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WO2000043543A1 - Detection des differences entre polynucleotides - Google Patents

Detection des differences entre polynucleotides Download PDF

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Publication number
WO2000043543A1
WO2000043543A1 PCT/US1999/029222 US9929222W WO0043543A1 WO 2000043543 A1 WO2000043543 A1 WO 2000043543A1 US 9929222 W US9929222 W US 9929222W WO 0043543 A1 WO0043543 A1 WO 0043543A1
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Prior art keywords
nucleic acid
complex
tailed
target
sequence
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English (en)
Inventor
Nurith Kurn
Yen Ping Liu
Alla Lishanski
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Dade Behring Inc
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Dade Behring Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids Hybridization is based on complementary base pairing When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules
  • ssDNA single stranded deoxy ⁇ bonucleic acid
  • RNA ⁇ bonucleic acid
  • Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria molds, fungi and viruses Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis
  • One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours After the above time period, the solid support is washed several times at a controlled temperature to remove unhyb ⁇ dized probe The support is then dried and the hybridized material is detected by autoradiography or by spectromet ⁇ c methods
  • PCR polymerase chain reaction
  • a displacement polynucleotide assay method and polynucleotide complex reagent therefor is discussed in U S Patent No 4, 766,062 (Diamond, et al.,).
  • a strand displacement assay and complex useful therefor is discussed in PCT application WO 94/06937 (Eadie, et al.,).
  • One method in accordance with the present invention is directed to detecting the presence of a difference between two related nucleic acid sequences.
  • a complex is formed comprising both of the nucleic acid sequences in double stranded form.
  • the complex comprises at least one pair of non- complementary strands and each of the non-complementary strands within the complex has a label.
  • the complex is subjected to conditions wherein, if a difference between the two related nucleic acid sequences is present, strand exchange in the complex ceases and wherein, if no difference between the two related nucleic acid sequences is present, strand exchange in the complex continues until complete strand exchange occurs.
  • a first signal is detected from the association of the labels as part of the complex.
  • the association of the labels is related to the presence of the difference.
  • a complex is also formed comprising the nucleic acid sequence suspected of comprising a difference in double stranded form and a predetermined amount of a non-relevant reference polynucleotide in double stranded form.
  • the complex comprises at least one pair of non- complementary strands and each of the non-complementary strands within the complex has a label.
  • a second signal from the association of the labels as part of the above complex is detected.
  • a ratio of the first signal to the second signal is determined and related to the presence of the difference.
  • a tailed target partial duplex A' is formed from the target sequence and comprises a duplex of two nucleic acid strands of the target sequence, a label and at one end of the duplex, two non-complementary oligonucleotides, one linked to each of the strands A combination is provided comprising the tailed target partial duplex A' and a tailed first reference partial duplex B' lacking the mutation and having a label as a part thereof
  • the tailed reference partial duplex B' is comprised of two nucleic acid strands Each of the strands is complementary, respectively, to a strand in the tailed target partial duplex A but for the possible presence of a mutation
  • the labels are present in non-complementary strands of the tailed target and tailed reference partial duplexes, respectively
  • the combination is subjected to conditions wherein, if a mutation is present strand exchange in the complex ceases and
  • Another aspect of the present invention is a method of detecting a mutation within a target nucleic acid sequence
  • the target sequence is amplified by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA
  • One of the primers P1 and P2 comprises a label
  • the primer P1 is comprised of a 3'-end portion Pa that can hybridize with the target sequence and 5'-end portion B1 that cannot hybridize with the target sequence
  • a primer P3 is extended by chain extension along one strand of amplicon AA to produce a tailed target partial duplex
  • A' Primer P3 is comprised of the 3'-end portion Pa and a 5'-end portion A1 that cannot hybridize to the target sequence or its complement
  • a first reference nucleic acid sequence is amplified using the primer P2 and the primer P3, by polymerase chain reaction to produce amplicon BB
  • the first reference sequence is identical to the target sequence but lacks a possible mutation
  • Primer P2 comprises a label when
  • the tailed target partial duplex A' is allowed to bind to the tailed first reference partial duplex B'
  • a first signal is detected from the labels as a result of the formation of a complex between the tailed partial duplexes, the formation thereof being directly related to the presence of the mutation.
  • a second reference nucleic acid sequence is amplified, using a set of primers, by polymerase chain reaction to produce amplicon CC
  • the reference sequence is non-relevant to the target sequence
  • the primers comprise priming sequences for the second reference and respectively, the 5'-end portion B1 and the 5'-end portion A1 wherein one of the primers comprises a label.
  • Primer P1 is extended by chain extension along one strand of amplicon CC to produce a tailed reference partial duplex C.
  • the tailed target partial duplex A' is allowed to bind to the tailed reference partial duplex C
  • a second signal is detected from the labels as a result of the formation of a complex comprising a four-stranded structure between the tailed partial duplexes above
  • a ratio of the first signal to the second signal is determined and related to the presence of the mutation.
  • Fig. 1A is a schematic diagram of an embodiment wherein a mutation is present in the target nucleic acid sequence and branch migration is stopped.
  • Fig B is a schematic diagram showing complete strand exchange in the absence of a mutation in the target nucleic acid sequence, there being no difference between target and reference nucleic acid sequences
  • Fig 2A is a schematic diagram of an embodiment wherein a mutation is present in the target nucleic acid sequence and branch migration is stopped
  • Fig 2B is a schematic diagram showing complete strand exchange in the absence of a mutation in the target nucleic acid sequence, there being no difference between target and reference nucleic acid sequences
  • Fig 3 is a schematic diagram of an embodiment wherein a mutation is present in the target nucleic acid sequence and branch migration is stopped
  • Fig 4 is a schematic diagram of an embodiment in accordance with the present invention wherein a mutation is present in the target nucleic acid sequence and a non-relevant nucleic acid sequence is employed in a branch migration, which is stopped because of the differences between the target nucleic acid sequence and the non-relevant nucleic acid sequence
  • Fig 5 is a depiction of the M tuberculosis (tb) rpoB gene sequence
  • the present invention provides an improvement in the method of Lishanski, et al
  • the method is universal and permits detection of any difference in two related nucleic acid sequences whether or not such difference is known Such differences include any mutation including single base substitution, deletion or insertion within a sequence that can be defined by a pair of primers for conducting the polymerase chain reaction
  • the method may be homogeneous or heterogeneous and non-radioactive
  • the present method is fast, provides a normalized result and is amenable to automation It is ideally suited for rapid mutation pre-screenmg
  • the invention also has application in the area of amplification by polymerase chain reaction
  • the present invention permits PCR and subsequent steps such as detection of the PCR products, to be conducted without the need for additional probes in a single container without a separation step
  • the present method involves formation of a four-strand DNA structure or complex from DNA
  • the formation involves producing two partial duplexes by amplification by using three different primers in the polymerase chain reaction and allowing the amplified products to anneal
  • the complex dissociates into normal duplex structures by strand exchange by means of branch migration when the hybridized portions of each partial duplex are identical
  • the complex does not dissociate and can be detected as an indication of the presence of a difference between the nucleic acids
  • a particularly attractive feature of the present invention is that the reactions may be carried out simultaneously in the same medium without a separation step
  • the normalization method is based on formation of stable four stranded polynucleotide structures when amplification product from test polynucleotide is mixed with similarly produced products of amplification of a non- relevant reference sequence
  • Nucleic acid a compound or composition that is a polymeric nucleotide or polynucleotide
