nucleic acid sequence is present in the sample, the first and second probes will, under appropriate conditions, hybridize to adjacent regions on the target nucleic acid sequence (see, e.g., probes 2 and 3 hybridized to target nucleic acid sequence 1 in Fig.
  • the target nucleic acid sequence (1 ) is depicted as hybridized with a first probe (2), for illustration purposes shown here as comprising a 5' primer-specific portion (25) and a target-specific portion (15a), and a second probe (3) comprising a 3' primer- specific portion (35), a target-specific portion (15b) and a free 5' phosphate group ("P") for ligation.
  • a first probe (2) for illustration purposes shown here as comprising a 5' primer-specific portion (25) and a target-specific portion (15a)
  • a second probe (3) comprising a 3' primer- specific portion (35), a target-specific portion (15b) and a free 5' phosphate group ("P") for ligation.
  • the adjacently hybridized probes may, under appropriate conditions, be ligated together to form a ligation product (see, e.g., ligation product 6 in Fig 2B).
  • Figure 2B depicts the ligation product (6), generated from the ligation of the first probe (2) and the second probe (3).
  • the ligation product (6) is shown comprising the 5' primer-specific portion (25) and the 3' primer-specific portion (35).
  • the duplex comprising the target nucleic acid sequence (1) and the ligation product (6) is denatured, for example, by heating, the ligation product (6) is released.
  • one forms an amplification reaction composition comprising the ligation product 6, at least one primer set 7, a polymerase 8, and a double-stranded-dependent label (see, e.g., Fig. 2C).
  • one carries out an amplification reaction with the amplification reaction composition and determines if the target nucleic acid is present in view of a determined Ct value.
  • one carries out an amplification reaction with the amplification reaction composition and determines if there is a threshold difference in signal value during and/or after the amplification reaction to determine whether the target nucleic acid sequence is present.
  • ligation products may form even if the appropriate target nucleic acid sequence is not in the sample, but such ligation occurs to a measurably lesser extent than when the appropriate target nucleic acid sequence is in the sample.
  • Certain embodiments may be substantially the same as those depicted in Figures 2A to 2C, except that two sets of ligation probes are used for detecting a given nucleic acid sequence.
  • the first set of ligation probes is the same as the set depicted in Figure 2.
  • the second set of ligation probes comprises a first probe that comprises a target-specific portion that hybridizes to the complement of the target nucleic acid sequence shown in Figure 2.
  • the first probe of the second set of ligation probes may have the same 5' primer-specific portion as the first probe of the first set of ligation probes or may have a different 5' primer-specific portion.
  • the second set of ligation probes comprises a second probe that comprises a target-specific portion that hybridizes to the complement of the target nucleic acid sequence shown in Figure 2.
  • the second probe of the second set of ligation probes may have the same 3' primer-specific portion as the second probe of the first set of ligation probes or may have a different 3' primer-specific portion.
  • the initial target nucleic acid sequence is an RNA, and mRNA is used to generate a cDNA copy.
  • the cDNA serves as a target nucleic acid sequence to which the first and second probes of the ligation probe set hybridize.
  • the amplification reaction is carried out in a manner that will result in such a threshold difference if the target sequence that is being sought is included in the sample.
  • the amplification reaction is carried out in a manner that will result in such an appropriate time threshold value and/or an appropriate cycle threshold value if the target sequence that is being sought is included in the sample.
  • one employs a ligation probe set that comprises: a first probe that comprises a 5' primer specific portion and a target- specific portion; and a second probe that comprises a target specific portion and a 3' primer-specific portion. If the target nucleic acid is present in the sample, the first and second probes are ligated together to form a ligation product during a ligation reaction.
  • the ligation product comprises the 5' primer-specific portion, the two target-specific portions, and the 3' primer-specific portion.
  • one forms an amplification reaction composition
  • amplification reaction composition comprising the ligation product, a double-stranded-dependent label, and a set of appropriate primers for the 5' and 3' primer-specific portions.
  • the double-stranded-dependent label has a first detectable signal value when it is not exposed to double-stranded nucleic acid sequences.
  • PCR is used as the amplification reaction.
  • no threshold difference is detected during and/or after the first cycle. No threshold difference is detected in such embodiments, because, whether or not the sought ligation product is present, the first cycle of amplification will not result in sufficient detectable signal from the double- stranded-dependent label, since there will be insufficient double-stranded nucleic acid after just one cycle.
  • a threshold difference in detectable signal value will result in such subsequent cycles of amplification when amplification products with both the 5' primer-specific portion and the 3' primer-specific portion increase exponentially when the ligation product is amplified.
