WO2002040126A2 - Procedes d'identification de nucleotides dans des positions definies dans des acides nucleiques cibles au moyen de la polarisation de fluorescence - Google Patents

Procedes d'identification de nucleotides dans des positions definies dans des acides nucleiques cibles au moyen de la polarisation de fluorescence Download PDF

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WO2002040126A2
WO2002040126A2 PCT/US2001/030743 US0130743W WO0240126A2 WO 2002040126 A2 WO2002040126 A2 WO 2002040126A2 US 0130743 W US0130743 W US 0130743W WO 0240126 A2 WO0240126 A2 WO 0240126A2
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nucleic acid
nueleotide
odnp
target nucleic
irers
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PCT/US2001/030743
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WO2002040126A3 (fr
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Jeffrey Van Ness
David J. Galas
Lori K. Garrison
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Keck Graduate Institute
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Application filed by Keck Graduate Institute filed Critical Keck Graduate Institute
Priority to AU2002239228A priority patent/AU2002239228A1/en
Priority to US10/398,101 priority patent/US20040038256A1/en
Publication of WO2002040126A2 publication Critical patent/WO2002040126A2/fr
Publication of WO2002040126A3 publication Critical patent/WO2002040126A3/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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • This invention relates to the field of molecular biology, more particularly to methods and compositions involving nucleic acids, and still more particularly to methods and compositions for identifying a particular nueleotide in a target nucleic acid.
  • Mutations occurring in somatic cells may induce disease if the mutations affect genes involved in cellular division control, resulting in, for example, tumor formation.
  • loss-of-function mutations in many genes can give rise to a detectable phenotype in humans.
  • the number of cell generations in the germline, from one gamete to a gamete in an offspring, may be around 20-fold greater in the male germline than in the female.
  • an egg is formed after a second meiotic division and lasts for 40 years. Therefore the incidence of different types of germline mutations and chromosomal aberrations depends on the parent of origin.
  • Molecular genetic techniques have not been employed to a significant extent in the diagnosis of chromosomal aberrations in genetic and malignant disease; cytogenetics remains the preferred technique to investigate these important genetic mechanisms.
  • the remaining normal allele may be replaced by a second copy of the mutant allele in one cell per 10 3 -10 4 .
  • Mechanisms causing this replacement include chromosomal nondisjunction, mitotic recombination, and gene conversion.
  • independent mutations destroying the function of the remaining gene copy are estimated to occur in one cell out of 10 6 .
  • Sensitive mutation detection techniques offer extraordinary possibilities for mutation screening. For example, analyses may be performed even before the implantation of a fertilized egg.
  • allele-specific PCR methods can rapidly and preferentially amplify mutant alleles.
  • multiple mismatch primers have been used to detect H-ras mutations at a sensitivity of one mutant in 10 5 wild-type alleles and sensitivity as high as one mutant in 10 wild-type alleles have been reported.
  • Haliassos et al. Nucleic Acids Res. 17:8093-8099 (1989) and Chen et al., Anal. Biochem., 244:191-194 (1997)).
  • RFLP detection methods are limited by the requirement that the location of the mutations must coincide with restriction endonuclease recognition sequences.
  • primers that introduce a restriction site have been employed in "primer-mediated RFLP.”
  • “primer-mediated RFLP” Jacobson et al., PCR Methods Applicat. 1:299 (1992); Chen et al, Anal. Biochem. 195:51-56 (1991); Di Giuseppe et al., Am. J. Pathol. 144:889-895 (1994); Kahn et al., Oncogene 6:1079-1083 (1991); Levi et al., Cancer Res.
  • nueleotide analogs that are designed to base pair with more than one of the four natural bases are termed "convertides”. Base incorporation opposite different convertides has been tested. (Hoops et al., Nucleic Acids Res. 25:4866-4871 (1997)). For each analog, PCR products were generated using Taq DNA polymerase and primers containing an internal nueleotide analog. The products generated showed a characteristic distribution of the four bases incorporated opposite the analogs.
  • the present invention fulfills this and other related needs by providing methods for parallel measurement of genetic variations that, inter alia, display increased speed, convenience and specificity.
  • methods according to the present invention are based on the incorporation of unique restriction endonuclease restriction sites flanking and/or encompassing genetic variation loci. These methods exploit the high degree of specificity afforded by restriction endonucleases and employ readily available detection techniques.
  • the present invention provides various compounds, compositions and kits useful for, and method of, identifying genetic variations at defined positions in target nucleic acids.
  • the present invention provides a method for identifying a nueleotide at a defined position in a single-stranded target nucleic acid, comprising
  • the first ODNP comprises a nueleotide sequence that is complementary to a nueleotide sequence of the target nucleic acid at a location 3' to the defined position
  • the second ODNP comprises a nueleotide sequence that is complementary to a nueleotide sequence of the complement of the target nucleic acid at a location 3' to the complementary nueleotide of the nueleotide at the defined position
  • the first and second ODNPs further comprise a first constant recognition sequence (CRS) of a first strand and a second CRS of a second strand of an interrupted restriction endonuclease recognition sequence (IRERS), respectively, but not a complete IRERS, the complete IRERS being a double-stranded nucleic acid having the first and the second strands and comprising the first and the second constant recognition sequences (CRS) of a first oligonucleotide primer (ODNP), a second ODNP, and the target nucleic acid
  • the first ODNP
  • the defined position is polymorphic.
  • a mutation at the defined position is associated with a disease, such as a human genetic disease that includes, but is not limited to, bladder carcinoma, colorectal tumors, sickle-cell anemia, thalassemias, al-antitrypsin deficiency, Lesch-Nyhan syndrome, cystic fibrosis/mucoviscidosis, Duchenne/Becker muscular dystrophy, Alzheimer's disease, X-chromosome-dependent mental deficiency, and Huntington's chorea, phenylketonuria, galactosemia, Wilson's disease, hemochromatosis, severe combined immunodeficiency, alpha- 1-antitrypsin deficiency, albinism, alkaptonuria, lysosomal storage diseases, Ehlers-Danlos syndrome, hemophilia, glucose-6-phosphate dehydrogenase disorder, agammaglobulimenia, diabetes insipidus
  • the single-stranded target nucleic acid is one strand of a denatured double-stranded nucleic acid.
  • the double-stranded nucleic acid may be genomic nucleic acid, cDNA or synthetic nucleic acid.
  • the single-stranded target nucleic acid is derived from the genome of a pathogenic virus.
  • the single-stranded target nucleic acid is derived from the genome or episome of a pathogenic bacterium.
  • the target nucleic acid is synthetic nucleic acid.
  • either the nueleotide sequence of the first ODNP complementary to the target nucleic acid, or the nueleotide sequence of the second ODNP complementary to the complement of the target nucleic acid is at least 6, 8, 10, 12, 14 or 16 nucleotides in length.
  • either the first ODNP, or the second ODNP, or both ODNPs are 8-100 nucleotides in length, more preferably 15-85 nucleotides in length.
  • the first ODNP may further comprise one or more nucleotides complementary to the target nucleic acid at the 3' terminus of the first CRS.
  • the second ODNP may further comprise one or more nucleotides complementary to the target nucleic acid at the 3' terminus of the second CRS.
  • the 5' terminus of either the first ODNP or the second ODNP is linked to a biotin molecule.
  • step (b) is carried out by performing a polymerase chain reaction.
  • step (d) is carried out with a RNA polymerase.
  • step (d) may be carried out with either deoxynucleoside triphosphate or dideoxynucleoside triphosphate using a DNA polymerase.
  • the fluorescence labeled dideoxynucleoside triphosphate is any one selected from the group consisting of FAM-ddA, FAM-ddU, FAM-ddC, FAM-ddG, BODIPY-fluorescein-ddA (BFL-ddA), tetramethylrhodamine- ddC (TMR-ddC), b-caboxy-x-prhodamine-ddG (ROX-ddG), and BODIPY-Texas Red- ddU (BTR-ddU).
  • the present method further comprises separating products of step (d) before the detection of step (e).
  • the IRERS is recognizable by a restriction endonuclease selected from the group consisting of PflF I, EcoN I, Fnu4H I, ScrF I, and Tthll I I.
  • an oligonucleotide primer comprising: (a) a first CRS of a first strand of an IRERS, but not the first strand of a complete IRERS, wherein the complete IRERS is a double-stranded oligonucleotide having the first strand and a second strand, the complete IRERS comprises the first CRS and a second CRS linked by a VRS having a number n of variable nucleotides, and the digestion at the IRERS produces a 5' overhang consisting of a single nueleotide; and
  • oligonucleotide sequence (b) is at least 6, 8, 10, 12, 14, or 16 nucleotides in length.
  • the primer is 8-200 nucleotides in length.
  • the primers are 15-85 or 18-32 nueleotide in length.
  • the primer may further comprise one or more nucleotides complementary to the target nucleic acid at the 3' terminus of the first CRS.
  • the 5' terminus of the primer is linked to a biotin molecule.
  • the IRERS may be recognizable by EcoN I.
  • the defined position in the target nucleic acid is polymorphic.
  • a mutation at the defined position in the target nucleic acid is associated with a disease.
  • the target nucleic acid may one strand of a denatured double-stranded nucleic acid, including genomic nucleic acid and cDNA.
  • an oligonucleotide primer pair for producing a portion of a single-stranded target nucleic acid containing a nueleotide to be identified at a defined position, comprising first and second ODNPs wherein the first ODNP comprises a nueleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 3' to the defined position; the second ODNP comprises a nueleotide sequence complementary to a nueleotide sequence of the complement of the target nucleic acid at a location 3' to the complementary nueleotide of the nueleotide to be identified; the first and second ODNPs further comprise a first constant recognition sequence (CRS) of a first strand and a second CRS of a second strand of an interrupted restriction endonuclease recognition sequence (IRERS), respectively, but not a complete IRERS, the complete IRERS being a double-stranded nucleic acid having the first and the second ODNPs wherein
  • either the nueleotide sequence complementary to the target nucleic acid of the first ODNP, or the nueleotide sequence complementary to the complement of the target nucleic acid of the second ODNP, or both are at least 6, 8, 10, 12, 14, or 16 nucleotides in length.
  • the IRERS is recognizable by EcoN I.
  • either the first ODNP, or the second ODNP, or both ODNPs are 8-100 nucleotides in length, preferably 15-85 nucleotides in length.
  • the first ODNP may further comprise one or more nucleotides complementary to the target nucleic acid at the 3' terminus of the first CRS.