  • the nucleic acids include both nucleic acids and fragments thereof from any source in purified or unpu ⁇ fied form including DNA (dsDNA and ssDNA) and RNA, including t-RNA, m-RNA, r-RNA, mitochondnal DNA and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomes of biological material such as microorganisms, e g bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals, humans, and the like
  • the nucleic acid can be only a minor fraction of a complex mixture such as a biological sample
  • the nucleic acid can be obtained from a biological sample by procedures well known in the art Also included are genes, such as hemoglobin gene for sickle-cell anemia cystic fibrosis gene, onc
  • Sample the material suspected of containing the nucleic acid
  • biological fluids such as blood, serum, plasma, sputum, lymphatic fluid, semen vaginal mucus, feces urine, spinal fluid and the like
  • biological tissue such as hair and skin and so forth
  • Other samples include cell cultures and the like, plants food forensic samples such as paper fabrics and scrapings, water, sewage, medicinals, etc
  • the sample may be pretreated with reagents to liquefy the sample and release the nucleic acids from binding substances
  • reagents to liquefy the sample and release the nucleic acids from binding substances
  • Amplification of nucleic acids any method that results in the formation of one or more copies of a nucleic acid (exponential amplification)
  • PCR polymerase chain reaction
  • This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing and primer extension by thermophilic template dependent polynucleotide polymerase, resulting in the exponential increase in copies of the desired sequence of the nucleic acid flanked by the primers
  • the two different PCR primers are designed to anneal to opposite strands of the DNA at positions that allow the polymerase catalyzed extension product of one primer to serve as a template strand for the other, leading to the accumulation of a discrete double stranded fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers
  • Primer length can vary from about 10 to 50
  • Chain extension of nucleic acids extension of the 3'-end of a polynucleotide in which additional nucleotides or bases are appended
  • Chain extension relevant to the present invention is template dependent that is, the appended nucleotides are determined by the sequence of a template nucleic acid to which the extending chain is hybridized
  • the chain extension product sequence that is produced is complementary to the template sequence
  • chain extension is enzyme catalyzed preferably, in the present invention, by a thermophilic DNA polymerase
  • Target nucleic acid sequence a sequence of nucleotides to be studied either for the presence of a difference from a related sequence or for the determination of its presence or absence
  • the target nucleic acid sequence may be double stranded or single stranded
  • the method of the present invention produces a nucleic acid duplex comprising the single stranded target nucleic acid sequence
  • the target sequence usually exists within a portion or all of a nucleic acid, the identity of which is known to an extent sufficient to allow preparation of various primers necessary for introducing one or more priming sites flanking the target sequence or conducting an amplification of the target sequence or a chain extension of the products of such amplification in accordance with the present invention Accordingly other than for the sites to which the primers bind, the identity of the target nucleic acid sequence may or may not be known In general, in PCR, primers hybridize to, and are extended along (chain extended), at least the target sequence, and thus, the target sequence acts as a template
  • the target sequence usually contains from about 30 to 20,000 or more nucleotides, more frequently, 100 to 10 000 nucleotides, preferably, 50 to 1 ,000 nucleotides
  • the target nucleic acid sequence is generally a fraction of a larger molecule or it may be substantially the entire molecule The minimum number of nucleotides in the target sequence is selected to assure that a determination of a difference between two
  • Reference nucleic acid sequence a nucleic acid sequence that is related to the target nucleic acid in that the two sequences are identical except for the presence of a difference, such as a mutation Where a mutation is to be detected, the reference nucleic acid sequence usually contains the normal or "wild type" sequence. In certain situations the reference nucleic acid sequence may be part of the sample as, for example, in samples from tumors, the identification of partially mutated microorganisms, or identification of heterozygous carriers of a mutation.
  • both the reference and the target nucleic acid sequences are subjected to similar or the same amplification conditions
  • the identity of the reference nucleic acid sequence need be known only to an extent sufficient to allow preparation of various primers necessary for introducing one or more priming sites flanking the reference sequence or conducting an amplification of the target sequence or a chain extension of the products of such amplification in accordance with the present invention. Accordingly, other than for the sites to which the primers bind, the identity of the reference nucleic acid sequence may or may not be known.
  • the reference nucleic acid sequence may be a reagent employed in the methods in accordance with the present invention This is particularly the situation where the present method is used in PCR amplification for detection of a target nucleic acid sequence. Depending on the method of preparation of this reagent it may or may not be necessary to know the identity of the reference nucleic acid.
  • the reference nucleic acid reagent may be obtained from a natural source or prepared by known methods such as those described below in the definition of oligonucleotides.
  • Non-relevant reference nucleic acid sequence a sequence of nucleotides that differs from the reference nucleic acid sequence, e.g , wild type sequence, in that there is no correspondence between the non-relevant sequence and the target nucleic acid.
  • the non-relevant reference nucleic acid reagent may be obtained from a natural source or prepared by known methods such as those described below in the definition of oligonucleotides
  • the non-relevant reference nucleic acid sequence may be a nucleic acid sequence from another organism or a different gene sequence for the same organism
  • Holliday junction the branch point in a four way junction in a complex of two identical nucleic acid sequences and their complementary sequences The junction is capable of undergoing branch migration resulting in dissociation into two double stranded sequences where sequence identity and complementarity extend to the ends of the strands
  • Complex a complex of four nucleic acid strands containing a Holliday junction, which is inhibited from dissociation into two double stranded sequences because of a difference in the sequences and their complements Accordingly, the complex is quadramolecular
  • nucleic acid sequences ⁇ two nucleic acid sequences are related when they contain at least 15 nucleotides at each end that are identical but have different lengths or have intervening sequences that differ by at least one nucleotide Frequently related nucleic acid sequences differ from each other by a single nucleotide Such difference is referred to herein as the "difference between two related nucleic acid sequences " A difference can be produced by the substitution, deletion or insertion of any single nucleotide or a series of nucleotides within a sequence
  • Mutation - a change in the sequence of nucleotides of a normally conserved nucleic acid sequence resulting in the formation of a mutant as differentiated from the normal (unaltered) or wild type sequence
  • Mutations can generally be divided into two general classes namely base-pair substitutions and frameshift mutations The latter entail the insertion or deletion of one to several nucleotide pairs
  • a difference of one nucleotide can be significant as to phenotypic normality or abnormality as in the case of, for example, sickle cell anemia
  • Partial duplex - a fully complementary double stranded nucleic acid sequence wherein one end thereof has non-complementary oligonucleotide sequences, one linked to each strand of the double stranded molecule, each non- complementary sequence having 8 to 60, preferably, 10 to 50, more preferably, 15 to 40, nucleotides.
  • the partial duplex is said to be "tailed” because each strand of the duplex has a single
  • Duplex a double stranded nucleic acid sequence wherein all of the nucleotides therein are complementary.
  • Oligonucleotide - a single stranded polynucleotide, usually a synthetic polynucleotide.
  • the oligonucleotide(s) are usually comprised of a sequence of 10 to 100 nucleotides, preferably, 20 to 80 nucleotides, and more preferably, 30 to 60 nucleotides in length.
  • oligonucleotide utilized in the present invention.
  • Such oligonucleotide can be obtained by biological synthesis or by chemical synthesis.
  • chemical synthesis will frequently be more economical as compared to the biological synthesis.
  • chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during the synthesis step.
  • chemical synthesis is very flexible in the choice of length and region of the target polynucleotide binding sequence.
  • the oligonucleotide can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers.