  • a threshold difference in detectable signal value will result in such subsequent cycles of amplification when amplification products with both the 5' primer-specific portion and the 3' primer-specific portion increase exponentially when the ligation product is amplified.
  • amplification products will only increase linearly from the presence of the unligated probes.
  • Such linear amplification occurs, since, unlike the ligation product, the unligated probes do not comprise 5' primer-specific portions.
  • a threshold difference in detectable signal value may result after one or more cycles of amplification if the system can detect a difference in signal based on the different lengths of the double-stranded nucleic acids.
  • the double-stranded- dependent label may result in a higher detectable signal value for longer length double-stranded nucleic acids than for shorter length double-stranded nucleic acids.
  • the double-stranded nucleic acid resulting from amplification of unligated primers are shorter than the double-stranded nucleic acid resulting from amplification of ligation products.
  • a threshold difference in detectable signal value may result after one or more cycles of amplification in view of the different detectable signal values resulting from the different sizes of the double-stranded nucleic acids.
  • a positive control which is a separate amplification reaction, that is known to contain the target nucleic acid sequence and which comprises the same probe set and primers as the sample being tested.
  • a negative control which is a separate amplification reaction, that is known not to contain the target nucleic acid sequence and which comprises the same probe set and primers as the sample being tested.
  • the reaction volume may comprise: the sample, a ligation probe set, a ligation agent, a polymerase, a double-stranded-dependent label, a primer set, and dNTPs.
  • a high temperature for a short cycle period during a ligation reaction such that the ligation reagent activity is not substantially destroyed, and after the ligation reaction, hold the reaction volume at the high temperature for a longer period of time that destroys a substantial amount of the ligation reagent activity.
  • destroying a substantial amount of ligation reagent activity means destroying at least 90% of the ligation reaction activity. In certain embodiments, at least 95% of the ligation reaction activity is destroyed.
  • 100% of the ligation reaction activity is destroyed.
  • one may employ amplification primers that do not interfere with hybridization and ligation of ligation probes during the ligation reaction.
  • substantially inactive means that at least 90% of the polymerase is inactive.
  • at least 95% of the polymerase is inactive.
  • 100% of the polymerase is inactive.
  • the polymerase may be substantially inactive at the temperatures that are employed for the ligation reaction.
  • a polymerase may not be substantially active at a lower temperature that is employed for a ligation reaction and the ligation reagent is active at such lower temperatures.
  • agents that may be used in such embodiments to inhibit polymerases at a lower temperature include, but are not limited to, aptamers. See, e.g., Lin et al., J. Mol. Biol., 271 :100-111 (1997).
  • a polymerase that is not substantially activated when held at a high temperature for a short period, but is activated if held at the high temperature for a longer period.
  • polymerase An exemplary, but nonlimiting, example of such a polymerase is AmpliTaq Gold ® (Applied Biosystems, Foster City, CA).
  • AmpliTaq Gold ® Applied Biosystems, Foster City, CA.
  • one may employ double-stranded-dependent labels that do not interfere with hybridization and ligation of ligation probes during the ligation reaction.
  • the first and second probes in each ligation probe set are designed to be complementary to the sequences immediately flanking the pivotal nucleotide of the target sequence (see, e.g., probes A, B, and Z in Fig. 8(A)).
  • two first probes A and B of a ligation probe set will comprise a different nucleotide at the pivotal complement and a different primer-specific portion (P-SPA and P-SPB, respectively) for each different nucleotide at the pivotal complement.
  • P-SPA and P-SPB primer-specific portion
  • the first and second probes will hybridize, under appropriate conditions, to adjacent regions on the target (see, e.g., Fig. 8(B)).
  • the pivotal complement is base-paired to the target, in the presence of an appropriate ligation agent, two adjacently hybridized probes may be ligated together to form a ligation product (see, e.g., Fig 8(C)).
  • the pivotal complement of a first probe is not base-paired to the target, no ligation product comprising that mismatched probe will be formed (see, e.g., probe B in Figs. 8(B) to 8(D).
  • the first probe B is not hybridized to a target.
  • the failure of a probe with a mismatched terminal pivotal complement to ligate to a second probe may arise from the failure of the probe with the mismatch to hybridize to the target under the conditions employed.
  • the failure of a probe with a mismatched terminal pivotal complement to ligate to a second probe may arise when that probe with the mismatch is hybridized to the target, but the nucleotide at the pivotal complement is not base-paired to the target.
  • the reaction volume that is subjected to the ligation reaction forms a test composition.
  • one then forms an amplification reaction composition comprising at least a portion of the test composition, a primer set comprising at least one primer comprising at least a portion of the sequence of one of the optional primer-specific portions P-SPA or P-SPB, a polymerase, and a double-stranded-dependent label (see, e.g., Fig. 8(D)).