  • the second ODNP may further comprise one or more nucleotides complementary to the target nucleic acid at the 3' terminus of the second CRS.
  • the 5' terminus of either the first or the second ODNP is linked to a biotin molecule.
  • the defined position in the target nucleic acid may be polymorphic or associated with a disease.
  • the target nucleic acid may be one strand of a denatured double-stranded nucleic acid, such as genomic nucleic acid and cDNA.
  • the present invention provides a composition comprising the primer and the target nucleic acid as described above. It further provides a kit comprising the above primer pair. The kit may further comprise a restriction endonuclease that recognizes the IRERS a portion of which constitutes partial sequences of the primer pair. The kit may also further comprise instruction of use thereof.
  • the present invention provides a set of two ODNP pairs, comprising first and second ODNP pairs each comprising first and second ODNPs wherein:
  • the first ODNP in the first ODNP pair comprises an oligonucleotide sequence complementary to a nueleotide sequence of a single-stranded target nucleic acid at a location 3' to a defined position in the target nucleic acid, and a first CRS of a first strand of an IRERS, but not the first strand of a complete IRERS, the complete IRERS being a double-stranded nucleic acid having first and second strands and comprising the first CRS and a second CRS linked by a VRS;
  • the second ODNP in the first ODNP pair comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 5' to the defined position, and a second CRS of the first strand of the IRERS, but not the first strand of the complete IRERS;
  • the first ODNP in the second ODNP pair comprises an oligonucleotide sequence complementary to a nueleotide sequence of the complement of the target nucleic acid at a location 5' to the position in the complement corresponding to the defined position in the target nucleic acid, and a first CRS of the second strand of the IRERS, but not the second strand of the complete IRERS;
  • the second ODNP in the second ONDP pair comprises an oligonucleotide sequence complementary to a nueleotide sequence of the complement of the target nucleic acid at a location 3' to the position in the complement corresponding to the defined position in the target nucleic acid, and a second CRS of the second strand of the IRERS, but not the second strand of the complete IRERS;
  • a fragment resulting from an extension and ligation of the first and second ODNPs in each ODNP pair comprises the complete IRERS, wherein digestion at the IRERS produces a 5' overhang consisting of either the nueleotide at the defined position or the complement thereof.
  • the present invention provides a method comprising: (a) providing a double-stranded nucleic acid molecule comprising an interrupted restriction endonuclease recognition sequence (IRERS), wherein the IRERS comprises a first constant recognition sequence (CRS) and a second CRS linked by a variable recognition sequence (VRS);
  • IRERS interrupted restriction endonuclease recognition sequence
  • CRS constant recognition sequence
  • VRS variable recognition sequence
  • step (a) comprises:
  • step (a) comprises:
  • step (a) comprises:
  • the first ODNP comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 3' to a defined position in the target nucleic acid, and a first CRS of a first strand of an IRERS, but not the first strand of a complete IRERS, ' the complete IRERS being a double-stranded nucleic acid having first and second strands and comprising the first CRS and a second CRS linked by a VRS
  • the second ODNP comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 5' to the defined position, and a second CRS of the first strand of the IRERS, but not the first strand of the complete IRERS;
  • step (ii) extending the first and second ODNPs; (iii) ligating the extended products of step (ii); (iv) denaturing the ligation product of step (iii); and
  • step (v) annealing the denatured ligation product of step (iv) that contains said first and second ODNPs with an oligonucleotide that has a universe nueleotide at the position corresponding to the defined position in the double stranded nucleic acid molecule, wherein the resulting double-stranded nucleic acid molecule comprises an complete IRERS.
  • the defined position is polymorphic.
  • a mutation at the defined position is associated with a disease, such as a human genetic disease that includes, but is not limited to, bladder carcinoma, colorectal tumors, sickle-cell anemia, thalassemias, al-antitrypsin deficiency, Lesch-Nyhan syndrome, cystic fibrosis/mucoviscidosis, Duchenne/Becker muscular dystrophy, Alzheimer's disease, X-chromosome-dependent mental deficiency, and Huntington's chorea, phenylketonuria, galactosemia, Wilson's disease, hemochromatosis, severe combined immunodeficiency, alpha- 1-antitrypsin deficiency, albinism, alkaptonuria, lysosomal storage diseases, Ehlers-Danlos syndrome, hemophilia, glucose-6-phosphate dehydrogenase disorder, agammaglobulimenia, diabetes insipidus
  • step (d) is carried out with a RNA polymerase.
  • step (d) may be carried out with either deoxynucleoside triphosphate or dideoxynucleoside triphosphate using a DNA polymerase.
  • the fluorescence labeled dideoxynucleoside triphosphate is any one selected from the group consisting of FAM-ddA, FAM-ddU,
  • the present method further comprises separating products of step (d) before the detection of step (e).
  • the IRERS is recognizable by a restriction endonuclease selected from the group consisting of PflF I, EcoN I, Fnu4H I, ScrF I, and Tthl ll I.
  • the present invention provides a method comprising the steps:
  • the first ODNP comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 3' to a defined position in the a target nucleic acid, and a first CRS of a first strand of an IRERS, but not the first strand of a complete IRERS, the complete IRERS being a double-stranded nucleic acid having first and second strands and comprising the first CRS and a second CRS linked by a VRS
  • the second ODNP comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 5' to the defined position, and a second CRS of the first strand of the IRERS, but not the first strand of the complete IRERS;
  • the defined position is associated with a disease.
  • the single-stranded target nucleic acid is one strand of a denatured double-stranded nucleic acid.
  • the double-stranded nucleic acid may be genomic nucleic acid, cDNA or synthetic nucleic acid.
  • either the nueleotide sequence of the first ODNP complementary to the target nucleic acid, or the nueleotide sequence of the second ODNP complementary to the complement of the target nucleic acid is at least 6, 8, 10, 12, 14 or 16 nucleotides in length.
  • the first ODNP may further comprise one or more nucleotides complementary to the target nucleic acid at the 3' terminus of the first CRS.
  • the second ODNP may further comprise one or more nucleotides complementary to the target nucleic acid at the 3' terminus of the second CRS.
  • the 5' terminus of either the first ODNP or the second ODNP is linked to a biotin molecule.
  • step (b) is carried out by performing a polymerase chain reaction.
  • step (d) is carried out with a RNA polymerase.
  • step (d) may be carried out with either deoxynucleoside triphosphate or dideoxynucleoside triphosphate using a DNA polymerase.
  • the fluorescence labeled dideoxynucleoside triphosphate is any one selected from the group consisting of FAM-ddA, FAM-ddU,
  • FAM-ddC, FAM-ddG, BODIPY-fluorescein-ddA BFL-ddA
  • TMR-ddC tetramethylrhodamine- ddC
  • ROX-ddG b-caboxy-x-prhodamine-ddG
  • BTR-ddU BODIPY-Texas Red- ddU
  • the present method further comprises separating products of step (d) before the detection of step (e).
  • the IRERS is recognizable by a restriction endonuclease selected from the group consisting of PflF I, EcoN I, Fnu4H I, ScrF I, and Tthl l l I.
  • Figures 1A and IB together are a diagram of major steps of the present method for identifying a nueleotide at a defined position in a target nucleic acid using two ODNPs and the restriction endonuclease recognition sequence for EcoN I.
  • Figure 2 shows changes in fluorescence polarization for DNA samples genotyped with FP-EcoN I assay.
  • Figure 3 is a schematic diagram of the major components of the ODNPs and a resulting amplicon of the present invention.
  • Figure 4 is a schematic diagram of the major components of an interrupted restriction endonuclease recognition sequence.
  • a ⁇ A 2 ...A m is a specific nueleotide sequence consisting of m nucleotides, whereas A' ⁇ A' 2 ...A' m is the complement sequence of A ⁇ A 2 ...A m .
  • the double-stranded fragment comprised of A ⁇ A 2 ...A m and A' ⁇ A' 2 ...A' m forms the first CRS (also referred to as "Region A").
  • N ⁇ N 2 ...N n is a variable nueleotide sequence consisting of n nucleotides where any one of the nueleotide can contain any of the four bases (a, c, t, or g).
  • N' ⁇ N' 2 ...N' n is the complement of N ⁇ N 2 ...N n and forms a VRS (also referred to "Region B" where the number n is equal to the number B) in combination of N ⁇ N 2 ...N n.
  • C ⁇ C 2 ... is a specific nueleotide sequence consisting of i nucleotides, whereas C' ⁇ C 2 ...C'i is the complement of C ⁇ C 2 ...Cj.
  • the double-stranded fragment comprised of C ⁇ C 2 ... and C' ⁇ C' 2 ...C'j forms the second CRS (also referred to as "Region C").
  • Figure 5 is a schematic diagram of a set of two ODNP pairs.
  • Figure 6 is a schematic diagram of major steps in the present method for identifying a nueleotide at a defined position in a target nucleic acid using a set of two ODNP pairs and the exemplary restriction endonuclease recognition sequence for EcoN I.
  • Figure 7 is a schematic diagram of major steps of one embodiment of the present method for providing a double-stranded nucleic acid molecule containing an IRERS.
  • the present invention provides methods, compositions, and kits for determining sequence information at a defined genetic locus in a target nucleic acid.
  • the invention provides for the design, preparation and use of oligonucleotide primers (ODNPs) that can be extended in a manner that incorporates information about the nueleotide of interest into the extension product.
  • ODNPs oligonucleotide primers
  • the resulting product e.g., amplicon
  • fluorescence polarization also described in more detail below, to determine the identity of the nueleotide of interest.
  • This information is advantageously utilized in a variety of applications, as described herein, such as genetic analysis for hereditary diseases, tumor diagnosis, disease predisposition, forensics or paternity, crop cultivation and animal breeding, expression profiling of cell function and/or disease marker genes, and identification and/or characterization of infectious organisms that cause infectious diseases in plants or animals and/or that are related to food safety.
  • the ODNPs of the present invention each contain part of an interrupted restriction endonuclease recognition sequence (IRERS), defined in detail below.
  • the interrupted segment of the restriction endonuclease recognition site also referred to as “variable recognition sequence (VRS)
  • VRS variable recognition sequence
  • the interrupted segment of the restriction endonuclease recognition site may be one or more nucleotides in length and the sequence is variable (each position can contain any of the four bases (a, c, t, or g)).
  • VRS variable recognition sequence
  • the complete IRERS of the present invention is selected so that digestion of a nucleic acid fragment containing such an IRERS by a restriction endonuclease that recognizes the IRERS produces digestion products with 5' overhang.