  • oligonucleotide synthesis examples include phosphotriester and phosphodiester methods (Narang, ET aL (1979) Meth. Enzvmol 68: 90) and synthesis on a support (Beaucage, et a], (1981 ) Tetrahedron Letters 22: 1859- 1862) as well as phosphoramidate technique Caruthers, M H et al_, "Methods in Enzymology," Vol 154 pp 287-314 (1988) and others described in “Synthesis and Applications of DNA and RNA " S A Narang editor, Academic Press, New York, 1987, and the references contained therein Oligonucleotide pr ⁇ mer(s) -- an oligonucleotide that is usually employed in a chain extension on a polynucleotide template such as in, for example, an amplification of a nucleic acid
  • the oligonucleotide primer is usually a synthetic oligonucleotide that is
  • Nucleoside t ⁇ phosphates - nucleosides having a 5'-t ⁇ phosphate substituent
  • the nucleosides are pentose sugar derivatives of nitrogenous bases of either pu ⁇ ne or pyrimidine derivation, covalently bonded to the 1 '-carbon of the pentose sugar which is usually a deoxy ⁇ bose or a ⁇ bose
  • the pu ⁇ ne bases comprise aden ⁇ ne(A) guanine (G) inosine (I), and derivatives and analogs thereof
  • the pyrimidine bases comprise cytosine (C), thymine (T), uracil (U), and derivatives and analogs thereof
  • Nucleoside t ⁇ phosphates include deoxy ⁇ bonucleoside t ⁇ phosphates such as the four common t ⁇ phosphates dATP, dCTP, dGTP and dTTP and ⁇ bonucleoside t ⁇ phosphates such as the four
  • Modified nucleotide - is the unit in a nucleic acid polymer that results from the incorporation of a modified nucleoside t ⁇ phosphate during an amplification reaction and therefore becomes part of the nucleic acid polymer
  • Nucleoside -- is a base-sugar combination or a nucleotide lacking a phosphate moiety
  • the nucleotide polymerase is a template dependent polynucleotide polymerase and utilizes nucleoside t ⁇ phosphates as building blocks for extending the 3'-end of a polynucleotide to provide a sequence complementary with the polynucleotide template
  • the catalysts are enzymes, such as DNA polymerases, for example, prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase T7 DNA polymerase, Klenow fragment, and reverse transcriptase and are preferably thermally stable DNA polymerases such as Vent DNA polymerase, VentR DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and the like, derived from any source such as cells, bacteria, such as E. coli. plants, animals, virus, thermophilic
  • Hybridization hybridizing
  • binding-in the context of nucleotide sequences these terms are used interchangeably herein
  • the ability of two nucleotide sequences to hybridize with each other is based on the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs
  • the more nucleotides in a given sequence that are complementary to another sequence, the more stringent the conditions can be for hybridization and the more specific will be the binding of the two sequences Increased stringency is achieved by elevating the temperature, increasing the ratio of cosolvents, lowering the salt concentration,
  • Complementary-Two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3' -end of each sequence binds to the 5'-end of the other sequence and each A, T(U), G, and C of one sequence is then aligned with a T(U), A, C, and G, re- spectively, of the other sequence
  • Copy - means a sequence that is a direct identical copy of a single stranded polynucleotide sequence as differentiated from a sequence that is complementary to the sequence of such single stranded polynucleotide
  • Conditions for extending a primer -- includes a nucleotide polymerase, nucleoside triphosphates or analogs thereof capable of acting as substrates for the polymerase and other materials and conditions required for enzyme activity such as a divalent metal ion (usually magnesium), pH, ionic strength, organic solvent (such as formamide) and the like
  • gand and receptor members of an immunological pair such as antigen-antibody, or may be operator-repressor, nuclease-nucleotide biotin-avidm hormone-hormone receptor, IgG-protein A, DNA-DNA, DNA-RNA and the like
  • Receptor any compound for which a receptor naturally exists or can be prepared Receptor ("ant ⁇ l ⁇ gand")-any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e g , epitopic or determinant site
  • Illustrative receptors include naturally occurring and synthetic receptors, e g thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids repressors oligonucleotides, protein A, complement component C1 q or DNA binding proteins and the like
  • the small organic molecule can provide a means for attachment of a nucleotide sequence to a label or to a support
  • the support can be hydrophi c or capable of being rendered hydrophilic and includes inorganic powders such as silica magnesium sulfate, and alumina, natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e g , filter paper, chromatographic paper, etc , synthetic or modified naturally occurring polymers, such as nitrocellulose cellulose acetate poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate poly(ethylene terephthalate), nylon, poly(v ⁇ nyl butyrate), etc , either used by themselves or in conjunction with other materials, glass available as Bioglass, ceramics, metals, and the like Natural or synthetic assemblies such as liposomes phospho pid vesicles, and cells can also be employed
  • Binding of sbp members to a support or surface may be accomplished by well-known techniques commonly available in the literature See, for example, "Immobilized Enzymes.” Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970).
  • the surface can have any one of a number of shapes, such as strip : rod, particle, including bead, and the like.
  • Labels include reporter molecules that can be detected directly by virtue of generating a signal, and specific binding pair members that may be detected indirectly by subsequent binding to a cognate that contains a reporter molecule such as oligonucleotide sequences that can serve to bind a complementary sequence or a specific DNA binding protein; organic molecules such as biotin or digoxigenin that can bind respectively to streptavidin and antidigoxin antibodies, respectively; polypeptides; polysaccharides; and the like. In general, any reporter molecule that is detectable can be used.
  • the reporter molecule can be isotopic or nonisotopic, usually non- isotopic, and can be a catalyst, such as an enzyme, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme substrate, radioactive group, a particle such as latex or carbon particle, metal sol, crystallite, liposome, cell, etc., which may or may not be further labeled with a dye, catalyst or other detectable group, and the like.
  • the reporter group can be a fluorescent group such as fluorescein, a chemiluminescent group such as luminol, a terbium chelator such as N- (hydroxyethyl) ethylenediaminetriacetic acid that is capable of detection by delayed fluorescence, and the like.
  • the label is a member of a signal producing system and can generate a detectable signal either alone or together with other members of the signal producing system.
  • a reporter molecule can serve as a label and can be bound directly to a nucleotide sequence.
  • the reporter molecule can bind to a nucleotide sequence by being bound to an sbp member complementary to an sbp member that comprises a label bound to a nucleotide sequence. Examples of particular labels or reporter molecules and their detection can be found in U.S. Patent Application Serial No. 07/555,323 filed July 19, 1990, the relevant disclosure of which is incorporated herein by reference.
  • the signal producing system may have one or more components, at least one component being the label
  • the signal producing system generates a signal that relates to the presence of a difference between the target polynucleotide sequence and the reference polynucleotide sequence
  • the signal producing system includes all of the reagents required to produce a measurable signal
  • the reporter molecule is normally bound to an sbp member complementary to an sbp member that is bound to or part of a nucleotide sequence
  • Other components of the signal producing system can include substrates, enhancers, activators, chemilummescent compounds cofactors, inhibitors, scavengers, metal ions, specific binding substances required for binding of signal generating substances, coenzymes, substances that react with enzymic products, enzymes and catalysts, and the like
  • the signal producing system provides a signal detectable by external means, such as by use of electromagnetic radiation, electrochemical detection, desirably by spectr
  • buffers will normally be present in the assay medium, as well as stabilizers for the assay medium and the assay components Frequently, in addition to these additives, proteins may be included, such as albumins, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran sulfate, surfactants, particularly non-ionic surfactants, binding enhancers, e g , polyalkylene glycols, or the like
  • one aspect of the present invention concerns a method for detecting the presence of a difference between two related nucleic acid sequences
  • a stable quadramolecular complex is formed comprising both of the nucleic acid sequences in double stranded form
  • the complex comprises a Holliday junction
  • Both members of at least one pair of non- complementary strands within the complex have labels
  • a first signal is obtained from the association of the labels as part of the complex as an indication of the presence of the difference between the two related sequences
  • a complex is also formed comprising the nucleic acid sequence suspected of comprising a difference in double stranded form and a predetermined amount of a non-relevant reference polynucleotide in double stranded form
  • the complex comprises at least one pair of non-complementary strands and each of the non-complementary strands within the complex has a label
  • a second signal from the association of the labels as part of the above complex is detected A ratio of the first
  • quadramolecular complex C comprises partial duplex A and partial duplex B Partial duplexes A' and B' are related in that their hybridized portions are identical except for mutation M in partial duplex A Additionally, partial duplex A' has a label L1 , which may or may not differ from label L2 in partial duplex B' Oligonucleotide tail A1 of partial duplex A' is hybridized to corresponding oligonucleotide tail B2 of partial duplex B' and, similarly, oligonucleotide tail A2 of partial duplex A' is hybridized to oligonucleotide tail B1 of partial duplex B' Accordingly complex C is quadramolecular and contains a four way junction H Because oligonucleotide tails A1 and B1 are
  • Fig. 2A concerns a mutation within a target nucleic acid sequence A that contains mutation M
  • a tailed target partial duplex A' is formed from the target sequence and is comprised of a duplex of the target sequence, a label L1 and at one end of the duplex, two non-complementary oligonucleotides A1 and A2, one linked to each strand of duplex A'
  • Oligonucleotides A1 and A2 have from 8 to 60 nucleotides, preferably, 15 to 30 nucleotides
  • the tailed target partial duplex is provided in combination with a labeled tailed reference partial duplex B' lacking mutation M.
  • the tailed reference partial duplex B' is comprised of two nucleic acid strands that are complementary to the strands in A' but for mutation M Accordingly, one terminus of the tailed reference partial duplex B' has, as the end part of each strand, a sequence of nucleotides B1 and B2, respectively, that are complementary to A2 and A1 , respectively, of A' and are not complementary to each other. Labels L1 and L2 are present in non-complementary strands of the tailed target and tailed reference partial duplexes A' and B', respectively, where L1 and L2 may be the same or different
  • a complex C is formed as described above for Fig. 1A.