  • the amplification reaction composition is subjected to an amplification reaction.
  • no target nucleic acid sequence in the sample has a pivotal nucleotide (C) that is complementary to the nucleotide of the pivotal complement of probe B.
  • C pivotal nucleotide
  • no ligation product comprising both 5' primer-specific portion P-SPB and the 3' primer-specific portion P-SP2 is formed.
  • the amplification reaction comprising the primer set PB and P2 should result in a ⁇ Ct that indicates that no target nucleic acid sequence is present.
  • the amplification reaction comprising the primer set PB and P2 should result in no threshold difference in signal value, which indicates that no target nucleic acid sequence is present.
  • ligation of probes with a pivotal complement that is not complementary to the pivotal nucleotide may occur, but such ligation occurs to a measurably lesser extent than ligation of probes with a pivotal complement that is complementary to the pivotal nucleotide.
  • ⁇ Ct Ct (amplification with primers PB and P2) minus Ct
  • Figure 8 can be modified to include an additional probe set for detecting the presence or absence of a nucleic acid sequence complementary to the target nucleic acid sequence sought to be detected in Figure 8.
  • the pivotal nucleotide of such a complementary target nucleic acid sequence in Figure 8 will be either (T) or (G).
  • the first probes of the additional probe set comprise a target-specific portion complementary to a portion of the complementary target nucleic acid sequence and will have either (A) or (C) as the pivotal complement.
  • probe C the first probe with (C) as the pivotal complement
  • the first probes A and C may share the same primer-specific portion P-SPA, and the first probes B and D may share the same primer-specific portion P-SPB.
  • each of the two separate amplification reactions as shown in Figure 8 would amplify the ligation products for one of the two different target nucleic sequences and its complement.
  • each of the different probes A, B, C, and D may have different 5' primer-specific portions, and four different amplification reactions with four different primer sets may be performed.
  • the methods of the invention comprise universal primers, universal primer sets, or both.
  • the methods of the present invention may comprise universal primers or universal primer sets that decrease the number of different primers that are added to the reaction composition, reducing the cost and time required.
  • the ligation reaction composition may comprise more than one first probe or more than one second probe for each potential allele in a multiallelic target locus.
  • Each of the first probes of each probe set comprises a target-specific portion that is complementary to a portion of the given locus and includes a different nucleotide at the pivotal complement (A or G for the first locus; T or G for the second locus; G or C for the third locus), and a different 5' primer-specific portion (P-SP(A) or P-SP(B)) corresponding to one of the two allelic nucleotide options for each locus.
  • the same set of 5' primer- specific portions (P-SP(A) or P-SP(B)) can be used on the two first probes of each of the three different probe sets.
  • Each of the second probes of each probe set comprises the same 3' primer-specific portion (P-SP(Z)) and a different target-specific portion for each different locus.
  • P-SP(Z) 3' primer-specific portion
  • a different target-specific portion for each different locus one can perform six separate amplification reactions.
  • the material from each of the three separate ligation reactions is split into two separate amplification reactions; one with primer set (PA) and (PZ), and one with primer set (PB) and PZ).
  • the amplification reactions each include a double-stranded- dependent label.
  • Figure 10 illustrates certain such embodiments in which there are three biallelic loci.
  • a ligation probe set comprising two first probes.
  • Each probe set comprises two first probes for the two different alleles at each locus.
  • Each of the first probes of each probe set comprises a target- specific portion that is complementary to a portion of the given locus and includes a different nucleotide at the pivotal complement (A or G for the first locus; T or G for the second locus; G or C for the third locus), and a different 5' primer-specific portion (P-SP(1 ) and P-SP(2) for the first locus; P-SP(3) and P-SP(4) for the second locus; P-SP(5) and P-SP(6) for the third locus).
  • Each of the second probes of each probe set comprises the same 3' primer-specific portion (P- SP(Z)) and a different target-specific portion for each different locus.
  • the embodiment in Figure 9 can be modified such that one performs six separate ligations reactions, one for each allele at each of the three loci. In certain such embodiments, each of the six separate ligation reactions has one of the six different first probes depicted in Figure 9.
  • each of the six different first probes depicted in Figure 9 may modify each of the six different first probes depicted in Figure 9 by employing the same 5' primer-specific portion on each of the six different probes, since each of those six different probes will be subjected to separate ligation reactions.
  • each of the six separate ligation reactions includes the appropriate second probe for the particular locus.
  • employing six separate ligation reactions with different first probes one may include in the composition prior to ligation, the appropriate primer set for the probe set, the double-stranded- dependent label, and other components for the subsequent amplification reaction.