  • the primers are designed such that the nueleotide of interest in a target nucleic acid is located in the amplicon within both the variable segment of the restriction endonuclease recognition site and 5' overhangs produced by digestion at the IRERS.
  • the amplicon can then be digested to generate small fragments of nucleic acid that can be analyzed to determine the nueleotide of interest with great accuracy and sensitivity.
  • the oligonucleotide primers of the present invention are shown schematically in Figure 3.
  • FIG. 1 a diagram of the present invention is shown using the exemplary restriction endonuclease recognition sequence for EcoN I.
  • Any interrupted restriction endonuclease recognition sequence may be used at which digestion produces a 5' overhang (preferably a 5' overhang consisting of a single nueleotide) may be used.
  • the present invention provides assays for determining the identity of a base at a predetermined location in a target nucleic acid molecule.
  • assays for determining the identity of a base at a predetermined location in a target nucleic acid molecule.
  • compounds, compositions, and kits that are useful in performing such assays.
  • a location in a nucleic acid is "5' to" or "5' of a nueleotide of interest, this means that the location is between the nueleotide of interest and the 5' phosphate of that strand of nucleic acid.
  • a nueleotide in the complement of a single-stranded target nucleic acid at a position "corresponding to" a defined position in the target refers to a nueleotide complementary to the nueleotide at the defined position in the target.
  • the word “a” refers to one or more of the indicated items unless the context clearly indicates otherwise.
  • “a" polymerase refers to one or more polymerases.
  • the identity of a nueleotide of interest in a target nucleic acid molecule is determined by combining the target with two primers, where one molecule of the first primer hybridizes to, and extends from, a location 3' of the nueleotide of interest in the target, so as to incorporate the complement of the nueleotide of interest in a first extension product.
  • One molecule of the second primer then hybridizes to, and extends based on, the first extension product, at a location 3' of the complement of the nueleotide of interest, so as to incorporate the nueleotide of interest in a second extension product.
  • Another molecule of the first primer then hybridizes to, and extends from, a location 3' of the nueleotide of interest in the second extension product, so as to form, in combination with the second extension product, a nucleic acid fragment.
  • the first and second primers are designed to each incorporate a portion of the recognition sequence of an IRERS, the IRERS being selected so that digestion at the IRERS produces fragments with 5' overhangs.
  • the primers are also designed such that the nueleotide of interest in the target is located within both the VRS and a 5' overhang produced by digestion at the IRERS.
  • the 3' recessed termini corresponding to 5' overhangs produced by digestion at the IRERS are then filled in with one or more fluorescence labeled nucleoside triphosphates.
  • the incorporated nucleotide(s) can be detected using the technique of fluorescence polarization.
  • Target Nucleic Acid Molecules typically involve or include a target nucleic acid molecule.
  • the target nucleic acid of the present invention is any nucleic acid molecule about which nueleotide information is desired, and which can serve as a template for a primer extension reaction, e.g., can base pair with a primer.
  • the target nucleic acid may also be referred to as the template nucleic acid.
  • nucleic acid refers generally to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid or an analog thereof.
  • the template nucleic acid can be either single-stranded or double-stranded.
  • the template nucleic acid is DNA.
  • the template is RNA.
  • Suitable nucleic acid molecules are DNA, including genomic DNA, ribosomal DNA and cDNA.
  • Other suitable nucleic acid molecules are RNA, including mRNA, rRNA and tRNA.
  • the nucleic acid molecule may be naturally occurring, as in genomic DNA, or it may be synthetic, i.e., prepared based up human action or intervention, or may be a combination of the two .
  • a naturally occurring nucleic acid is obtained from a biological sample.
  • the biological sample can be any sample that contains biological material ⁇ e.g., cells, tissues) from any organisms, including but not limited to, animals, higher plants, fungi, bacteria, and viruses.
  • Mammalian tissue for example blood, plasma/serum, hair, skin, lymph node, spleen, liver, etc
  • the biological sample may comprise one or more different human tissues and/or cells. Mammalian and/or human tissues and/or cells may further comprise one or more tumor tissues and/or cells.
  • Another type of preferred biological sample is or contains a virus, such as pathogenic viruses ⁇ e.g., hepatitis A, B, or C), herpes virus ⁇ e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein Barr virus), adenovirus, influenza virus, echovirus, rhinovirus, cornovirus, respiratory syncytical virus, mumps virus, measles virus, rubella virus, parvovirus, poliovirus, rabies virus, and flaviviruses.
  • pathogenic viruses ⁇ e.g., hepatitis A, B, or C
  • herpes virus ⁇ e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein Barr virus
  • adenovirus influenza virus, echovirus, rhinovirus, cornovirus, respiratory syncytical virus, mumps virus, measles virus, rubella virus, parvovirus, poliovirus,
  • Such bacteria include, but are not limited to, chlamydia, rickettsial bacteria, mycobacteria, staphylococci, treptocci, pneumonococci, meningococci, conococci, klebsiella, proteus, serratia, pseudomonas, bacilli, cholera, tetanus, anthrax, plague, and Lymes disease bacteria.
  • Methodology for isolating populations of nucleic acids from biological samples is well known and readily available to those skilled in the art of the present invention.
  • a synthetic nucleic acid is produced by human intervention.
  • many companies are in the business of making and selling synthetic nucleic acids that may be useful as the template nucleic acid molecule in the present invention. See, e.g., Applied Bio Products Bionexus (www.bionexus.net); Blue Heron Biotechnology (Bothell, WA; www.blueheron.com); Commonwealth Biotechnologies, Inc.
  • the synthetic nucleic acid template may be prepared using an amplification reaction.
  • the amplification reaction may be, for example, the well- known polymerase chain reaction (PCR).
  • the synthetic nucleic acid template may be prepared using recombinant DNA means through production in one or more prokaryotic or eukaryotic organism such as, e.g., E. coli, yeast, Drosophila or mammalian tissue culture cell line.
  • prokaryotic or eukaryotic organism such as, e.g., E. coli, yeast, Drosophila or mammalian tissue culture cell line.
  • the nucleic acid molecule may, and typically will, contain one or more of the 'natural' nueleotide bases, i.e., adenine (A), guanine (G), cytosine (C), thymine (T) and, in the case of an RNA, uracil (U).
  • the target nucleic acid may include "unnatural" nucleotides.
  • Unnatural nucleotides are chemical moieties that can be substituted for one or more natural nucleotides in a nueleotide chain without causing the nucleic acid to lose its ability to serve as a template for a primer extension reaction.
  • the substitution may include either sugar and/or phosphate substitutions, in addition to base substitutions.
  • Such moieties are very well known in the art, and are known by a large number of names including, for example, abasic nucleotides, which do not contain a commonly recognized nueleotide base, such as adenine, guanine, cytosine, uracil or thymine ⁇ see, e.g., Takeshita et al. "Oligonucleotides containing synthetic abasic sites" The Journal of Biological Chemistry, vol. 262, pp. 10171-10179 1987; Iyer et al. "Abasic oligodeoxyribonucleoside phosphorothioates: synthesis and evaluation as anti- HIV-1 agents" Nucleic acids Research, vol. 18, pp.
  • Other exemplary unnatural bases include universal mismatch base analogs, (such as the abasic 3-nitropyrrole); convertides (see, e.g., Hoops et al, Nucleic Acids Res.
  • nucleotides see, e.g., Millican et al., "Synthesis and biophysical studies of short oligodeoxynucleotides with novel modifications: A possible approach to the problem of mixed base oligodeoxynucleotide synthesis," Nucleic Acids Research 12:7435-7453 (1984); nueleotide mimetics; nucleic acid related compounds; spacers ⁇ see, e.g., Nielsen et al. Science, 254:1497-1500 (1991); and specificity spacers ⁇ see, e.g., PCT International Publication No. WO 98/13527).
  • base-pair mismatch refers to all single and multiple nueleotide substitutions that perturb the hydrogen bonding between conventional base pairs, e.g., G:C, A:T, or A:U, by substitution of a nueleotide with a moiety that does not hybridize according to the standard Watson-Crick model to a corresponding nueleotide on the opposite strand of the oligonucleotide duplex.
  • Such base-pair mismatches include, e.g., G:G, G:T, G:A, G:U, C:C, C:A, C:T, C:U, T:T, T:U, U:U and A:A.
  • base-pair mismatches are single or multiple nueleotide deletions or insertions that perturb the normal hydrogen bonding of a perfectly base-paired duplex.
  • base-pair mismatches arise when one or both of the nucleotides in a base pair has undergone a covalent modification ⁇ e.g., methylation of a base) that disrupts the normal hydrogen bonding between the bases.
  • Base-pair mismatches also include non-covalent modifications such as, for example, those resulting from incorporation of intercalating agents such as ethidium bromide and the like that perturb hydrogen bonding by altering the helicity and/or base stacking of an oligonucleotide or polynucleotide duplex.
  • intercalating agents such as ethidium bromide and the like that perturb hydrogen bonding by altering the helicity and/or base stacking of an oligonucleotide or polynucleotide duplex.
  • nueleotide and, optionally, nueleotide analogs that form the target nucleic acid there is one or more natural nucleotide(s) of unknown identity.
  • the present invention provides kits, compositions and methods whereby the identity of the unknown nucleotide(s) becomes known.
  • the nueleotide of unknown identity at the "nueleotide loci" (or the "defined position") refers to a specific nueleotide having a precise location on a target nucleic acid.
  • the nueleotide to be identified in the target nucleic acid may be a mutation.
  • the term "mutation" refers to an alteration in a wild-type nucleic acid sequence. Mutations may be in non-coding regions (introns or 5' and 3' flanking regions) or may be in regions encoding proteins (exons) of a target nucleic acid. Exemplary mutations in non-coding regions include regulatory mutations that alter the amount of gene product, localization of protein and/or timing of expression. Exemplary mutations in coding regions include nonsense mutations and missense mutations.
  • a "nonsense mutation” is a single nueleotide change resulting in a triplet codon (where mutation occurs) being read as a "STOP" codon causing premature termination of peptide elongation, i.e., a truncated peptide.
  • a “missense mutation” is a mutation that results in one amino acid being exchanged for a different amino acid in the gene's peptide expression product. Such a mutation may cause a change in the folding (3- dimensional structure) of the peptide and/or its proper association with one or more other peptides in a multimeric structure.
  • the nueleotide of interest i.e., the nueleotide to be identified, may be a "single-nucleotide polymorphism" (SNP), which refers to any nueleotide sequence variation, preferably one that is common in a population of organisms and is inherited in a Medelian fashion.