  • Oligonucleotide tail A1 of A is hybridized to corresponding oligonucleotide tail B2 of B' and, similarly, oligonucleotide tail A2 of A' is hybridized to oligonucleotide tail B1 of B'. Because oligonucleotide tails A1 and B1 are different, branch migration can only proceed away form these tails and then only until mutation M is reached, at which point branch migration stops Thus, when a mutation is present, complex C is stable and can be detected by determining whether both labels L1 and L2 have become associated. The association of the labels indicates the presence of complex C. The formation of complex C is directly related to the presence of the mutation.
  • Fig 3 depicts by way of example and not limitation the production of tailed target partial duplex A' from target nucleic acid duplex A having mutation M and the production of tailed reference partial duplex B from reference nucleic acid duplex B
  • PCR polymerase chain reaction
  • the amplification may be carried out separately from that of the reference nucleic acid sequence or in the presence of the reference nucleic acid sequence
  • the amplification of the target nucleic acid sequence should be carried out separately from that of the reference nucleic acid sequence
  • Primer P2 contains a label L1 and primer P1 is comprised of a 3'-end portion Pa that can hybridize with the target sequence and 5 -end portion B1 that
  • the above amplification is carried out by PCR utilizing temperature cycling to achieve denaturation of duplexes, oligonucleotide primer annealing, and primer extension by thermophilic template dependent nucleotide polymerase
  • the medium is cycled between two to three temperatures
  • the temperatures for the present method for the amplification by PCR generally range from about 50°C to about 100°C, more usually, from about 50°C to about 95°C Relatively low temperatures of from about 50°C to about 80°C are employed for the hybridization steps, while denaturation is carried out at a temperature of from about 80°C to about 100°C and extension is carried out at a temperature of from about 70°C to about 80°C, usually about 72°C to about 74°C
  • the amplification is conducted for a time sufficient to achieve a desired number of copies for an accurate determination of whether or not two related nucleic acids have a difference Generally, the time period for conducting the method is from about 10 seconds to about 10 minutes
  • the medium is subjected to multiple temperature cycles of heating at about 90°C to about 100°C for about 10 seconds to about 3 minutes and cooling to about 70°C to about 80°C for a period of about 10 seconds to about 3 minutes
  • a chain extension of primer P3 along the labeled strand of amplicon AA occurs to produce tailed target partial duplex
  • Primer P3 is comprised of a 3'-end portion Pa which is identical to Pa of primer P1 and which binds to the labeled strand of AA P3 has 5'-end portion A1 that is not complementary to amplicon AA
  • the chain extension occurs in the presence of a nucleotide polymerase and nucleoside triphosphates under appropriate temperature conditions to produce the complementary strand of the labeled strand
  • copies thereof may be produced, these are not shown in Fig 3 for purposes of simplicity It has been found that such copies do not interfere with the branch migration procedures described herein To avoid production of such copies
  • the medium is subjected to heating at about 90°C to about 100°C for a period of about 10 seconds to about 3 minutes, cooling to about 50°C to about 65°C for a period of about 10 seconds to about 2 minutes and heating to about 70°C to about 80°C for a period of about 30 seconds to about 5 minutes
  • reference nucleic acid sequence B is amplified by PCR preferably in a separate medium
  • Primer P2 and primer P3 are employed in a polymerase chain reaction to produce amplicon BB
  • the amplification is carried out using temperature cycling under the conditions described above in the presence of a nucleotide polymerase and nucleoside triphosphates B is comprised of a sequence identical to A except for mutation M
  • primer P2 used for this amplification contains a label L2 that may be the same as or different than L1
  • Amplicon BB has two strands, a labeled strand derived from primer P2 and an unlabeled strand derived from primer P3
  • the unlabeled strand has end portion A1 of primer P3 and the labeled strand has corresponding end portion B2, which is the complement of A1
  • a chain extension of primer P1 along the labeled strand of amplicon BB is carried out, under the conditions mentioned above for the chain extension of primer P
  • reaction mixtures above for the PCR amplification reactions for target nucleic acid sequence A and reference nucleic acid sequence B are combined where the above amplifications were carried out separately
  • the combined reaction mixtures are subjected to conditions for branch migration
  • the strands of partial duplexes A' and B' are allowed to bind and undergo branch migration by combining the mixtures containing partial duplexes A' and B' and incubating the combination at a temperature of about 30°C to about 75°C, preferably about 60°C to about 70°C, for at least about one minute, preferably, about 20 to about 60 minutes, wherein complex C is formed as described above for Figs 1 and 2
  • Oligonucleotide tail A1 of A is hybridized to corresponding oligonucleotide tail B2 of B' and, similarly, oligonucleotide tail A2 of A' is hybridized to oligonucleotide tail B1 of B'
  • Branch migration within complex C continues under the above temperature conditions with separation of the
  • labels L1 and L2 are incorporated into the partial duplexes that comprise complex C and provide a means for detection of complex C
  • L1 or L2 which comprises an sbp member or a reporter molecule
  • a receptor for the sbp member and a receptor that can bind to complex C by virtue of a feature other than L1 or L2 can both bind to complex C and provide a means for detection
  • the above reactions are carried out independently to produce tailed partial duplexes A' and B', respectively, in separate reaction mixtures Then, the reaction mixtures can be combined to allow the respective strands of A and B' to bind to one another to form complex C If a normalization method in accordance with the present invention was not to be carried out, the above reactions can be carried out in the same reaction medium and many or all of the reactions preferably are carried out simultaneously. In this approach a combination is provided in a single medium.
  • the combination comprises (i) a sample containing a target nucleic acid sequence suspected of having a mutation, (ii) a reference nucleic acid sequence, which may be added separately if it is not known to be present in the sample and which corresponds to the target nucleic acid lacking the mutation, which as explained above may be the wild type nucleic acid, (iii) a nucleotide polymerase, (iv) nucleoside triphosphates, and (v) primers P1 , P2 and P3, wherein P2 may include primer P2 labeled with L1 and primer P2 labeled with L2, or P2 may be unlabeled and primers P1 and P3 may be labeled respectively with L1 and L2.
  • each cycle includes heating the medium at about 90 C C to about 100°C for about 10 seconds to about 3 minutes, cooling the medium to about 60°C to about 70°C for a period of about 10 seconds to about 3 minutes, and heating the medium at about 70°C to about 75°C for a period of about 10 seconds to about 3 minutes although different temperatures may be required depending on the lengths of the primer sequences.
  • the medium is subjected to heating for a period of time sufficient to denature double stranded molecules, preferably, at about 90°C to about 99°C for about 10 seconds to about 2 minutes, and cooled to about 40°C to about 80°C, preferably about 60°C to about 70°C, and held at this temperature for at least about one minute, preferably for about 20 minutes to about 1 hours.
  • partial and complete duplexes are formed that can form from 1 ) single strands that have any combination of reference or mutant sequences and 5'-ends A2 and B2, and 2) single strands having any combination of reference or mutant sequences and 5'-ends A1 or B1 wherein the strands may further be labeled with either L1 or L2 when L1 and L2 are different
  • the partial duplexes that are formed are the tailed partial duplexes A' and B', which can bind to each other to form complex C, which does not dissociate into duplexes D and E when a mutation is present
  • a determination of the presence of such a complex is then made to establish the presence of a mutation in the target nucleic acid sequence
  • primers P1 and P3 are labeled instead of primer P2
  • the labels L1 and L2 in partial duplexes A and B' are attached to tails A1 and B1 , respectively, which still provides for detection of complex C when a mutation is present
  • PCR amplification of target sequence A and target sequence B each using primers P1 , P2 and P3, can be conducted in separate solutions
  • the solution can then be combined, heated to about 90°C to about 100°C to denature strands and then incubated as before at about 40°C to about 80°C to permit formation of duplexes and complex C when a mutation is present
  • Detection of complex C can then be carried out directly in the combined solutions or by adding reagents required for detection or by separating the complex C, for example, on a solid surface, and detecting its presence on the surface
  • the normalization method is based on formation of stable four stranded polynucleotide structures when amplification product from target polynucleotide is mixed with similarly produced products of amplification of a non-relevant reference sequence As explained above, such a sequence differs from the above reference sequence, e g , wild type sequence in that there is no correspondence between the non-relevant sequence and the test polynucleotide Thus, any non-relevant polynucleotide may be used in the normalization procedure.