  • each of the first probes of each of the two probe sets for each locus comprises a target- specific portion that is complementary to a portion of one of the 48 different loci and includes a different nucleotide at the pivotal complement.
  • the second probes of the two probe sets for each locus are the same, and the second probes in probe sets for different loci are complementary to a portion of one of the 48 different loci.
  • the two first probes of each of the 96 probe sets may further comprise the same primer- specific portion.
  • each of the second probes of each of the 96 probe sets may further comprise another primer-specific portion.
  • Figure 11 shows certain exemplary embodiments.
  • the first probe comprises a target-specific portion T-SP1.
  • the second probe comprises a 3' primer-specific portion P-SP 42 and a target-specific portion T-SP2.
  • the primer set included in the amplification reaction composition may only comprise one primer 42' that comprises a sequence that is complementary to the sequence of the 3' primer-specific portion P-SP 42 of the second probe.
  • a cycle of amplification with that primer results in an amplification product that comprises a sequence complementary to the ligation product (see Figure 11 C).
  • the primer P-SP 42' again results in an amplification product that comprises a sequence complementary to the ligation product (see Figure 11 D).
  • excess first probe serves as a primer that interacts with the sequence that is complementary to the ligation product to form an amplification product that comprises the sequence of the ligation product (see Figure 11 D).
  • the first probe may contain additional nucleotides at the 5' end that do not hybridize to the target nucleic acid sequence.
  • Certain embodiments that employ excess first probe as a primer for subsequent amplification reactions can be used in the various embodiments of ligation and amplification that are discussed throughout this application. Examples include, but are not limited to, the embodiments depicted in Figure 7. According to certain such embodiments, one may modify the first probes Z that are shown in Figure 7 by not including a primer-specific portion P-SP1. In a subsequent amplification reaction, one may employ excess first probes to serve as primers rather than employing primers that correspond to a P-SP1 sequence on the first probe shown in Figure 7.
  • ligation probes can be designed with a pivotal complement at any location in either the first probe or the second probe. Additionally, in certain embodiments, ligation probes may comprise multiple pivotal complements.
  • the target-specific portions of each of the different first probes for a given locus may have the same sequence except for a different nucleotide at the pivotal complement.
  • the target-specific portions of each of the first probes for a given locus may have a different nucleotide at the pivotal complement and may have different length sequences 5' to the pivotal complement.
  • such target-specific portion sequences 5' to the pivotal complement may all be complementary to a portion of the same locus nucleic acid sequence adjacent to the pivotal nucleotide, but may have different lengths.
  • the target-specific portion sequences 5' to the pivotal complement may be the same except one of them may have one or more additional nucleotides at the 5' end of the target- specific portion.
  • the target-specific portions of each of the different second probes for a given locus may have the same sequence except for a different nucleotide at the pivotal complement.
  • the target-specific portions of each of the second probes for a given locus may have a different nucleotide at the pivotal complement and may have different length sequences 3' to the pivotal complement.
  • such target-specific portion sequences 3' to the pivotal complement may all be complementary to a portion of the same locus nucleic acid sequence adjacent to the pivotal nucleotide, but may have different lengths.
  • the target-specific portion sequences 3' to the pivotal complement may be the same except one of them may have one or more additional nucleotides at the 3' end of the target-specific portion.
  • one may add additional nucleotides to the end of a target specific portion of a ligation probe to affect its melting temperature.
  • the different nucleotide at the pivotal nucleotide of two first probes of a ligation probe set may result in different melting temperatures for such probes if they have the same length target-specific portion.
  • such a spacer nucleotide may be included to affect the melting temperature of a ligation probe.
  • one or more nucleotides of a primer-specific portion may be complementary to the target nucleic acid sequence in the region adjacent to the sequence that hybridizes to the target-specific portion of a ligation probe.
  • the end of a target-specific portion (TSP) adjacent to a primer-specific portion (PSP), and the end of the primer-specific portion adjacent to the target- specific portion may hybridize to a target nucleic acid as follows: PSP/TSP (hybridizing portions shown with double underlining) ACG/ATC (ligation probe) TGC/TAG (target nucleic acid)
  • PSP/TSP hybridizing portions shown with double underlining
  • ACG/ATC ligation probe
  • TGC/TAG target nucleic acid
  • the hybridization of the one or more nucleotides of the primer-specific portion to the target influences the melting temperature of the probe.
  • TGC/TAG target nucleic acid
  • one or more spacer nucleotides may be included between different portions of a ligation probe.
  • one or more spacer nucleotides may be included between a primer-specific portion and a target-specific portion.