  • SNP single-nucleotide polymorphism
  • the SNP is either of two possible nucleotides, and there is little or no possibility of finding a third or fourth nueleotide identity at an SNP site.
  • polymorphism or “genetic variation,” as used herein, refers to the occurrence of two or more genetically determined alternative sequences or alleles at a defined position in a population. The allelic form occurring most frequently in a selected population is referred to as the wild type form. Other allelic forms are designated as variant forms. Diploid organisms may be homozygous or heterozygous for allelic forms.
  • the genetic variation may be associated with or cause diseases or disorders.
  • associated with refers to the correlation between the occurrence of the genetic variation and the presence of a particular disease or a disorder.
  • Such diseases or disorders may be human genetic diseases or disorders that include, but are not limited to, bladder carcinoma, colorectal tumors, sickle-cell anemia, thalassemias, al-antitrypsin deficiency, Lesch-Nyhan syndrome, cystic fibrosis/mucoviscidosis, Duchenne/Becker muscular dystrophy, Alzheimer's disease, X-chromosome-dependent mental deficiency, Huntington's chorea, phenylketonuria, galactosemia, Wilson's disease, hemochromatosis, severe combined immunodeficiency, alpha- 1-antitrypsin deficiency, albinism, alkaptonuria, lysosomal storage diseases, Ehlers-Danlos syndrome, hemophilia, glucose-6-phosphate dehydrogenase disorder, agammaglobulimenia, diabetes insipidus, Wiskott-Aldrich syndrome, Fabry's disease, fragile X-syndrome,
  • Target nucleic acids may be amplified before being combined with ODNPs as described below. Any known method for amplifying nucleic acids may be used. A few exemplary methods, including Qbeta Replicase, Strand Displacement Amplification, transcription-mediated amplification, RACE, and one-sided PCR, are summarized elsewhere herein.
  • Methods, kits and compositions of the present invention typically involve or include one or more ODNPs which generally contain a partial IRERS and a region of complementarity with a target nucleic acid.
  • the target nucleic acid is described as a single-stranded nucleic acid below.
  • the ODNP pair(s) of the present invention wherein the target nucleic acids are double-stranded.
  • oligonucleotide refers to a nucleic acid fragment (typically DNA or RNA) obtained synthetically as by a conventional automated nucleic acid ⁇ e.g., DNA) synthesizer. Oligonucleotide is used synonymously with the term polynucleotide.
  • oligonucleotide primer refers to any polymer having two or more nucleotides used in a hybridization, extension, and/or amplification reaction.
  • the ODNP may be comprised of deoxyribonucleotides, ribonucleotides, or an analog of either.
  • ODNPs are generally between 8 and 200 bases in length. More preferred are ODNPs of between 12 and 50 bases in length and still more preferred are ODNPs of between 18 and 32 bases in length.
  • the present invention provides an ODNP useful for producing a portion of a target nucleic acid containing a nueleotide of interest at a defined position.
  • the ODNP comprises an oligonucleotide sequence complementary to a nueleotide sequence of a target nucleic acid at a location 3' to the defined position.
  • the complementarity between the ODNP and its target need not be exact, but must be sufficient for the ODNP to selectively hybridize with the target, so that the ODNP is able to function as a primer for extension and/or amplification using the target as a template.
  • the ODNP further comprises a first CRS of a first strand of an IRERS at a location 3' to the oligonucleotide sequence complementary to a portion of the target.
  • a complete IRERS is a double-stranded oligonucleotide sequence comprising a first CRS and a second CRS linked with a VRS ( Figure 4).
  • the complete IRERS is selected so that digestion at the IRERS produces a 5' overhang.
  • the 5' overhang consists of a single nueleotide.
  • the ODNP is so designed that when it anneals to the target, the distance between the nueleotide corresponding to the 3' terminal nueleotide of the ODNP and the defined position in the target is within the range 0 to n- 1.(preferably 0 to (n-l)/2) where n is the number of variable nucleotides in the IRERS.
  • n is the number of variable nucleotides in the IRERS.
  • the ODNP is further designed so that when typically used in combination with another ODNP to produce a fragment containing a complete IRERS and a nucleotides of interest (described in detail below), the nueleotide of interest is within both the VRS and the 5' overhang produced by digestion at the IRERS.
  • the ODNP further comprises one or more nucleotides complementary to the target nucleic acid at the 3' terminus of the first CRS.
  • the presence of such nucleotides facilitates extension of the primer as the sequence of the first CRS in the ODNP may or may not be exactly complementary to the corresponding nueleotide sequence of the target.
  • the ODNP with its 5 '-terminus linked to a biotin molecule.
  • the present invention provides an ODNP pair for producing a portion of a target nucleic acid containing a nueleotide to be identified at a defined position ( Figure 3).
  • One primer of the ODNP pair (“the first ODNP” or “the forward primer”) comprises a nucleic acid sequence complementary to a nueleotide sequence of a target nucleic acid at a location 3' to the defined position ("the first region of the target nucleic acid”)
  • the other primer (“the second ODNP” or “the reverse primer” comprises a nucleic acid sequence complementary to a nueleotide sequence of the complement of the target nucleic acid at a location 3' to the complementary nueleotide of the nueleotide at the defined position ("the first region of the complement”).
  • each ODNP contains at least 6, preferably 8, more preferably 10, most preferably 12, 14, or 16 nucleotides that are complementary to the target nucleic acid or the complement thereof.
  • each ODNP of the ODNP pair hybridizes to a target nucleic acid, or the complement thereof, at a location 3' to the defined position in the target or the complementary position in the complement of the target, the resulting extension and/or amplification products from the ODNP pair contains the nueleotide to be identified at the defined position.
  • Each ODNP in the ODNP pair of the present invention further comprises a partial IRERS, but not a complete IRERS, at a location 3' to, or preferably at the 3' terminus of, its nucleic acid sequence described above ⁇ i.e., the sequence complementary to the target nucleic acid or the complement thereof).
  • the first ODNP and the second ODNP comprise the first CRS of the first strand of the IRERS and the second CRS of the second strand of the IRERS, respectively.
  • the IRERS is selected so that digestion at the IRERS with a restriction endonuclease that recognizes the IRERS produces a 5' overhang.
  • the 5' overhang consists of a single nueleotide.
  • the first ODNP and the second ODNP are so spaced that (1) the extension and/or amplification product with the ODNP pair as primers and the target nucleic acid as a template contains a complete IRERS.
  • the number of nucleotides between the first and the second CRS is the exact number of nucleotides in the NRS so that the extension and/or amplification product from both OD ⁇ Ps can be digested by a RE that recognizes the complete IRERS.
  • the nucleic acid to be identified in the extension and/or amplification product is within both the NRS and the 5' overhang produced by digestion at the IRERS.
  • the partial IRERS in each OD ⁇ P may or may not be complementary to the target nucleic acid.
  • each OD ⁇ P of the OD ⁇ P pair further contains one or more nucleotides that is complementary to the target nucleic acid or the complement thereof ("the second region of the target nucleic acid” and “the second region of the complement,” respectively) at a location 3' to, or preferably the 3' terminus of, the CRS.
  • Such nucleotides are a portion of the NRS ( Figure 3).
  • the number of the nucleotides between first and second regions of the target nucleic acid or the complement thereof may be larger or smaller, but preferably equal to, the number of nucleotides of ODNPs between their two regions that are complementary to the target nucleic acids or the complement thereof.
  • one ODNP of the ODNP pair has its 5' terminus linked to a biotin molecule.
  • a linkage facilitates separation of a digestion product containing the ODNP from others before such a product is filled at its 3' recessed terminus with a fluorescence labeled nucleoside triphosphate and subsequently characterized by fluorescence polarization as described in detail below.
  • the present invention provides a set of two ODNP pairs for producing a portion of a target nucleic acid containing a nueleotide to be identified at a defined position ( Figure 5).
  • Each pair of the set contain a first ODNP and a second ODNP.
  • the first ODNP of the first ODNP pair comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 3' to the defined position. It further comprises a first CRS of a first strand of an IRERS at a location 3' to, preferably at the 3' terminus of, the above oligonucleotide sequence.
  • the second ODNP of the first ODNP pair comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 5' to the defined position. It further comprises a second CRS of the first stand of the IRERS at a location 5' to, preferably at the 5' terminus, of the above oligonucleotide sequence.
  • the first ODNP of the second ODNP pair comprises an oligonucleotide sequence complementary to a nueleotide sequence of the complement of the target nucleic acid at a location 5' to the position in the complement corresponding to the defined position in the target nucleic acid.
  • the second ODNP of the second ODNP pair comprises an oligonucleotide sequence complementary to a nueleotide sequence of the complement of the target nucleic acid at a location 3' to the position in the complement corresponding to the defined position in the target. It further comprises the sequence of the second CRS of the second strand of the IRERS at a location 3' to, preferably at the 3' terminus of, the above oligonucleotide sequence.
  • the IRERS is selected to produce a 5' overhang upon digestion.
  • the set of two ODNP pairs are so spaced that the nueleotide to be identified in an extension and ligation fragment from such a set is within the 5' overhang resulting from digestion at the IRERS.
  • the first ODNP of the first ODNP pair and the second ODNP of the second ODNP pair each further contains one or more nucleotides that are complementary to a nueleotide sequence of the target nucleic acid or the complement thereof at the 3' terminus of the first or the second CRS. Such complementarity at the 3' termini of the ODNPs increases the extension and/or amplification efficiency from the ODNPs.
  • General techniques for designing sequence-specific primers are well known.
  • Primer Master ⁇ see, Proutski and Holmes, Primer Master: A new program for the design and analysis of PCR primers. Comput. Appl. Biosci. 12: 253-5, 1996) and OLIGO Primer Analysis Software from Molecular Biology Insights, Inc. (Cascade, CO, USA).
  • OLIGO Primer Analysis Software from Molecular Biology Insights, Inc. (Cascade, CO, USA).
  • suitably designed primers may be synthesized by techniques known in the art, or more conveniently, may be purchased from one of the many supply houses that will prepare oligonucleotide primers having a custom sequence.
  • Methods, kits and compositions of the present invention may involve or include ODNP that are hybridized to the target nucleic acid, where the ODNP facilitates the production and/or amplification of a defined nueleotide locus within the target nucleic acid.
  • the ODNP and target nucleic acid are thus preferably combined under base-pairing condition. Selection of suitable nucleic acid hybridization and/or amplification conditions are available in the art by, e.g., reference to the following laboratory research manuals: Sambrook et al., "Molecular Cloning” (Cold Spring Harbor Press, 1989) and Ausubel et al, "Short Protocols in Molecular Biology” (1999) (incorporated herein by reference in their entirety).