  • the non- relevant reference sequence be treated to incorporate oligonucleotide tails that are the same tails as used for the target polynucleotide
  • the primers used in the PCR amplification of the non-relevant reference sequence comprise the same tails as the tails in the primers employed in the PCR amplification of the target polynucleotide
  • the primers for the PCR amplification of the non-relevant reference sequence also comprise appropriate priming sequences that are capable of hybridizing to the non-relevant reference sequence in accordance with standard PCR methodology
  • the length of the primers for the non-relevant reference sequence is determined employing considerations that are similar to those for the relevant reference sequence as discussed above
  • the first signal is obtained as described above, with reference to Fig 3, for the target polynucleotide and the relevant reference sequence or wild type sequence Ahquots of the PCR amplification products from the target polynucleotide and the relevant reference sequence are combined and subjected to branch migration conditions
  • a signal, i e the first signal, is obtained
  • the signal is representative of both the specific difference such as genotype, of the target polynucleotide and the amount of the target polynucleotide
  • an aliquot from the PCR amplification of the target polynucleotide is combined with an aliquot that contains the PCR amplification product of a predetermined amount of the non-relevant reference sequence
  • the combination is subjected to branch migration conditions as discussed above for the target polynucleotide and the relevant reference
  • the PCR amplification products from the target polynucleotide and the non-relevant reference sequence both contain the same oligonucleotide tails
  • there is cross-hybridization of the tails between the two products leading to four-stranded structures between the two products Because of the difference in the sequences of the target polynucleotide and the non-relevant reference sequence, these four- stranded structures are stable and do not dissociate
  • signals are obtained from all target polynucleotides regardless of the specific genotype
  • the signal obtained, namely the second signal is indicative of only the amount of the target polynucleotide since
  • PCR amplification of target nucleic acid sequence A is shown on the left
  • the product of the PCR amplification is A'
  • the PCR amplification of the non-relevant reference nucleic acid sequence E is shown on the right in Fig 4
  • the product of this PCR amplification is E'
  • these PCR amplification reactions are carried out separately
  • the reaction mixture containing the PCR amplification product of target nucleic acid sequence A is an aliquot of the reaction mixture discussed above with reference to Fig 3
  • An aliquot of this reaction mixture containing A' is combined with an aliquot of the PCR amplification reaction mixture of non-relevant reference nucleic acid sequence E, which is present in a predetermined amount
  • predetermined amount is meant that a known amount of the non-relevant reference E is employed in order that the ratio of the signals in accordance with the normalization procedure of the present invention provide for a sensitive and accurate determination Usually, this predetermined amount
  • non-relevant reference nucleic acid sequence E is amplified by PCR in a separate medium
  • Primer P4 and primer P5 are employed in a polymerase chain reaction to produce amplicon EE
  • the amplification is carried out using temperature cycling under the conditions described above in the presence of a nucleotide polymerase and nucleoside triphosphates E is comprised of a non-relevant sequence and thus the primers employed correspond to the non-relevant sequence
  • primer P4 used for this amplification contains a label L2 that may be the same as or different than L1
  • Amplicon EE has two strands, a labeled strand derived from primer P4 and an unlabeled strand derived from primer P5
  • the unlabeled strand has end portion A1 of primer P3 and the labeled strand has corresponding end portion B2, which is the complement of A1
  • a chain extension of primer P6 along the labeled strand of amplicon EE is carried out, under the conditions mentioned above for the chain extension of primer P3 along the labeled strand in duplex AA, to produce tailed non-relevant reference partial duplex E'
  • primer P6 is comprised of portion Pb, which binds to the labeled strand of EE and portion B1 that does not bind to amplicon EE
  • the chain extension is carried out in the presence of a nucleotide polymerase and nucleoside triphosphates under appropriate temperature conditions so that only the complement of the labeled strand is produced and not a copy
  • the extended primer P6 has a 5'-end portion B1 , which is not complementary to end portion B2 of the labeled strand of E'
  • it is important A and E' be unrelated but that tails A1 and B1 be the same for primers the set or primers used for amplification of target nucleic acid sequence A and non-relevant nucleic acid sequence E
  • an aqueous medium is employed
  • Other polar cosolvents may also be employed, usually oxygenated organic solvents of from 1-6, more usually from 1 -4 carbon atoms, including alcohols, ethers and the like Usually these cosolvents if used, are present in less than about 70 weight percent, more usually in less than about 30 weight percent
  • the pH for the medium is usually in the range of about 4 5 to 9 5, more usually in the range of about 5 5 - 8 5, and preferably in the range of about 6 - 8, usually about 8
  • the pH and temperature are chosen and varied, as the case may be so as to cause, either simultaneously or sequentially, dissociation of any internally hybridized sequences, hybridization of the oligonucleotide primer with the target nucleic acid sequence, extension of the primer, and dissociation of the extended primer.
  • Various buffers may be used to achieve the desired pH and maintain the pH during the determination.
  • Illustrative buffers include borate, phosphate, carbonate, Tris, barbital and the like. .
  • the particular buffer employed is not critical to this invention but in individual methods one buffer may be preferred over another.
  • the buffer employed in the present methods normally contains magnesium ion (Mg 2+ ), which is commonly used with many known polymerases, although other metal ions such as manganese have also been used.
  • magnesium ion is used at a concentration of from about 1 to about 20mM, preferably, from about 4 to about 10mM.
  • the magnesium can be provided as a salt, for example, magnesium chloride and the like.
  • the primary consideration is that the metal ion permit the distinction between different nucleic acids in accordance with the present invention.
  • the concentration of the nucleotide polymerase is usually determined empirically.
  • a concentration is used that is sufficient such that further increase in the concentration does not decrease the time for the amplification by over 5-fold, preferably 2-fold.
  • the primary limiting factor generally is the cost of the reagent.
  • the amount of the target nucleic acid sequences that is to be examined in accordance with the present invention can be as low as one or two molecules in a sample.
  • the priming specificity of the primers used for the detection of a difference between two related nucleic acids and other factors will be considered with regard to the need to conduct an initial amplification of the target nucleic acid.
  • the amplification can be by any convenient method such as PCR, amplification by single primer, NASBA, and so forth, but will preferably be by PCR.
  • the amount of the target nucleic acid sequence to be subjected to subsequent amplification using primers in accordance with the present invention may vary from about 1 to about 101 °, more usually from about 103 to about 108 molecules, preferably at least about 10-21 M in the medium and may be about 10- 10 to about 10-19M, mor e usually about 10-14 to about 10-19M
  • an initial amplification of the target nucleic acid sequence is carried out to increase the number of molecules it may be desirable, but not necessary, to remove, destroy or inactivate the primers used in the initial amplification depending on the nature of the protocol utilized Accordingly, when the present method is carried out using step-wise addition of reagents for each separate reaction, such as, for example, in the embodiment of Fig 3, primer P1 should be removed prior to the extension of primer P3
  • primer P1 should be removed prior to the extension of primer P3
  • An example, by way of illustration and not limitation, of an approach to destroy the primers is to employ an enzyme that can digest only single stranded DNA
  • an enzyme may be employed that has both 5' to 3' and 3' to 5 exonuclease activities, such as, e g , exo VII
  • the medium is incubated at a temperature and for a period of time sufficient to digest the primers Usually incubation at about 20°C to
  • the amount of the oligonucleotide p ⁇ mer(s) used in the amplification reaction in the present invention will be at least as great as the number of copies desired and will usually be about 10-9 to about 10-3 M, preferably, about 10-7 to about 10-4 M
  • the concentration of the oligonucleotide p ⁇ mer(s) is substantially in excess over, preferably at least about 100 times greater than, more preferably, at least 1000 times greater than, the concentration of the target nucleic acid sequence
  • concentration of the nucleoside triphosphates in the medium can vary widely, preferably, these reagents are present in an excess amount for both amplification and chain extension
  • the nucleoside triphosphates are usually present in about 10-6 to about 10-2M, preferably about 10-5 to about 10-3M
  • the order of combining the various reagents may vary
  • the target nucleic acid may be combined with a pre-prepared combination of primers unlabeled P2, labeled P2, and P1 , nucleoside triphosphates and nucleotide polymerase
  • the target nucleic acid for example, can be combined with only unlabeled primer P2 together with the nucleoside triphosphates and polymerase After temperature cycling is carried out, the reaction mixture can be combined with the remaining primers P1 and labeled P2
  • the identity of the target nucleic acid sequence does not need to be known except to the extent to allow preparation of the necessary primers for carrying out the above reactions
  • the present invention permits the determination of the presence or absence of a mutation in a nucleic acid in a sample without the need to fully identify the sequence of the nucleic acid Accordingly, one is able to determine the presence of a mutation in a nucleic acid between two sequences of nucleotides for which primers can be made
  • one means of detecting the quadramolecular complex involves the use of two labels on non-complementary strands
  • the labels become associated by virtue of both being present in the quadramolecular complex if a difference is present between the related sequences
  • Detection of the two labels in the complex provides for detection of the complex
  • the association of the labels within the complex is detected This association may be detected in many ways
  • one of the labels can be an sbp member and a complementary sbp member is provided attached to a support Upon the binding of the complementary sbp members to one another, the complex becomes bound to the support and is separated from the reaction medium
  • the other label employed is a reporter molecule that is then detected on the support The presence of the reporter molecule on the support indicates the presence of the complex on the support, which in turn indicates the presence of the mutation in the target nucleic acid sequence.