  • one or more ligation probes may include an addressable portion or an addressable support-specific portion as discussed, e.g., in U.S. Patent No. 6,027,889, PCT Published Patent Application No. WO 01/92579, and U.S. Patent Application Nos. 09/584,905; 10/011,993; and 60/412,225.
  • the target-specific portions of two ligation probes that are intended to hybridize to the same portion of a target nucleic acid sequence may include different nucleotides as long as such differences do not prevent appropriate ligation.
  • two probes that comprise target-specific portions that are designed to hybridize to an identical portion of a target, but have different pivotal complements A and C at their 3' ends may include variation within the target-specific portion as follows (see lower case nucleotide): 5' CATGCcAATGACGGA-3' 5' CATGCgAATGACGGC-3'
  • the number of ligation probes used to detect any number of target sequences is the product of the number of targets to be detected times the number of alleles to be detected per target plus one (i.e., (number of target sequences x [number of alleles + 1]).
  • numbers of target sequences x [number of alleles + 1] are used to detect 3 biallelic sequences.
  • nine probes are used (3 x [2 + 1]).
  • 16 probes are used (4 x [3 + 1]), and so forth.
  • certain embodiments of the present invention can effectively reduce this number to as few as one amplification primer.
  • as few as two "universal" primers can be used to amplify one or more ligation or amplification products, since the probes may be designed to share primer-specific portions.
  • a sample containing 100 possible biallelic loci would require 200 primers in certain conventional detection methods, yet only one universal primer can be used in certain embodiments of the present invention.
  • the present invention may be used to detect the presence or absence of (or to quantitate) splice variants in a target nucleic acid sequence.
  • genes the DNA that encodes for a protein or proteins, may contain a series of coding regions, referred to as exons, interspersed by non-coding regions referred to as introns.
  • introns are removed and exons are juxtaposed so that the final RNA molecule, typically a messenger RNA (mRNA), comprises a continuous coding sequence.
  • mRNA messenger RNA
  • a gene may comprise five exons each separated from the other exons by at least one intron, see Figure 12.
  • the hypothetical gene that encodes the primary transcript, shown at the top of Figure 12, codes for three different proteins, each encoded by one of the three mature mRNAs, shown at the bottom of Figure 12. Due to alternate splicing, exon 1 may be juxtaposed with (a) exon 2a-exon 3, (b) exon 2b-exon 3, or (c) exon 2c-exon 3, the three splicing options depicted in Figure 12, which result in the three different versions of mature mRNA.
  • the rat muscle protein, troponin T is but one example of alternate splicing.
  • the gene encoding troponin T comprises five exons (W, X, ⁇ , ⁇ , and Z), each encoding a domain of the final protein. The five exons are separated by introns.
  • Two different proteins, an ⁇ -form and a ⁇ -form are produced by alternate splicing of the troponin T gene.
  • the ⁇ -form is translated from an mRNA that contains exons W, X, ⁇ , and Z.
  • the ⁇ -form is translated from an mRNA that contains exons W, X, ⁇ , and Z.
  • first exon and second exon are not limited to the actual first exon and the actual second exon of a given nucleic acid sequence, unless such terms are explicitly used in that manner. Rather, those terms are used to differentiate between any adjoining exons.
  • first exon and second exon are not limited to the actual first exon and the actual second exon of a given nucleic acid sequence, unless such terms are explicitly used in that manner. Rather, those terms are used to differentiate between any adjoining exons.
  • Exon 2 of Sequence A would be the "first exon” and Exons 3 and 5 of Sequence A would be two "second exons.”
  • a method for detecting the presence or absence of (or quantitating) at least one splice variant of at least one given nucleic acid sequence in a sample, wherein the at least one splice variant comprises a sequence that corresponds to a juncture between a first exon and one of a plurality of second exons.
  • the method comprises forming a ligation reaction composition comprising the sample and a ligation probe set for each given nucleic acid sequence.
  • the ligation probe set for each given nucleic acid sequence comprises: (1 ) a first probe that comprises (a) a target-specific portion that is complementary to a portion of the given nucleic acid sequence that corresponds to a portion of the first exon and (b) a 5' primer-specific portion, and (2) at least one a second probe that comprises: (a) a splice-specific portion that is complementary to a portion of the given nucleic acid sequence that corresponds to a portion of one of the plurality of second exons; (b) a 3' primer-specific portion, wherein the 3' primer- specific portion is specific for the one of the plurality of second exons.
  • the first probe and the second probe which comprises the splice-specific portion that is complementary to the portion of the given nucleic acid sequence that corresponds to the portion of the one of the plurality of second exons, hybridize to the given nucleic acid sequence adjacent to one another so that they are suitable for ligation together.