  • hybrids may vary conditions of hybridization to achieve desired degrees of selectivity of ODNP towards target sequence.
  • relatively stringent conditions may be employed to form the hybrids, such as e.g., low salt and/or high temperature conditions, such as from about 0.02 M to about 0.15 M salt at temperatures of from about 50°C to about 70°C.
  • selective conditions are relatively intolerant of large mismatches between the ODNP target nucleic acid.
  • hybridization of the ODNPs may be achieved under moderately stringent buffer conditions such as, for example, in 10 mM Tris, pH 8.3; 50 mM KC1; 1.5 mM MgCl 2 at 60°C which conditions permit the hybridization of ODNP comprising nueleotide mismatches with the target nucleic acid.
  • moderately stringent buffer conditions such as, for example, in 10 mM Tris, pH 8.3; 50 mM KC1; 1.5 mM MgCl 2 at 60°C which conditions permit the hybridization of ODNP comprising nueleotide mismatches with the target nucleic acid.
  • the design of alternative hybridization conditions is well within the expertise of the skilled artisan.
  • the ODNPs are extended with the target or the complement thereof as a template using various methodologies known in the art, such as the polymerase chain reaction (PCR) and modified ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • LCR modified ligase chain reaction
  • the hybridization and extension/amplification process will be described below for the situation where the target nucleic acid is single stranded. It is well within the skill of one of ordinary skill in the art to use an analogous process when the target nucleic acid is double stranded.
  • the first run of extension is for the first ODNP having a first CRS to incorporate the complement of the nueleotide of interest in the first extension product.
  • the second ODNP having a second CRS then hybridizes to and extends using the first extension product as a template and thereby incorporate the nueleotide of interest and the first CRS in a second extension product.
  • first ODNP then hybridizes to and extends using the second extension product as a template and thereby form, in combination with the second extension product, a double-stranded nucleic acid fragment. Because the first ODNP and the second ODNP of the ODNP pair are spaced in a distance of the same number of base pairs as that of the VRS, the double-stranded nucleic acid fragment resulting from the three runs of extensions contains a complete IRERS.
  • the first primer can hybridize to and extend using any of the target nucleic acid, the second extension product and the complement of the third extension product, as a template.
  • the second primer can hybridize to and extend using either the first extension product or the third extension product as a template.
  • the third extension product and the complement thereof are shorter than any of the target nucleic acid, the first extension product and the second extension product, they are the preferred templates for subsequent extension reactions from either the first or the second ODNPs. This is because the extension efficiency with a short fragment as a template is higher than that with a large fragment as a template. With the increase of the number of extension reactions, the double stranded fragment containing both the nueleotide to be identified and a complete IRERS accumulates quickly than other molecules in the reaction mixture. Such accumulation increases the sensitivity of subsequent characterization of the fragment after being digested with a RE that recognizes the complete IRERS.
  • the extension/amplification reaction can be carried out known in the art, including PCR methods.
  • U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159 all describe PCR methods.
  • PCR methods are also described in several books, e.g., Gelfand et al., "PCR Protocols: A Guide to Methods and Application” (1990); Burke (ed), “PCR: Essential Techniques”; McPherson et al. "PCR (Basic: From Background to Bench)”.
  • PCR Two ODNPs are prepared that are complementary to regions on opposite complementary strands of the target nucleic acid sequence.
  • An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase ⁇ e.g., Taq or Pfu polymerase). If the target nucleic acid sequence is present in a sample, the ODNPs will bind to the target and the polymerase will cause the ODNPs to be extended along the target nucleic acid sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended ODNPs will dissociate from the target to form reaction products, excess ODNPs will bind to the target and to the reaction product and the process is repeated.
  • a DNA polymerase e.g., Taq or Pfu polymerase
  • Exemplary PCR conditions according to the present invention may include, but are not limited to, the following: 100 ⁇ l PCR reactions comprise 100 ng target nucleic acid; 0.5 ⁇ M of each first ODNP and second ODNP; 10 mM Tris, pH 8.3; 50 mM KC1; 1.5 mM MgCl 2; 200 ⁇ M each dNTP; 4 units TaqTM DNA Polymerase (Boehringer Mannheim; Indianapolis, IN), and 880 ng TaqStartTM Antibody (Clontech, Palo Alto, CA).
  • Exemplary thermocycling conditions may be as follows: 94°C for 5 minutes initial denaturation; 45 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute; final extension at 72°C for 5 minutes.
  • Exemplary nucleic acid polymerases may include one of the thermostable DNA polymerases that are readily available in the art such as, e.g., TaqTM , VentTM or PFUTM. Depending on the particular application contemplated, it may be preferred to employ one of the nucleic acid polymerases having a defective 3' to 5' exonuclease activity.
  • LCR uses both a nucleic acid polymerase enzyme and a nucleic acid ligase enzyme to drive the reaction.
  • exemplary nucleic acid polymerases may include one of the thermostable DNA polymerases that are readily available in the art such as, e.g., TaqTM , VentTM or PFUTM.
  • Exemplary nucleic acid ligases may include T4 DNA ligase, or the thermostable Tsc or Pfu DNA ligases.
  • T4 DNA ligase or the thermostable Tsc or Pfu DNA ligases.
  • U.S. Patent No. 4,883,750 incorporated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding ODNP pairs to a target sequence.
  • Exemplary gap-LCR conditions may include, but are not limited to, the following: 50 ⁇ l LCR reactions comprise 500 ng DNA; a buffer containing 50 mM EPPS, pH 7.8, 30 mM MgCl 2 , 20 mM K + , 10 ⁇ M NAD, 1-10 ⁇ M gap filling nucleotides, 30 nM each oligonucleotide primer, 1 U Thermusflavus DNA polymerase, lacking 3'-»5' exonuclease activity (MBR, Milwaukee, WI), and 5000 U T. thermophilus DNA ligase (Abbott Laboratories). Cycling conditions may consist of a 30 s incubation at 85°C and a 30 s incubation at 60°C for 25 cycles and may be carried out in a standard PCR machine such as a Perkin Elmer 9600 thermocycler.
  • the first ODNP comprises an oligonucleotide sequence complementary to a nueleotide sequence of a target nucleic acid at a location 3' to a nueleotide of interest in the target nucleic acid and a first CRSD of a first strand of an IRERS.
  • the second ODNP comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target at a location 5' to the nueleotide of interest and a second CRS of the first strand of the IRERS.
  • a DNA polymerase and a DNA ligase the two ODNPs extend and ligate with each other and the resulting product incorporates a nueleotide complementary to the nueleotide of interest in the target.
  • Such a product is then annealed to a single-stranded oligonucleotide having a sequence complementary to the amplification and ligation product at least within the region from the 5' terminus of the first ODNP and 3' terminus of the second ODNP and a universal nueleotide at the position complementary to the nueleotide of interest.
  • FIG. 7 Another way to provide a double-stranded nucleic acid fragment containing a nueleotide to be identified at a defined location in a target nucleic acid and a complete IRERS is illustrated in Figure 7.
  • a primer pair is mixed with the target.
  • One primer (“the first ODNP”) comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target nucleic acid at a location 3' to the defined position in the target and a first CRS of a first stand of an IRERS
  • the other primer (“the second ODNP”) comprises an oligonucleotide sequence complementary to a nueleotide sequence of the target at a location 5' to the defined position and a second CRS of the first strand of the IRERS.
  • the two primers are then extended using the target as the template to incorporate the complement of the nueleotide to be identified (also referred to as "nueleotide of interest").
  • the extension products from the two primers are ligated and subsequently disassociated from the target.
  • the disassociated, ligated extension product is then annealed to another nucleic acid molecule that contains the sequence complementary to the ligated extension product in the region from the 5' terminus of the first ODNP to the 3' terminus of the second ODNP.
  • This nucleic acid molecule contains a universal nueleotide at a position corresponding to the complement of the nueleotide of interest in the ligated extension product.
  • Such annealing produces a double stranded nucleic acid containing a complete IRERS and the complement of the nueleotide of interest.
  • the primer pair is so spaced that the complement of the nueleotide of interest in the ligated extension product is within the VRS of the IRERS and the 5' overhang produced by digestion at the IRERS.
  • a number of other template dependent methodologies may be used either to amply target nucleic acids before combining the target nucleic acids with the ODNPs of the present invention.
  • such methodologies may be used, in combination of the ODNP pair or the set of two ODNP pairs described above, to produce a fragment containing a portion of a target nucleic acid with a defined nueleotide locus and a complete IRERS.
  • Qbeta Replicase described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880, incorporated herein by reference in its entirety, may alternatively be used with methods of the present invention.
  • RNA polymerase a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • Strand Displacement Amplification SDA
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS) (also referred to as transcription-mediated amplification, or TMA) (Kwoh et al, 1989; PCT Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR.
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR nucleic acid sequence based amplification
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and application onto minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • RNAs are reverse transcribed into DNA, and transcribed once again with a polymerase such as T7 or SP6.
  • a polymerase such as T7 or SP6.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a first template for a first ODNP, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a second template for a second ODNP, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template.
  • This ODNP is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the ODNPs and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification.
  • Methods, kits and compositions of the present invention typically involve or include one or more interrupted restriction endonucleases.
  • restriction endonuclease refers to the class of nucleases that bind to unique double stranded nucleic acid sequences and that generate a cleavage in the double stranded nucleic acid that results in either blunt, double stranded ends, or single stranded ends with either a 5' or a 3' overhang.
  • IRERS interrupted restriction endonuclease recognition sequence
  • first CRS also referred to as “Region A”
  • second CRS a restriction endonuclease recognition site
  • VRE variable recognition sequence
  • “Second CRS” (also referred to as “Region C”) is defined as that region of the IRERS that contains the constant (not variable) nucleotides of the IRERS that are located 3' of the VRE of the IRERS.
  • the "VRE” (also referred as “Region B”) is defined as the stretch of one or more variable nucleotides that are located between the first and second CRSs.
  • EcoN I refers to an exemplary RE that binds to, and cleaves, a unique double-stranded nucleic acid sequence shown below:
  • N designates an undefined nueleotide, that is, any one of the four nucleotides: A, T, G, and C.
  • the bottom and top strands are cleaved 6 bases in from the 3' -OH ends ("/" indicates the cleavage sites).