  • ELISA enzyme-linked immunosorbent assay
  • the reporter molecule is an enzyme, additional members of the signal producing system would include enzyme substrates and so forth.
  • the product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electrometric means.
  • the reporter molecule is a fluorescent molecule, the medium can be irradiated and the fluorescence determined Where the label is a radioactive group, the medium can be counted to determine the radioactive count.
  • the association of the labels within the complex may also be determined by using labels that provide a signal only if the labels become part of the complex.
  • Such systems include enzyme channeling immunoassay, fluorescence energy transfer immunoassay, electrochemiluminescence assay, induced luminescence assay, latex agglutination and the like.
  • detection of the complex is accomplished by employing at least one suspendable particle as a support, which may be bound directly to a nucleic acid strand or may be bound to an sbp member that is complementary to an sbp member attached to a nucleic acid strand.
  • Such a particle serves as a means of segregating the bound target polynucleotide sequence from the bulk solution for example by settling, electrophoretic separation or magnetic separation
  • a second label which becomes part of the complex if a mutation is present is a part of the signal producing system that is separated or concentrated in a small region of the solution to facilitate detection
  • Typical labels that may be used in this particular embodiment are fluorescent labels, particles containing a sensitizer and a chemiluminescent olefin (see U S Serial No 07/923,069 filed July 31 , 1992 the disclosure of which is incorporated herein by reference), chemiluminescent and electroluminescent labels
  • the particle itself can serve as part of a signal producing system that can function without separation or segregation
  • the second label is also part of the signal producing system and can produce a signal in concert with the particle to provide a homogeneous assay detection method
  • a variety of combinations of labels can be used for this purpose When all the reagents are added at the beginning of the reaction
  • an oligonucleotide or polynucleotide analog attached to a reporter molecule or particle can bind to its complementary polynucleotide analog or oligonucleotide separated by an abasic site that has become incorporated into partial duplexes A' and B' as labels during amplification If the partial duplexes become part of a quadramolecular complex, the reporter molecule or particle becomes part of the complex
  • the reporter molecule or particle becomes part of the complex
  • L1 and L2 two different reporter molecules or particles can become part of the complex
  • the particles for example, may be simple latex particles or may be particles comprising a sensitizer chemiluminescer, fluorescer, dye, and the like
  • Typical particle/reporter molecule pairs include a dye crystallite and a fluorescent label where binding causes fluorescence quenching or a t ⁇ tiated reporter molecule and
  • detection of the quadramolecular complex using the induced luminescence assay as applied in the present invention involves employing a photosensitizer as part of one label and a chemiluminescent compound as part of the other label If the complex is present the photosensitizer and the chemiluminescent compound come into close proximity The photosensitizer generates singlet oxygen and activates the chemiluminescent compound when the two labels are in close proximity The activated chemiluminescent compound subsequently produces light The amount of light produced is related to the amount of the complex formed
  • a particle which comprises the chemiluminescent compound associated therewith such as by incorporation therein or attachment thereto
  • the particles have a recognition sequence usually an oligonucleotide or polynucleotide analog, attached thereto with a complementary sequence incorporated into one of the nucleic acid strands as a label, L1
  • Another particle is employed that has the photosensitizer associated therewith
  • These particles have a recognition sequence attached thereto, which is different than that attached to the chemiluminescent particles
  • a complementary sequence is incorporated as a label L2 in the nucleic acid strand in complex C that is not complementary to the nucleic acid strand carrying label L1
  • kits can be varied widely to provide for concentrations of the reagents that substantially optimize the reactions that need to occur during the present method and to further substantially optimize the sensitivity of the method in detecting a mutation
  • one or more of the reagents in the kit can be provided as a dry pow- der, usually lyophilized including excipients which on dissolution will provide for a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present invention
  • Each reagent can be packaged in separate containers or some or all of the reagents can be combined in one con- tamer where cross-reactivity and shelf life permit
  • the kits may also include a written description of a method in accordance with the present invention as described above
  • Tris - T ⁇ s(hydroxymethyl)am ⁇ nomethane-HCI (a 10X solution) from BioWhittaker, Walkersville, MD
  • Buffer A 10mM T ⁇ s-HCI (pH8 3) 50mM KCI, 1 5mM MgCI 2 200 ⁇ g/ml BSA
  • Buffer B 100mM T ⁇ s-HCI (pH8 3), 500mM KCI, 15mM MgCI 2 2 mg/ml BSA Buffer C - 0 1 M Tris 0 3M NaCl 25 mM EDTA, 0 1 % BSA, 0 1 % dextran T- 500, a 1 320 dilution of mouse IgG (HBR-1 from Scantibodies Laboratory Inc , Los Angeles, CA), 0 05% Kathon (Rohm and Haas, Philadelphia, PA), and 0 01 % gentamycm sulfate RLU - relative light units nt - nucleotides
  • the appropriated immunogen was injected into a host, usually a mouse or other suitable animal, and after a suitable period of time the spleen cells from the host were obtained Alternatively, unsensitized cells from the host were isolated and directly sensitized with the immunogen in vitro Hybrid cells were formed by fusing the above cells with an appropriate myeloma cell line and cultu ⁇ ng the fused cells The antibodies produced by the cultured hybrid cells were screened for their binding affinity to the particular antigen, dig-BSA conjugate A number of screening techniques were employed such as, for example, ELISA screens Selected fusions were then recloned Beads
  • Acc-AbDig - Acceptor beads coupled (MAD) to the anti-Dig antibody were prepared as follows
  • Hydroxypropylaminodextran (1 NH 2 / 7 glucose) was prepared by dissolving Dextran T-500 (Pharmacia, Uppsala Sweden) (50g) in 150 mL of H 2 O in a 3-neck round-bottom flask equipped with mechanical stirrer and dropping funnel To the above solution was added 18 8g of Zn (BF 4 ) 2 and the temperature was brought to 87°C with a hot water bath Epichlorohyd ⁇ n (350mL) was added dropwise with stirring over about 30 mm while the temperature was maintained at 87-88°C The mixture was stirred for 4 hr while the temperature was maintained between 80°C and 95°C, then the mixture was cooled to room temperature Chlorodextran product was precipitated by pouring slowly into 3L of methanol with vigorous stirring, recovered by filtration and dried overnight in a vacuum oven
  • the chlorodextran product was dissolved in 200mL of water and added to 2L of concentrated aqueous ammonia (36%) This solution was stirred for 4 days at room temperature, then concentrated to about 190mL on a rotary evaporator The concentrate was divided into two equal batches, and each batch was precipitated by pouring slowly into 2L of rapidly stirring methanol. The final product was recovered by filtration and dried under vacuum.