  • one forms a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridized probes are ligated together to form a ligation product comprising the 5' primer-specific portion, the target-specific portion, the splice- specific portion, and the 3' primer-specific portion.
  • one forms an amplification reaction composition
  • amplification reaction composition comprising: (1 ) the test composition; (2) a polymerase; (3) at least one double-stranded-dependent label, wherein the at least one double-stranded- dependent label has a first detectable signal value when it is not exposed to double-stranded nucleic acid sequence; and (4) a primer set comprising at least one first primer comprising the sequence of the 5' primer-specific portion of the ligation product and at least one second primer comprising a sequence complementary to the sequence of the 3' primer-specific portion of the ligation product.
  • one subjects the amplification reaction composition to an amplification reaction.
  • one detects a second detectable signal value from the at least one double-stranded-dependent label at least one of during and after the amplification reaction.
  • a threshold difference between the first detectable signal value from the at least one double-stranded-dependent label and the second detectable signal value from the at least one double-stranded-dependent label indicates the presence of the at least one splice variant of the at least one given target nucleic acid sequence.
  • no threshold difference between the first detectable signal value from the at least one double-stranded- dependent label and the second detectable signal value from the at least one double-stranded-dependent label indicates the absence of the at least one splice variant of the at least one given target nucleic acid sequence.
  • one may employ Ct values to determine the presence or absence of the at least one splice variant of the at least one given target nucleic acid sequence.
  • the quantity of the at least one splice variant in the at least one target nucleic acid sequence is determined.
  • a method for detecting the presence or absence of (or quantitating) at least one splice variant of at least one given nucleic acid sequence in a sample comprising forming a ligation reaction composition comprising the sample and a ligation probe set for each given nucleic acid sequence.
  • the ligation probe set for each given nucleic acid sequence comprises: (1) at least one first probe that comprises: (a) a 5' primer-specific portion, and (b) a splice-specific portion that is complementary to a portion of the given nucleic acid sequence that corresponds to a portion of one of the plurality of second exons, wherein the 5' primer-specific portion is specific for the one of the plurality of second exons; and (2) a second probe that comprises: (a) a target-specific portion that is complementary to a portion of the given nucleic acid sequence that corresponds to the first exon and (b) a 3' primer-specific portion.
  • the first and second probe of the probe set hybridize to the given nucleic acid sequence adjacent to one another so that they are suitable for ligation together.
  • one forms a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridized probes are ligated together to form a ligation product comprising the 5' primer-specific portion, the splice-specific portion, the target- specific portion, and the 3' primer-specific portion.
  • one forms an amplification reaction composition
  • amplification reaction composition comprising: (1) the test composition; (2) a polymerase; (3) at least one double-stranded-dependent label, wherein the double-stranded-dependent label has a first detectable signal value when it is not exposed to double-tranded nucleic acid sequence; and (4) a primer set comprising at least one first primer comprising the sequence of the 5' primer-specific portion of the ligation product and at least one second primer comprising a sequence complementary to the sequence of the 3' primer-specific portion of the ligation product.
  • one subjects the amplification reaction composition to an amplification reaction.
  • one detects a second detectable signal value from the at least one double-stranded-dependent label at least one of during and after the amplification reaction.
  • a threshold difference between the first detectable signal value from the at least one double-stranded-dependent label and the second detectable signal value from the at least one double-stranded-dependent label indicates the presence of the at least one splice variant of the at least one given target nucleic acid sequence.
  • no threshold difference between the first detectable signal value from the at least one double-stranded- dependent label and the second detectable signal value from the at least one double-stranded-dependent label indicates the absence of the at least one splice variant of the at least one given target nucleic acid sequence.
  • one may employ Ct values to determine the presence or absence of the at least one splice variant of the at least one given target nucleic acid sequence.
  • the quantity of the at least one splice variant in the at least one target nucleic acid sequence is determined.
  • the at least one target nucleic acid sequence comprises at least one complementary DNA (cDNA) generated from an RNA.
  • the at least one cDNA is generated from at least one messenger RNA (mRNA).
  • the at least one target nucleic acid sequence comprises at least one RNA target sequence present in the sample.
  • cDNA complementary DNA
  • mRNA messenger RNA
  • the at least one target nucleic acid sequence comprises at least one RNA target sequence present in the sample.
  • FIG. 13 Certain nonlimiting embodiments for identifying splice variants are illustrated by Figure 13. With such embodiments, one detects the presence or absence of (or quantitates) two different splice variants.
  • One splice variant includes exon 1 , exon 2, and exon 4.
  • the other splice variant includes exon 1 , exon 3, and exon 4.