  • nucleic acid fragments are produced that include this EcoN I binding sequence, where the nueleotide to be identified is positioned at the 6 th nueleotide (in bold face, also referred to as "the central nueleotide") from the 5' end of the top strand.
  • restriction endonuclease that recognizes an interrupted restriction endonuclease recognition sequence and produces a 5' overhang containing one or more variable nucleotides upon digestion can be used in the present invention.
  • Exemplary restriction endonucleases suitable for use in the present invention includes, but is not limited to, BIP I, BsaJ I, BssK I, BstE II, Bsu36 I, Dde I, EcoN I, Fnu4H I, Hinf I, Mae III, PflF I, Sau96 I, ScrF I and Tthl 11 1.
  • Some of such enzymes are commercially available from numerous companies such as, e.g., New England Biolabs Inc.
  • Non-commercially available restriction enzymes may be isolated and/or purified based on the teaching available in the art.
  • a nucleic acid fragment containing a portion of target nucleic acid with a defined nueleotide locus and a complete IRERS is digested (or cleaved) by a RE that recognizes the IRERS.
  • Conditions for storage and use of restriction endonucleases used according to the present invention are readily available in the art, for example, by reference to one of the laboratory manuals such as Sambrook et al., supra and Ausubel et al., supra.
  • the number of units of RE added to a reaction may be calculated and adjusted according to the varying cleavage rates of nucleic acid substrates.
  • 1 unit of restriction endonuclease will digest 1 ⁇ g of substrate nucleic acid in a 50 ⁇ l reaction in 60 minutes. Generally, fragments ⁇ e.g., amplicons) may require more than 1 unit/ ⁇ g to be cleaved completely.
  • the restriction enzyme buffer is typically used at IX concentration in the reaction.
  • Some restriction endonucleases require bovine serum albumin (BSA) (usually used at a final concentration of 100 ⁇ g/ml for optimal activity). Restriction endonucleases that do not require BSA for optimal activity are not adversely affected if BSA is present in the reaction.
  • BSA bovine serum albumin
  • restriction enzymes are stable when stored at -20°C in the recommended storage buffer. Exposure to temperatures above -20°C should be minimized whenever possible. All restriction endonucleases should be kept on ice when not otherwise being stored in the freezer. Enzymes should always be the last component added to a reaction.
  • restriction endonucleases The recommended incubation temperature for most restriction endonucleases is about 37°C. Restriction endonucleases isolated from thermophilic bacteria require higher incubation temperatures, typically ranging from 50°C to 65°C. Incubation time may often be shortened if an excess of restriction endonuclease is added to the reaction. Longer incubation times are often used to allow a reaction to proceed to completion with fewer units of restriction endonuclease.
  • Methods, kits and compositions of the present invention may involve or include incorporation of fluorescence labeled nucleotides into an EcoN I digestion product as described above.
  • the incorporation of the labeled nucleotides facilitates the identification of a nueleotide of interest in a target nucleic acid.
  • an extension product or an amplicon using a target nucleic acid as a template and the ODNPs of the present invention as primers contains a complete IRERS.
  • the ODNPs are so designed in relation to the target nucleic acid that the nueleotide of interest is within both the VRS of the IRERS and a 5' overhang produced by digestion at the IRERS.
  • the 3' recessed termini corresponding , to the 5' overhang may be subsequently filled in with fluorescence labeled nucleotides.
  • the incorporation of fluorescence labeled nucleotides into a 3' recessed terminus of a digestion product may be catalyzed by a DNA polymerase in the presence of one or more fluorescent labeled deoxyribonucleoside triphosphates (dNTPs) or dideoxyribonucleoside triphosphates (ddNTPs).
  • dNTPs deoxyribonucleoside triphosphates
  • ddNTPs dideoxyribonucleoside triphosphates
  • alkaline phosphatase may be used to dephosphorylate all the existing non-labeled dNTPs from the extension/amplification reaction.
  • the incorporation of fluorescence labeled nucleotides is carried out using an RNA polymerase in the presence of one or more fluorescence labeled ribonucleoside triphosphates (rNTPs).
  • rNTPs fluorescence labeled ribonucleoside triphosphates
  • the ODNPs of the present invention are so designed in relation to a target nucleic acid that the nueleotide to be identified is within the VRS of an IRERS ⁇ e.g., EcoN I recognition site) and is the only nueleotide within a 5' overhang produced by digestion at the IRERS ⁇ e.g., the central nueleotide with the EcoN I recognition site).
  • an embodiment is particularly useful for identifying a nueleotide at a known SNP site.
  • an SNP is either of two possible nucleotides and there is no possibility of finding a third or fourth nueleotide identity at an SNP site.
  • digestion products having 3 ' recessed termini may be filled in using the two nucleotides each attached to a different fluorophore ⁇ e.g., BFL-ddATP and ROX-ddGTP).
  • the signals from the two fluorophores attached to the nucleotides after the nucleotides are incorporated into the digestion products can be distinguished using fluorescence polarization described in detail below. The presence of both signals indicates that the biological sample from which the target nucleic acid is isolated is heterozygous at the SNP site.
  • the digestion products may be divided into two portions: one portion filled in using a fluorophore linked to a first possible nueleotide, the other portion filled in using a fluorophore linked to a second possible nueleotide.
  • the fluorophore linked to the first possible nueleotide may or may not be the same as that linked to the second possible nueleotide.
  • the two possible nucleotides at an SNP site are complementary to each other, it may be necessary to separate digestion products from each other and then fill in one of the products using one or both possible nucleotides for the SNP site linked to fluorophores.
  • the separation of the digestion products allows the distinction between the incorporation of the nueleotide at the SNP site from that of its complement nueleotide, which is the other possible nueleotide at the SNP site.
  • the digestion of a nucleic acid fragment containing both the IRERS and the SNP by a restriction endonuclease that recognizes the IRERS produces two double-stranded oligonucleotides: one with a 5' overhang consisting of the nueleotide "A,” the other with a 5' overhang consisting of the nueleotide "T.”
  • the two types of 5' overhangs are present after digestion no matter whether the nueleotide at the SNP site is the nueleotide "A" or the nueleotide "T.”
  • both fluorescence labeled nueleotide "A” and fluorescence labeled nueleotide "T” will be incorporated in a fill-in reaction, thus the identity of the nueleotide at the SNP site is not determined.
  • the two digestion products are separated from each other and each product is filled in
  • the method of the present invention may be used to determine the identity of a nueleotide at a defined position in a target nucleic acid where the nueleotide can be any one of the four nucleotides ⁇ i.e., A, T or U, G and C). However, it may be necessary to separate digestion products from each other prior to a fill-in reaction.
  • the separation of digestion products from one another may be accomplished by any method suitable for separating small double-stranded oligonucleotides, including capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC).
  • CE capillary electrophoresis
  • HPLC high-performance liquid chromatography
  • the first ODNP is linked to, at its 5' terminus, a molecule that can be subsequently attached to a solid support ⁇ e.g., a biotin molecule) so the digestion product with a 5' overhang consisting of a nueleotide of interest is also linked to this molecule via the first ODNP.
  • This digestion product can be attached to a solid support ⁇ e.g., avidin- or streptavidin-coated beads) via this molecule and readily separable from the other digestion product ⁇ i.e., the product with a 5' overhang consisting of the complement nueleotide of the nueleotide of interest) in solution.
  • a solid support e.g., avidin- or streptavidin-coated beads
  • restriction endonucleases that produce 5' overhangs consisting of single variable nucleotides
  • other restriction endonucleases that produce 5' overhangs consisting of more than one nucleotides may also be used in the present application as long as the nueleotide to be identified is not identical, or complementary, to the other nucleotide(s) within the 5' overhangs.
  • the 3' recessed termini corresponding to the 5' overhangs may be filled in with unlabeled nucleoside triphosphate(s) at position(s) corresponding to those other than that of the nueleotide of interest, and with fluorescence labeled nucleoside triphosphate(s) at the position corresponding to that of the nueleotide of interest.
  • an amplification product using a target nucleic acid with a SNP at a defined position contains a recognition sequence for BssK I ⁇ i.e., /CCNGG where "/" indicates the cleavage site) and the SNP is the "N" of the BssK I recognition sequence and may be either an "A" or a "T,"
  • the two double strand digestion products may be first separated from each other and filled in with unlabeled cytidine triphosphate, unlabeled guanosine triphosphate, and fluorescence labeled adenosine triphosphate or deoxythymidine triphosphate.
  • Various fluorescent dyes or probes may be employed in the present invention.
  • Fluorescent dyes are identified and quantified most directly by their absorption and fluorescence emission wavelengths and intensities. Emission spectra (fluorescence and phosphorescence) are much more sensitive and specific than absorption spectra. Other photophysical characteristics (like fluorescence anisotropy) are less widely used.
  • the useful intensity parameters are quantum yield (QY) for fluorescence, and the molar extinction coefficient ( ⁇ ) for absorption. QY is a measure of the total photon emission over the entire fluorescence spectral profile and the value of ⁇ is specified at a given wavelength (usually the absorption maximum of the probe).
  • a narrow optical bandwidth ( ⁇ 25 nm) is usually used for fluorescence excitation (via absorption), whereas the fluorescence detection bandwidth is more variable, ranging from full spectrum for maximal sensitivity to narrow band (-20 nm) for maximal resolution.
  • Fluorescence intensity per probe molecule is proportional to the product of ⁇ and QY.
  • Commercially important and exemplary fluorochromes that are widely used are fluorescein, tetramethylrhodamine, lissamine, Texas Red and BODIPYs.
  • Fluorescent labels are now commonly used for the detection of small nucleic acid fragments that have been separated by CE and HPLC.
  • One group of labels that may be employed for this purpose are those based on near-infrared (near-IR) fluorescent dyes.
  • these types of tags In aqueous solution, these types of tags have a maximum absorption of light at >680 nm, followed by the emission of fluorescence at near-IR wavelengths (emission maximum, >700 nm).
  • emission maximum >700 nm.
  • One advantage of using this type of fluorescence for detection is that it occurs in a spectral region where there is relatively little absorption or emission due to other compounds that might be present in biological samples. This, plus the fact that most near IR probes can be excited with commercially available lasers, provides this approach with low background signals and limits of detection that extend into the attomole range.
  • fluorescence spectroscopy is the phenomenon of autofluorescence.
  • fluorochromes that possess significantly longer delay times to emission ⁇ see Fernandes for review). These fluorochromes are usually luminescent metal chelates that are attached at the 5'- end of an ODN probe or primer. Terbium deoxynucleoside triphosphates are available that allow the incorporation of time-resolved fluorochromes into "natural" nucleic acids. These probes have the advantage of the large Stokes shift, narrow emission bands and long lifetimes.