  • Hydroxypropylaminodextran (1 NH 2 / 7 glucose), prepared above, was dissolved in 50mM MOPS, pH 7.2, at 12.5 mg/mL. The solution was stirred for 8 hr at room temperature, stored under refrigeration and centrifuged for 45 min at 15,000 rpm in a Sorvall RC-5B centrifuge immediately before use to remove a trace of solid material. To 10mL of this solution was added 23.1 mg of Sulfo-SMCC in 1 mL of water. This mixture was incubated for 1 hr at room temperature and used without further purification.
  • C-28 thioxene was prepared as follows: To a solution of 4-bromoaniline (30g, 174mmol) in dry DMF (200mL) was added 1- bromotetradecane (89.3mL, 366mmol) and N,N-diisopropylethylamine (62.2mL, 357mmol). The reaction solution was heated at 90°C for 16 hr under argon before being cooled to room temperature. To this reaction solution was again added 1- bromotetradecane (45mL, 184mmol) and N,N-diisopropylethylamine (31 mL, 178mmol) and the reaction mixture was heated at 90°C for another 15 hr.
  • Carboxyl chemiluminescer (acceptor) beads (TAR beads)
  • the following dye composition was employed 20% C-28 thioxene (prepared as described above), 1 6% 1 -chloro-9 10-b ⁇ s(phenylethynyl)anthracene (1 -CI-BPEA) (from Ald ⁇ ch Chemical Company) and 2 7% rubrene (from (from Ald ⁇ ch Chemical Company)
  • the particles were latex particles (Seradyn Particle Technology, Indianapolis IN)
  • the dye composition (240-250 mM C-28 thioxene, 8-16 mM 1-CI- BPEA, and 20-30 mM rubrene) was incorporated into the latex beads in a manner similar to that described in U S Patent 5,340,716 issued August 23, 1994 (the 716 patent), at column 48 lines 24-45 which is incorporated herein by reference
  • the dyeing process involved the addition of the latex beads (10% solids) into a mixture of ethylene glycol (65 4%
  • the beads were removed form the oil bath and are allowed to cool to 40 C C ⁇ 10°C
  • the beads were then passed through a 43-m ⁇ cron mesh polyester filter and washed
  • the dyed particles were washed using a Microgon (Microgon Inc , Madison Hills, CA)
  • the beads were first washed with a solvent mixture composed of ethylene glycol and 2-ethoxyethanol (70%/30%).
  • the beads were washed with 500 ml of solvent mixture per gram of beads.
  • Monoclonal anti-digoxm Ab (prepared as described above) was purified by ABx resin (Baker Chemical Company Phillipsburg, NJ) and was dialyzed into 0 15 M NaCl, 5mM Na 2 HP0 4 pH 7 4
  • the anti-digoxm Ab was thiolated by mixing 622 ⁇ L (4 28mg) with 10 2 uL of SATA (1 25 mg/mL in ethanol, 2 eq ) incubating for 1 hr at room temperature and dialyzing cold against 2x2 L of 150 mM NaCl, 10mM Na 2 HPO , 1 mM EDTA pH7
  • the thioacetylated antibody was deacetylated by adding 62 2 ⁇ L of hydroxylamme (1 M H 2 NOH, 50 mM MOPS, 25 mM EDTA, pH 7), bubbling with argon and incubating for 1 hr at room temperature
  • the product was applied to a Pharmacia PD-10 column (
  • Sens-Sav - Sensitizer beads coupled to Streptavidin (2300 Sav/bead).
  • the sensitizer beads were prepared placing 600mL of carboxylate modified beads (Seradyn) in a three-necked, round-bottom flask equipped with a mechanical stirrer, a glass stopper with a thermometer attached to it in one neck, and a funnel in the opposite neck
  • the flask had been immersed in an oil bath maintained at 94+ /-1°C
  • the beads were added to the flask through the funnel in the neck and the bead container was rinsed with 830mL of ethoxyethanol, 1700mL of ethylene glycol and 60mL of 0 1 N NaOH and the rinse was added to the flask through the funnel
  • the funnel was replaced with a 24-40 rubber septum
  • the beads were stirred at 765 rpm at a temperature of 94+ /-1 °C for 40m ⁇ n
  • Silicon tetra-t-butyl phthalocyanme (10 Og) was dissolved in 300mL of benzyl alcohol at 60+/-5°C and 85mL was added to the above round bottom flask through the septum by means of a syringe heated to 120+/-10°C at a rate of 3mL per min.
  • the remaining 85mL of the phthalocyanme solution was then added as described above
  • the syringe and flask originally containing the phthalocyanme was rinsed with 40mL of benzyl alcohol and transferred to round-bottom flask After 15 mm 900mL of deionized water and 75mL of 0 1 N NaOH was added dropwise over 40 m
  • the temperature of the oil bath was allowed to drop slowly to 40+/-10°C and stirring was then discontinued
  • the beads were then filtered through a 43 micron polyester filter and subjected to a Microgon tangential flow filtration apparatus (Microgon Inc Website, CA) using ethanol water, 100 0 to 10 90, and then filtered through a 43 micron polyester filter
  • Sulfo-SMCC (11 55mg) was dissolved in 0 5mL distilled water Slowly, during 10 sec the above solution was added to 5mL of stirring aminodextran (Molecular Probes, Eugene Oregon) solution (12 5 mg/mL in 50mM MOPS, pH 7 2) The mixture was incubated for 1 hr at room temperature
  • the resulting thiolated streptavidin was purified on a Pharmacia PD10 column and washed with a column buffer containing 50mM MOPS, 50mM EDTA, pH 7.2. The volume of the sample was brought to 2.5mL by adding 1 ,5mL of the above column buffer. The sample was loaded on the column and eluted with 3.5mL of the column buffer. The thiolated streptavidin was diluted to 5mL by adding 1.5mL of 50mM MOPS, 50mM EDTA, 0.1 % Tween-20, pH 7.2. 5mL of the thiolated streptavidin solution was added to 5mL of the sensitizer beads, under argon, and mixed well. The beads were topped with argon for 1 min, the tube was sealed and the reaction mixture was incubated overnight at room temperature in the dark.
  • PCR primer sequences used in the branch migration are set forth in Table 1; the primers have been modified as indicated for the purpose of conducting a "hot start ' procedure as discussed below.
  • Table 1 Specific primers for M tuberculosis rpoB gene mutation detection Forward primers
  • Tail 1 5'-ACCATGCTCGAGATTACGAG-3' (SEQ ID NO 9)
  • Tail 2 5'-GATCCTAGGCCTCACGTATT-3' (SEQ ID NO 10)
  • N etheno dA modification
  • the position of hybridization of the primers to the rpoB gene sequence is indicated by the primer number
  • the number indicates the target nucleotide complementary to the 5'-end of the primer (shown in bold)
  • the position is related to the complementary sequence only, not including tail 1 or tail 2
  • the positions of the forward and reverse PCR primers are denoted in the detail for the full sequence of the M tuberculosis rpoB gene (GenBank Accession No. U12205) (SEQ ID NO 1 1 ) as depicted in Fig 5 where the single line and double lines under a portion of the gene in bold type is the primer sequence employed and the direction of the arrow indicates forward (->) primers and reverse ( ⁇ -) primers
  • the forward PCR primers are 5 -labeled with biotin or digoxigenin (dig)
  • the reverse PCR primers are composed of two parts
  • the 3'-parts of the primers are identical and are complementary to the target DNA (shown in bold)
  • the 5'-parts of the two primers are different and are not related to the target DNA sequence
  • These latter sequences (designated as tail 1 and tail 2) are designed to form the tails of the heteroduplexes, which upon annealing result in the formation of a four stranded DNA structure used in the mutation analysis
  • All forward and reverse primers are also modified at the 3'-end by the addition of two etheno A residues and an additional nucleotide so that a "hot start" procedure may be carried out using an antibody specific for the modifications
  • the 3'-most residue is added for convenience of oligonucleotide synthesis PCR amplification of the rpoB gene sequence PCR amplification of the rpoB gene sequence was
  • PCR amplification of test target was carried out using 5'-b ⁇ ot ⁇ n labeled forward p ⁇ mer and two related reverse primers
  • the reference target, wild type was amplified using the corresponding 5'-d ⁇ g labeled forward primer and the same set of reverse p ⁇ mers as for the test target amplification PCR amplification with wax bead based hot start was carried out as follows
  • thermocycle program was as follows 4 mm at 95 C followed by 40 cycles of 45 sec at 95 C, and 2 mm at 70 C
  • PCR amplification using the antibody-based hot start procedure was carried out as follows a master reaction mixture containing 10 mM T ⁇ s-HCI pH 8.3, 50 mM KCI, 1 5 mM MgCI 2 , 200 uM of each of four dNTPs, 0 2 mg/ml BSA, 0 5 ⁇ M anti etheno A monoclonal antibody (from the Inst fur Zellbiologie, Dr Petra Lorenz), and 1.25 U/25 ⁇ l Pfu DNA polymerase was prepared 2 5 ⁇ l of test or reference target was added to 22 5 ⁇ l of the master reaction mix, in PCR tubes PCR amplification was carried out using conditions similar to the above
  • thermocycler 50 ⁇ l of a bead mixture (2 5 ⁇ g of streptavid - coated sensitizer beads and 1 25 ⁇ g of anti-Dig-coated acceptor beads) were added to each tube, and the tubes were incubated at 37°C for 30 mm and signal was read (3 cycles of 1 sec illumination and 1 sec read) Genotypic detection of ⁇ fampin resistance
  • M tuberculosis clinical isolates grown in culture were suspended in Buffer B (lOOmMT ⁇ s HCI pH 8 3 500mM KCI 15mM MgCI2 and 2mg/ml BSA) and heat inactivated by boiling for 30 m at 95°C
  • Direct analysis was achieved following sonication, (12-15 pulse on Branson Sonifier 450 ), boiling ( 98°C for 15-m ⁇ n , using a thermocycler) or treatment in a microwave (45 sec to 1-m ⁇ n at high power setting).