  • one employs a ligation probe set that comprises a first probe (Probe EX1 ) that comprises a 5' primer-specific portion (PSPa) and a target-specific portion that corresponds to at least a portion of exon 1 (TSP).
  • the probe set further comprises two different second probes (Probe EX2 and Probe EX3).
  • Probe EX2 comprises a 3' primer-specific portion PSP2, and a splice-specific portion (SSP-EX2) that corresponds to at least a portion of exon 2.
  • Probe EX3 comprises a 3' primer-specific portion PSP3, and a splice- specific portion (SSP-EX3) that corresponds to at least a portion of exon 3.
  • the first and second probes corresponding to that splice variant hybridize adjacent to one another and are ligated together to form a ligation product.
  • two separate amplification reactions using a double-stranded-dependent label are performed; one with the primer set Pa and P2; and one with the primer set Pa and P3.
  • ligation products corresponding to both exon 2 and exon 3 are present. With such results, one concludes that the sample comprises both splice variants.
  • a gene expression profile for that sample can be compiled and compared with other samples.
  • samples may be obtained from two aliquots of cells from the same cell population, wherein one aliquot was grown in the presence of a chemical compound or drug and the other aliquot was not.
  • the protein insulin regulates the level of blood glucose.
  • the amount of insulin that is produced in an individual can determine whether that individual is healthy or not. Insulin deficiency results in diabetes, a potentially fatal disease. Diabetic individuals typically have low levels of insulin mRNA and thus will produce low levels of insulin, while healthy individuals typically have higher levels of insulin mRNA and produce normal levels of insulin.
  • Tay-Sachs disease Another human disease typically due to abnormally low gene expression is Tay-Sachs disease. Children with Tay-Sachs disease lack, or are deficient in, a protein(s) required for sphingolipid breakdown. These children, therefore, have abnormally high levels of sphingolipids causing nervous system disorders that may result in death.
  • cancer and certain other known diseases or disorders may be detected by, or are related to, the over- or under-expression of certain genes.
  • PSA prostate specific antigen
  • proteins from tumor suppressor genes are believed to play critical roles in the development of many types of cancer.
  • minute amounts of a biological sample can typically provide sufficient material to simultaneously test for many different diseases, disorders, and predispositions. Additionally, there are numerous other situations where it would be desirable to quantify the amount of specific target nucleic acids, in certain instances mRNA, in a cell or organism, a process sometimes referred to as "gene expression profiling.” When the quantity of a particular target nucleic acid within, for example, a specific cell-type or tissue, or an individual is known, in certain cases
  • kits designed to expedite performing certain methods serve to expedite the performance of the methods of interest by assembling two or more components used in carrying out the methods.
  • kits may contain components in pre-measured unit amounts to minimize the need for measurements by end-users.
  • kits may include instructions for performing one or more methods of the invention.
  • the kit components are optimized to operate in conjunction with one another.
  • kits for detecting at least one target nucleic acid sequence in a sample are provided. In certain embodiments, the kits comprise:
  • At least one first probe comprising a target-specific portion, a 5' primer-specific portion, wherein the 5' primer-specific portion comprises a sequence
  • At least one second probe comprising a target-specific portion, a 3' primer-specific portion, wherein the 3' primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target nucleic acid sequence;
  • kits for detecting at least one target nucleic acid sequence in a sample are provided.
  • the kits comprise:
  • At least one first probe comprising a target-specific portion, a 5' primer-specific portion, wherein the 5' primer-specific portion comprises a sequence
  • At least one second probe comprising a target-specific portion, a 3' primer-specific portion, wherein the 3' primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target nucleic acid sequence;
  • compositions for a ligation reaction comprising a ligase and poly-deoxy-inosinic-deoxy-cytidylic acid are provided.
  • kits further comprise primers.
  • kits further comprise at least one primer set comprising (i) at least one first primer comprising the sequence of the 5' primer-specific portion of the at least one first probe, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3' primer-specific portion of the at least one second probe.
  • kits comprise one or more additional components, including, without limitation, at least one of: at least one polymerase, at least one transcriptase, at least one ligation agent, oligonucleotide triphosphates, nucleotide analogs, reaction buffers, salts, ions, and stabilizers.
  • kits comprise one or more reagents for purifying the ligation products, including, without limitation, at least one of dialysis membranes, chromatographic compounds, supports, and oligonucleotides.
  • UA1 5TGATGCTACTGGATCGCT3'
  • UA2 5'TTGCCTGCTCGACTTAGA3'
  • UL 5 ⁇ CGTCGCTATCCAGTGAT3'
  • a ligation probe set for each target nucleic acid sequence comprised first and second ligation probes designed to adjacently hybridize to the appropriate target nucleic acid sequence. These adjacently hybridized probes were, under appropriate conditions, ligated to form a ligation product.