  • Time-resolved fluorescence spectroscopy is particularly useful in structural biology and is used to monitor molecular interactions and motions that occur in the picosecond-nanosecond time range. Time-resolved fluorescence spectroscopy is beginning to dominate the analysis of biomolecular structure and dynamics.
  • Deoxyribonucleoside analogs that may be incorporated into a small nucleic acid fragment of the present invention, to thereby afford an effective characterization means for the small nucleic acid, include but are not limited to: Fluorescein- 12-dUTP, Coumarin-5-dUTP, Tetramethylrhodamine-6-dUTP, Texas Red ® -5-dUTP, Napthofluorescein-5-dUTP, Fluorescein Chlorotriazinyl-4-dUTP, Pyrene-8-dUTP, Diethylaminocoumarin-5-dUTP, Cyanine 3-dUTP, Cyanine 5-dUTP, Coumarin-5-dCTP, Fluorescein- 12-dCTP, Tetramethylrhodamine-6-dCTP, Texas Red ® -5-dCTP, LissamineTM-5-dCTP, Napthofluorescein-5-dCTP, Fluorescein Chlorotriazinyl-4-dCTP, Py
  • Ribonucleoside analogs include but are not limited to: Fluorescein- 12- UTP, Coumarin-5-UTP, Tetramethylrhodamine-6-UTP, Texas Red ® -5-UTP, LissamineTM-5-UTP, Napthofluorescein-5-UTP, Fluorescein Chlorotriazinyl-4-UTP, Pyrene-8-UTP, Cyanine 3-UTP, Cyanine 5-UTP, Coumarin-5-CTP, Fluorescein- 12- CTP, Tetramethylrhodamine-6-CTP, Texas Red ® -5-CTP.
  • LissamineTM-5-CTP Napthofluorescein-5-CTP, Fluorescein Chlorotriazinyl-4-CTP, Pyrene-8-CTP, Cyanine 3-CTP, Cyanine 5-CTP, Coumarin-5-ATP, Fluorescein- 12- ATP, Tetramethyhhodamine-6-ATP, Texas Red ® -5-ATP, LissamineTM-5-ATP, Coumarin-5- GTP, Fluorescein-12-GTP, Tetramethylrhodamine-6-GTP, Texas Red ® -5-GTP, and LissamineTM-5-GTP.
  • Dideoxy analogs include but are not limited to: Fluorescein- 12-ddUTP, FAM-ddUTP, ROX-ddUTP, R6G-ddUTP, TAMRA-ddUTP, JOE-ddUTP, R110- ddUTP, Fluorescein-12-ddCTP, FAM-ddCTP, ROX-ddCTP, R6G-ddCTP, TAMRA- ddCTP, JOE-ddCTP, RllO-ddCTP, Fluorescein- 12-ddGTP, FAM-ddGTP, ROX- ddGTP, R6G-ddGTP, TAMRA-ddGTP, JOE-ddGTP, Rl lO-ddGTP, Fluorescein- 12- ddATP, FAM-ddATP, ROX-ddATP, R6G-ddATP, TAMRA-ddATP, JOE-ddATP, and RllO
  • Analogs can also be un-natural nucleoside analogs including, but not limited to, the following: 8-Bromo-2'-deoxyadenosine-TTP, 8-Oxo-2'-deoxyadenosine, Etheno-2'-deoxyadenosine-TTP, Etheno-2'-deoxyadenosine-TTP, N 6 -Methyl- 2'deoxyadenosine-TTP, 2,6-Diaminopurine-2'-deoxyriboside-TTP, 8-Bromo-2'- deoxyguanosine-TTP, 7-Deaza-2'-deoxyguanosine-TTP, 2'-Deoxyisoguanosine-TTP, - Oxo-2'-deoxyguanosine-TTP, O 6 -Methyl-2'-deoxyguanosine-TTP, S 6 -DNP-2'- deoxythioguanosine-TTP, 3-Nitropyrrole-2
  • FP is based on the property that when a fluorescent molecule is excited by plane-polarized light, it emits polarized fluorescent light into a fixed plane if the tagged-molecules do not significantly rotate between excitation and emission. If the molecule is small enough and rotates and tumbles in space, however, fluorescence polarization is not observed fully by the detector.
  • the fluorescence polarization of a molecule is proportional to the molecule's rotational relaxation time (usually the time it takes to rotate through an angle of 68.5°), which is related to properties of the solution such as the viscosity, temperature, and molecular volume of the analyte or biomolecule. Therefore, if the viscosity and temperature are held constant, fluorescence polarization is directly proportional to molecular volume, which, in turn, is directly proportional to molecular weight. Larger tagged molecules rotate and tumble slowly in space and, accordingly, fluorescence polarization values can be obtained. In contrast, smaller molecules rotate and tumble faster and fluorescence polarization cannot be measured.
  • the present invention uses fluorescence polarization to detect fluorescence labeled nucleotides upon their incorporation into double-stranded oligonucleotides ⁇ i.e., EcoN I digestion products) and thus determines the identity of the nueleotide of interest.
  • Fluorescence labeled nucleoside triphosphates are small and rotate rapidly in solution. Thus, absent incorporation into a small nucleic acid fragment, fluorescent-tagged-nucleoside triphosphates are undetectable by fluorescence polarization.
  • the fill-in reaction described above that incorporates a fluorescence labeled nucleoside triphosphate into a nucleic acid fragment increases by about 20-fold the molecular weight of the fluorophore. Because of the increase in the molecular weight, the fluorescence polarization of the oligonucleotide can be detected and measured.
  • polarizing fluorometers and more than 50 fluorescence polarization immunoassays are commercially available, many of which are routinely used in clinical laboratories for the measurement of therapeutics, metabolites, and drugs of abuse in biological fluids.
  • FPIAs fluorescence polarization immunoassays
  • Many polarizing fluorometers and FPIAs are capable of measuring samples in mitrotiter plates, thus the identity of nucleotides at defined position in target nucleic acids can be determined in parallel.
  • the method of the present invention may optionally comprises the use of one or more computer algorithms for analyzing the derived sequence information.
  • Algorithms of the present invention may be encompassed within software packages that convert a detection signal, such as a fluorescence value of a given small nucleic acid fragment, to a genotyping call.
  • a genotyping system for use with a fluorescence based detection system may comprise one or more components as follows: a peak identification algorithm which identifies peaks above a certain threshold of intensity (area under the curve), an algorithm that identifies and records the retention time of the peaks between certain time intervals ⁇ e.g., between 1.75 and 3 minutes in a 5 minute run), an algorithm that calculates the intensity of peaks by measuring the area under the curve, an algorithm that calculates the number of peaks between a certain time interval, an algorithm that calculates the ratio of each set of two peaks, and an algorithm that calculates the allele calling from the ratiometric values.
  • the software package and algorithms record the sample identification (sample ID), source, primer name and sequence, length of expected fragment, estimation of expected retention time, chromatography details, sample plate ID, sample well ID, date and time, number of peaks observed, observed retention times, and calculated allele call.
  • the algorithms will also download the data to existed databases and check for accuracy of recording.
  • the software package that converts the presence of a label on the fragment to a genotyping call is composed of the following: an algorithm that calculates the allele calling from the ratiometric values of fluorescence or fluorescence polarization.
  • the software package and algorithms record the sample identification (sample ID), source, primer name and sequence, mass to charge ratio of expected fragment, estimation of expected fluorescence ratios, instrument details, sample plate ID, sample well ID, date and time, and calculated allele call.
  • the algorithms will also download the data to existing databases and check for accuracy of recording.
  • the present invention provides methodology for identifying nucleotides (including identifying mutations and/or SNPs) at defined nueleotide loci within target nucleic acids.
  • Methods according to the present invention will find utility in a wide variety of applications, including but not limited to genetic analysis for hereditary diseases, tumor diagnosis, disease predisposition, forensics or paternity, crop cultivation and animal breeding, expression profiling of cell function and/or disease marker genes, and identification and/or characterization of infectious organisms that cause infectious diseases in plants or animals and/or that are related to food safety. Described below are certain exemplary applications of the present invention.
  • mRNAs are transcribed from single copy sequences.
  • Another property of cDNAs is that they represent a longer region of the genome because of the introns present in the chromosomal version of most genes. The representation varies from one gene to another but can be very significant as many genes cover more than 100 kb in genomic DNA, represented in a single cDNA.
  • molecular profiling is the use of probes from one species to find clones made from another species. Sequence divergence between the mRNAs of mouse and man permits specific cross-reassociation of long sequences, but except for the most highly conserved regions, prevents cross-hybridization of PCR primers.
  • transcription promoter sequences were designed into primers for cDNA synthesis and complex antisense cRNAs were generated by in vitro transcription with bacteriophage RNA polymerases.
  • the method of the present invention is useful for determining whether a particular cDNA molecule is present in cDNAs from a biological sample and further determine whether genetic variation(s) exist in the cDNA molecule.
  • DNA sequence variation offers a number of practical advantages over such conventional criteria as fingerprints, blood type, or physical characteristics.
  • DNA analysis readily permits the deduction of relatedness between individuals such as is required in paternity testing.
  • Genetic analysis has proven highly useful in bone marrow transplantation, where it is necessary to distinguish between closely related donor and recipient cells.
  • Two types of probes are now in use for DNA fingerprinting by DNA blots.
  • Polymorphic minisatellite DNA probes identify multiple DNA sequences, each present in variable forms in different individuals, thus generating patterns that are complex and highly variable between individuals.
  • VNTR probes identify single sequences in the genome, but these sequences may be present in up to 30 different forms in the human population as distinguished by the size of the identified fragments.
  • the probability that unrelated individuals will have identical hybridization patterns for multiple VNTR or minisatellite probes is very low. Much less tissue than that required for DNA blots, even single hairs, provides sufficient DNA for a PCR-based analysis of genetic markers. Also, partially degraded tissue may be used for analysis since only small DNA fragments are needed.
  • the methods of the present invention are useful in characterizing polymorphism of sample DNAs, therefore useful in forensic DNA analyses. For example, the analysis of 22 separate gene sequences in a sample, each one present in two different forms in the population, could generate 1010 different outcomes, permitting the unique identification of human individuals.
  • v-oncogenes Viral oncogenes
  • c-oncogenes cellular counterparts
  • the cellular oncogenes can, however, be activated by specific modifications such as point mutations (as in the c-K-ras oncogene in bladder carcinoma and in colorectal tumors).