  • the direct analysis of 10 M. tuberculosis clinical isolates, obtained using cell suspensions pretreated as detailed above demonstrated direct genotypic detection (Table 3).
  • RLU-1 * relative luminescence unit for M. tuberculosis cells heat killed Buffer A
  • RLU-2 relative luminescence unit for M. tuberculosis cells heat killed in Buffer B
  • the following experiments demonstrate a method for normalization of the signals relative to input test target
  • the normalization method is based on formation of stable four stranded DNA structures when test target amplification products are mixed with similarly produced products of amplification of non-relevant reference sequence
  • the two sequences are not related signals are produced from all test samples, regardless of the specific genotype
  • the ratio of signals produced with relevant reference sequence to those produced with non-relevant reference sequence are the normalized signals and represent test genotype regardless of input target sequence
  • PCR amplification of non-relevant target was carried out using the antibody- based hot start procedure as described above All of the reagents were the same as stated above except the target was cystic fibrosis exonl 1 (wild type) and the primers were 5'-d ⁇ g labeled forward primer and a mixture of reverse primers modified with the same ta ⁇ l-1 and ta ⁇ l-2 as for M tuberculosis as described above The primers are identified in the CFTR Gene, Exon 11 Sequence below Analysis and detection were performed as follows (1 ) For genotype determination
  • 551 553 560 351 actgagtgga GGTcaaCGAq caagaatttc tttagcaAGG tgaataacta tgactcacct ccagttgctc grtcttaaag aaatcgttcc acttattgat
  • the f2/rl primer ser flanks 173 bases of the CFTR Exon 11 sequence, resulting in an amplicon which includes 217 bases from Exon 11 and 20 bases from the reverse primer tails, for a total of 237 bp.
  • the above discussion includes certain theories as to mechanisms involved in the present invention These theories should not be construed to limit the present invention in any way, since it has been demonstrated that the present invention achieves the results described

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Abstract

Cette invention se rapporte à un procédé permettant de détecter la présence d'une différence entre deux séquences d'acide nucléique mises en relation. A cet effet, on forme un complexe comprenant les deux séquences d'acide nucléique sous forme de double brin. Ce complexe contient au moins une paire de brins non complémentaires et chacun de ces brins non complémentaires à l'intérieur du complexe possède une étiquette. Le complexe est soumis à des conditions telles que, si une différence entre les deux séquences d'acide nucléique mises en relation est présente, alors l'échange des brins dans le complexe cesse et, si aucune différence n'est présente entre les deux séquences d'acide nucléique mises en relation, alors l'échange des brins dans le complexe se poursuit jusqu'à ce qu'un échange complet des brins soit réalisé. Un premier signal est détecté à partir de l'association des étiquettes comme faisant partie du complexe. L'association des étiquettes est mise en relation avec la présence de la différence recherchée. On forme également un complexe comprenant la séquence d'acide nucléique suspectée de présenter une différence sous forme de double brin et une quantité prédéterminée d'un polynucléotide de référence non pertinent sous forme de double brin. Ce complexe contient au moins une paire de brins non complémentaires et chacun de ces brins non complémentaires à l'intérieur du complexe possède une étiquette. Un second signal provenant de l'association des étiquettes comme faisant partie de ce complexe est détecté. Un rapport entre le premier signal et le second signal est déterminé et mis en relation avec la présence de la différence.
PCT/US1999/029222 1999-01-19 1999-12-09 Detection des differences entre polynucleotides Ceased WO2000043543A1 (fr)

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Cited By (7)

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WO2002024946A3 (fr) * 2000-09-19 2003-03-13 Ingeneus Corp Adn quadruple brin et systemes d'essai double brin
US6602665B2 (en) 2000-03-31 2003-08-05 Whitehead Institute For Biomedical Research Referenced amplification of small quantities of RNA
US6656692B2 (en) 1999-12-21 2003-12-02 Ingeneus Corporation Parallel or antiparallel, homologous or complementary binding of nucleic acids or analogues thereof to form duplex, triplex or quadruplex complexes
WO2002103051A3 (fr) * 2001-06-20 2004-04-29 Ingeneus Corp Formation de structures multiplex d'acide nucleique
US6911536B1 (en) 1999-12-21 2005-06-28 Ingeneus Corporation Triplex and quadruplex catalytic hybridization
US7052844B2 (en) 1999-12-21 2006-05-30 Ingeneus, Inc. Purification of DS-DNA using heteropolymeric capture probes and a triplex, quadruplex or homologous duplex binding mechanism
US10450597B2 (en) 2014-01-27 2019-10-22 The General Hospital Corporation Methods of preparing nucleic acids for sequencing

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WO1997023646A1 (fr) * 1995-12-22 1997-07-03 Behringwerke Aktiengesellschaft Detection de differences dans les acides nucleiques

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6656692B2 (en) 1999-12-21 2003-12-02 Ingeneus Corporation Parallel or antiparallel, homologous or complementary binding of nucleic acids or analogues thereof to form duplex, triplex or quadruplex complexes
US6911536B1 (en) 1999-12-21 2005-06-28 Ingeneus Corporation Triplex and quadruplex catalytic hybridization
US6927027B2 (en) 1999-12-21 2005-08-09 Ingeneus Corporation Nucleic acid multiplex formation
US7052844B2 (en) 1999-12-21 2006-05-30 Ingeneus, Inc. Purification of DS-DNA using heteropolymeric capture probes and a triplex, quadruplex or homologous duplex binding mechanism
US6602665B2 (en) 2000-03-31 2003-08-05 Whitehead Institute For Biomedical Research Referenced amplification of small quantities of RNA
WO2002024946A3 (fr) * 2000-09-19 2003-03-13 Ingeneus Corp Adn quadruple brin et systemes d'essai double brin
US6900300B1 (en) 2000-09-19 2005-05-31 Ingeneus Corporation Quadruplex DNA and duplex probe systems
CN1535318B (zh) * 2000-09-19 2010-10-06 英詹尼斯公司 四链体dna和双链体探针系统
WO2002103051A3 (fr) * 2001-06-20 2004-04-29 Ingeneus Corp Formation de structures multiplex d'acide nucleique
CN100560733C (zh) * 2001-07-20 2009-11-18 英詹尼斯公司 平行，反平行,同源或互补结合的核酸
US10450597B2 (en) 2014-01-27 2019-10-22 The General Hospital Corporation Methods of preparing nucleic acids for sequencing

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