  • This illustrative embodiment used three different ligation probe sets for detecting three biallelic loci.
  • Three different samples of genomic DNA were tested.
  • Table 1 shows the three probe sets that were used.
  • the ligation probes included a target-specific portion, shown with underlined letters in Table 1. As shown by bold letters in Table 1 , the ligation probes also included primer-specific portion sequences.
  • Each probe set included two ASO (allele-specific oligo) probes, AS01 and ASO2, which included a different nucleotide at the 3' end to differentiate between the two different alleles at the given locus.
  • Each probe set also included an LSO (locus-specific oligo) probe for the given locus.
  • the ligation probes were synthesized using conventional automated DNA synthesis chemistry.
  • Oligonucleotide Ligation Assay Oligonucleotide Ligation Assay
  • Ligation reactions were performed in separate reaction volumes with each of the three different ligation probe sets shown in Table 1.
  • the ligation reactions were performed in 96-well microtiter plates in 10 ⁇ L volumes with 2 nM (20 fmol) of each ASO probe (ASO1 and AS02), 4 nM (40 fmol) of LSO probe, 0.12 units/ ⁇ L (1.2 units) Taq Ligase (New England Biolabs, Inc., Beverly, MA), 10 ng/ ⁇ L genomic DNA (100 ng/reaction) (partially fragmented by boiling for 15 minutes at 99°C to an average size of 2 kb), and 1 X ligation buffer (1 OX OLA Buffer Mixture: 200 mM Sodium (3-[N-Morpholino]propanesulfonate) (MOPS), pH 7.5 at 50° C, 1 % (w/v) Triton X-100, 10 mM Dithiothreitol (DTT), 70 mM Magnes
  • the three genomic DNA samples were obtained from Coriell Cell Repositories (Camden, NJ) and were designated as follows: NA17140, NA17155, and NA17202.
  • the ligation reaction volumes were subjected to the reaction conditions shown in Table 2 below using an ABI GeneAmp ® PCR System 9700 Thermal Cycler (Applied Biosystems, Foster City, CA). The ligation reaction volumes were chilled until they were transferred for the amplification reaction. The ligation reaction tubes were transferred to an ABI PRISM ® 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) for amplification when the system reached the first hold temperature of 90°C. TABLE 2
  • SYBR ® Green Master Mix includes SYBR ® Green, PCR buffer, dNTPs, MgCI 2 , and TaqGold ® polymerase.
  • SYBR ® Green Master Mix contains dUTP instead of dTTP to allow AmpErase ® Uracil N-glycosylase (UNG) digestion prior to each new PCR reaction to reduce carryover contamination.
  • UNG P/N N8080096 Applied Biosystems, Foster City, CA
  • Genotype calls were made based on the allele-specific amplification rates monitored real-time by SYBR ® Green I fluorescence (See Figure 14). Threshold cycle (Ct) values were used as a measure for the input amount of allele 1 or allele 2 specific ligation product. The Ct value was the minimum number of cycles that resulted an intensity measurement of 1.
  • ⁇ Ct Ct (amplification with UA2/UL primers) - Ct (amplification with UA1/UL primers). [0303] For this example, it was determined that the sample: is homozygous for allele 1 if the ⁇ Ct is greater than or equal to 4.5; homozygous for allele 2 if the ⁇ Ct is less than or equal to -2; heterozygous if ⁇ Ct is greater than or equal to -1 and less than or equal to 3.5; and no call is made if ⁇ Ct is greater than -2 and less than -1 or greater than 3.5 and less than 4.5.
  • the ⁇ Ct values were set for the genotype calls because, with the primers and assay conditions that were employed, the average ⁇ Ct values for known heterozygotes are 1.25.
  • products with one of the allele specific primer-specific portions or its complement may result in more efficient PCR amplification than products with the other allele specific primer-specific portion or its complement. Accordingly, one may set the ⁇ Ct values as appropriate for making genotype calls.
  • the total ligation reaction volume may be less than 5 ⁇ L. In certain embodiments, certain robot pipetting may be employed. In certain embodiments, the genomic DNA in ligation reaction volume may be less than 10 ng/ ⁇ L.

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Abstract

L'invention concerne des procédés et des kits de détection de la présence ou de l'absence (ou de quantification) de séquences d'acides nucléiques cibles au moyen de ligation et d'amplification.
PCT/US2002/033801 2002-10-23 2002-10-23 Procedes et composition de detection de cibles WO2004040020A1 (fr)

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AU2002342093A AU2002342093A1 (en) 2002-10-23 2002-10-23 Methods and composition for detecting targets
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