  • point mutations as in the c-K-ras oncogene in bladder carcinoma and in colorectal tumors.
  • mutations may also inactivate the so-called “recessive oncogenes” and thereby leads to the formation of a tumor (as in the retinoblastoma (Rb) gene and the osteosarcoma). Accordingly, the present invention is useful in detecting or identifying mutations that activate oncogenes or inactivate recessive oncogenes, which in turn, cause cancers.
  • HLA histocompatibility antigens
  • the T-cells expressing a specific T-cell receptor which fits to the foreign antigen could therefore be eliminated from the T-cell pool.
  • Such analyses are possible by the identification of antigen-specific variable DNA sequences which are amplified by PCR and hence selectively increased.
  • the specific amplification reaction permits the single cell-specific identification of a specific T-cell receptor.
  • the present invention is useful for determining gene variations in T-cell receptor genes encoding variable, antigen-specific regions that are involved in the recognition of various foreign antigens.
  • Genome Diagnostics Four percent of all newborns are born with genetic defects; of the 3,500 hereditary diseases described which are caused by the modification of only a single gene, the primary molecular defects are only known for about 400 of them.
  • Hereditary diseases have long since been diagnosed by phenotypic analyses (anamneses, e.g., deficiency of blood: thalassemias), chromosome analyses (karyotype, e.g., mongolism: trisomy 21) or gene product analyses (modified proteins, e.g., phenylketonuria: deficiency of the phenylalanine hydroxylase enzyme resulting in enhanced levels of phenylpyruvic acid).
  • phenotypic analyses anamneses, e.g., deficiency of blood: thalassemias
  • chromosome analyses karyotype, e.g., mongolism: trisomy 21
  • gene product analyses modified proteins, e.g., phenylketonuria: deficiency of the phenylalanine hydroxylase enzyme resulting in enhanced levels of phenylpyruvic acid.
  • the additional use of nucleic acid detection methods considerably increases the range
  • the modification of just one of the two alleles is sufficient for disease (dominantly transmitted monogenic defects); in many cases, both alleles must be modified (recessively transmitted monogenic defects).
  • the outbreak of the disease is not only determined by the gene modification but also by factors such as eating habits (in the case of diabetes or arteriosclerosis) or the lifestyle (in the case of cancer). Very frequently, these diseases occur in advanced age. Diseases such as schizophrenia, manic depression or epilepsy should also be mentioned in this context; it is under investigation if the outbreak of the disease in these cases is dependent upon environmental factors as well as on the modification of several genes in different chromosome locations.
  • bladder carcinoma bladder carcinoma, colorectal tumors, sickle-cell anemia, thalassemias, al-antitrypsin deficiency, Lesch-Nyhan syndrome, cystic fibrosis/mucoviscidosis, Duchenne/Becker muscular dystrophy, Alzheimer's disease, X-chromosome-dependent mental deficiency, and Huntington's chorea, phenylketonuria, galactosemia, Wilson's disease, hemochromatosis, severe combined immunodeficiency, alpha- 1-antitrypsin deficiency, albinism, alkaptonuria, lysosomal storage diseases, Ehlers-Danlos syndrome, hemophilia, glucose-6-phosphate dehydrogenase disorder, agammaglobulimenia, diabetes insipidus, Wiskott-Aldrich syndrome, Fabry's disease, fragile X-syndrome
  • the present invention is useful to detect and/or measure genetic variations that are involved in infectious diseases, especially those in drug resistance genes.
  • the present invention facilitates the characterization and classification of organisms that cause infectious diseases and consequently the treatment of such diseases caused by these organisms.
  • This example discloses the use of fluorescence polarization (FP) and the EcoN I template-directed primer extension (fill-in) assay in assigning genotype.
  • Oligonucleotides used are listed in Table 1. Four synthetic 48-mers with identical sequence except for position 23 were prepared (CF508-48), the variant bases are shown as boldface letters. PCR and EcoN I primers and synthetic template oligonucleotides were obtained from Life Technologies (Grand Island, NY).
  • BFL, and BTR were obtained from NEN Life Science Products, Inc. (Boston, MA). Unlabeled ddNTPs were purchased from Pharmacia Biotech (Piscataway, NJ).
  • Fluorescence polarization value was calculated using the formula:
  • Ivv is the emission intensity measured when the excitation and emission polarizers are parallel and Ivh is the emission intensity measured when the emission and excitation polarizers are oriented perpendicular to each other.
  • the degree of polarization is expressed by the unit mP, or a 0.001 ratio between ⁇ Ivv — Ivh) and ⁇ Ivv + Ivh).
  • the average FP value and standard deviation of the negative control samples were determined for each set of experiment. The FP value of the test sample reactions was then compared to the average FP value of the control samples. If the net change is >40 mP (more than seven times the standard deviation of the controls), the test sample is scored as positive for the allele.
  • FP is a simple, highly sensitive and specific detection method in a fill-in reaction for measuring SNPs.
  • first set of experiments four synthetic oligonucleotide templates containing the four possible nucleotides at one particular site in the middle of otherwise identical sequence were used to establish the sensitivity and specificity of FP detection of labeled nucleoside incorporation.
  • second set of experiments several dyes were tested for their utility in the fill-in assay.
  • PCR products were used as templates in a dual-color FP assay to show that accurate genotyping data could be obtained for both alleles of a marker or mutation in a homogeneous assay.
  • FAM-ddA a FAM-ddC (mP) a FAM-ddG (mP) a FAM-ddU (mP) a
  • nucleoside complementary to the polymorphic base was incorporated and showed significant FP change, with net gains of FP of at least 50 mP, which is nine times standard deviation of the controls.
  • BFL-ddA BODIPY-fluorscein-ddA
  • TMR-ddC NNN ⁇ N'-tetramethyl- ⁇ -carboxyrhodamine
  • ROX-ddG b-carboxy- -rhodamine-ddG
  • BTR-ddU BODIPY-Texas Red-ddU
  • Marker D18S8 and the C282Y mutation in the human hereditary hemochromatosis ⁇ HFE gene implicated in hemochromatosis were used in FP-TDI assays designed to test for both alleles in the same reaction.
  • genomic DNA samples from 34 individuals were amplified and then cut with EcoN I and the 3 '-ends filled in, in the presence of BFL-ddA and ROX-ddG.
  • the FP values of the reaction mixtures were read at the BFL and ROX emission wavelengths, respectively, and the results are plotted and shown in Figure 2 as changes in fluorescence polarization. The results are plotted in mP units above the average polarization of the negative controls.
  • a change of 40 mP for a dye-terminator is scored as positive.
  • DNA samples from 34 individuals and 6 water blanks were used.
  • ⁇ Samples positive for the G allele but negative for the A allele (homozygous G);
  • A samples positive for the A allele but negative for the G allele (homozygous A);
  • ⁇ samples positive for both alleles (heterozygotes);
  • • negative controls;
  • O samples with failed PCR amplification.
  • the FP values cluster into four groups. In the upper left corner of the plot, the samples have high FP for ROX-ddG but low FP for BFL-ddA, signifying that they are of homozygous G genotype ( ⁇ ).
  • the heterozygous A/G samples ( ⁇ ) exhibit high FP values in both BFL-ddA and ROX-ddG and occupy the right upper corner of the plot.
  • the homozygous A/A samples (A) are found in the lower right corner, with low ROX-ddG but high BFL-ddA FP values.
  • the negative controls (•) and samples with failed PCR reactions (O) occupy the area near the origin with low FP values for both dyes.

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Abstract

L'invention concerne un procédé d'identification d'un nucléotide dans une position définie dans l'acide nucléique cible au moyen d'endonucléases de restriction et de polarisation de fluorescence. L'invention concerne également des composés, des compositions, et des nécessaires associés à ce procédé.
PCT/US2001/030743 2000-10-02 2001-10-01 Procedes d'identification de nucleotides dans des positions definies dans des acides nucleiques cibles au moyen de la polarisation de fluorescence WO2002040126A2 (fr)

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WO2003008642A2 (fr) * 2001-07-15 2003-01-30 Keck Graduate Institute Amplification de fragments d'acide nucleique au moyen d'agents de coupure
WO2003020984A2 (fr) * 2001-08-29 2003-03-13 Amersham Biosciences Corp Nucleotides marques sur le phosphate de terminaison et methodes d'utilisation
EP1427852A2 (fr) * 2001-08-29 2004-06-16 Amersham Biosciences Corp. Detection et amplification d'un nucleotide par polymerase
US7041812B2 (en) 2001-08-29 2006-05-09 Amersham Biosciences Corp Labeled nucleoside polyphosphates
US7223541B2 (en) 2001-08-29 2007-05-29 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides and methods of use
US7256019B2 (en) 2001-08-29 2007-08-14 Ge Healthcare Bio-Sciences Corp. Terminal phosphate blocked nucleoside polyphosphates
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EP1427852A4 (fr) * 2001-08-29 2005-12-28 Amersham Biosciences Corp Detection et amplification d'un nucleotide par polymerase
WO2003020984A2 (fr) * 2001-08-29 2003-03-13 Amersham Biosciences Corp Nucleotides marques sur le phosphate de terminaison et methodes d'utilisation
US7041812B2 (en) 2001-08-29 2006-05-09 Amersham Biosciences Corp Labeled nucleoside polyphosphates
US7052839B2 (en) 2001-08-29 2006-05-30 Amersham Biosciences Corp Terminal-phosphate-labeled nucleotides and methods of use
US7223541B2 (en) 2001-08-29 2007-05-29 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides and methods of use
US7256019B2 (en) 2001-08-29 2007-08-14 Ge Healthcare Bio-Sciences Corp. Terminal phosphate blocked nucleoside polyphosphates
US7452698B2 (en) 2001-08-29 2008-11-18 Ge Healthcare Bio-Sciences Corp. Terminal phosphate blocked nucleoside polyphosphates
US7560254B2 (en) 2001-08-29 2009-07-14 Ge Healthcare Bio-Sciences Corp. Allele specific primer extension

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WO2002046447A2 (fr) 2002-06-13
AU3922802A (en) 2002-05-27
AU4146002A (en) 2002-06-18
WO2002029006A3 (fr) 2002-08-29
WO2002046447A3 (fr) 2003-12-24
AU1183902A (en) 2002-04-15
AU2001296491A1 (en) 2002-04-15
WO2002040126A3 (fr) 2006-04-06
WO2002028501A1 (fr) 2002-04-11
WO2002029006A2 (fr) 2002-04-11

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