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WO1996012824A2 - Emploi de spermidine pour attenuer les effets inhibiteurs de reactions en chaine de ligase dans un echantillon d'essai clinique - Google Patents

Emploi de spermidine pour attenuer les effets inhibiteurs de reactions en chaine de ligase dans un echantillon d'essai clinique Download PDF

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Publication number
WO1996012824A2
WO1996012824A2 PCT/US1995/012874 US9512874W WO9612824A2 WO 1996012824 A2 WO1996012824 A2 WO 1996012824A2 US 9512874 W US9512874 W US 9512874W WO 9612824 A2 WO9612824 A2 WO 9612824A2
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target
spermidine
probes
probe
amplification
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PCT/US1995/012874
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WO1996012824A3 (fr
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Alan H. Davis
Jianli Cao
Eui H. Lee
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Abbott Laboratories
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Priority to JP51396296A priority Critical patent/JP2002501361A/ja
Priority to EP95938175A priority patent/EP0791075A2/fr
Publication of WO1996012824A2 publication Critical patent/WO1996012824A2/fr
Publication of WO1996012824A3 publication Critical patent/WO1996012824A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • This invention relates to the ligase chain reaction (LCR) and in particular, relates to an improved LCR method which employs spermidine to relieve LCR inhibition.
  • LCR ligase chain reaction
  • two sets of probe partners are used which include one set of primary probes (first and second probe partners) and a second set of secondary probes (third and fourth probe partners).
  • One probe partner hybridizes to a first segment of a target strand and the other probe partner hybridizes to a second segment of the same target strand, the first and second segments being contiguous so that the primary probes abut one another in 5 ' phosphate-3 ' hydroxyl relationship and so that a ligase enzyme or other reagent can covalently fuse or ligate the two probes of the partner set into a fused product.
  • a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion.
  • the secondary probes in the first instance will also hybridize to the target complement.
  • the fused strand of primary probes Once the fused strand of primary probes is separated from the target strand, the fused strand will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary fused product. It is important to realize that the fused products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described in K. Backman, et al.
  • EP-A-320 308 published June 14, 1989 and is incorporated by reference in its entirety. LCR variations have also been described in, for example, PCT Patent Application No. WO 90/01069, British Patent No. GB 2 225 1 1 2 A and European Patent Application EP-A-439 182.
  • inhibition may occur from the presence of reagents that sequester required cofactors, and/or reagents that block active regulatory enzymatic sites. If inhibition occurs while testing for an infectious agent, a target sequence (i.e. infectious agent nucleic acid) actually present in the sample will not be amplified.
  • the present invention provides an improved LCR method that minimizes pre-amplification sample preparation and allows greater flexibility with respect to amplification reagent addition. These advantages are made available by adding an inhibition reducing amount of spermidine to an LCR amplification reaction. Briefly, the improved method comprises the steps of:
  • the improved method comprises the steps of:
  • composition comprising two pairs of probes, each pair comprising a primary probe hybridizable to the target and a secondary probe hybridizable to the primary probe, wherein the two primary probes hybridize with the target at adjacent positions to form a reaction mixture and;
  • the final concentration of spermidine provided in the clinical test sample can be between about 0.5 mM and about 4 mM, more typically, between about 1 mM and about 3 mM.
  • the use of spermidine as taught herein allows effective LCR amplification in the presence of final MgCl2 concentrations less than 20 mM and greater than 35.5 mM such as between about 0.5 mM and less than 20 mM.
  • the target sequence prior to LCR amplification of a target nucleic acid sequence, the target sequence frequently is purified from the source material or crude sample by, for example, extraction or Cesium Chloride (CsCI) gradients. As a result, the target sequence is separated from contaminants found in the source material. After purification, the resulting test sample is contacted with the reagents necessary to perform the amplification reaction and amplification is performed. Accordingly, amplification is performed with a relatively "clean" sample.
  • the present invention arises out of the unexpected discovery that spermidine can be employed to relieve inhibition of amplification of a target nucleic acid sequence which may be present in a "clinical test sample".
  • Clinical sample is a sample, taken from a malian source, which has not undergone purification via nucleic acid extraction or CsCI gradients. Samples which have undergone crude separation techniques, such as filtration and centrifugation; or which contain reagents such as, for example, acids, bases, lysing agents, buffers and pH inidcators added thereto, shall not be excluded from the term "clinicl test sample” provided they otherwise meet the definintion of that term given above.
  • a clinical test sample can be blood, serum, sputum or other malian body fluid which is centrifuged and resuspended in a buffer contining a lysing agent.
  • LCR amplification occurs in the presence of final MgCl concentrations less than 20 mM and greater than 35.5 mM. Typically, final MgCl2 concentrations between 0.5 mM and less than 20 mM can be employed.
  • Modified LCR procedures which can be employed according to the present invention use either one or two partner sets herein designated A, B (primary probes), and A ' , B ' (secondary probes).
  • a partner set refers to two probes (eg.
  • a and B which are directed to the same target strand and which will ultimately be ligated to one another after annealing to the target.
  • Each probe in a partner set is designated as a probe partner.
  • a probe pair refers to one probe from one partner set and another probe from a second partner set, that are complementary to each other. (Eg. Probe partner A of AB is complementary to probe partner A' of A'B'). Probe pairs can hybridize to each other to form a "duplex", resulting for example, in the hybridization of A to A' to form the duplex AA' and B to B' to form the duplex BB'. At least one of the probes from a probe pair initially includes a "modified" end which renders the resultant duplex
  • nonblunt and/or not a suitable substrate for a ligase catalyzed fusion of the two probe duplexes.
  • a group on the probe that is obligatorily involved in the enzyme catalyzed step of the amplification reaction is masked or blocked with, for example, a chemical moiety.
  • the enzymatic steps may be ligation and gap-filling.
  • the probe is capable of hybridizing with the target and initiates the enzymatic reaction (and thus may be termed the
  • amplification product The blocking group is selected so that it can be removed by an enzyme substantially only when the probes are hybridized to the target or amplification product and not when hybridized to one another as a probe pair.
  • a probe may be modified to contain an overhang of additional bases at one end. The bases are later cleaved in a target dependent fashion allowing the amplification reaction to occur.
  • Each of the probes comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be routinely synthesized using conventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, CA); DuPont, (Wilmington, DE); or Milligen, (Bedford, MA).
  • Phosphorylation of the 5' ends of the appropriate probes is necessary for ligation by ligase, and may be accomplished enzymatically by a kinase or by any chemical synthetic method known to phosphorylate 5' ends. Commercial reagents are available for this purpose.
  • modified LCR methods useful in the practice of the present invention comprise the steps of: (a) hybridizing the modified probes to a target (and, if present, to the target complement); (b) correcting the modification(s) in a target dependent manner (e.g. filling a gap) to render the probes ligatable; (c) ligating the corrected probe(s) to its partner to form a fused or ligated product; and (d) dissociating the fused product(s) from the target.
  • the hybridization, correction and ligation steps can be repeated to further amplify the desired target sequence.
  • Steps (a), (c) and (d) are essentially the same for all of the embodiments and can be discussed together.
  • Step (b) varies depending on the type of modification employed.
  • Hybridization or “hybridizing” conditions are defined generally as conditions which promote annealing. It is well known in the art, however, that such annealing and hybridization is dependent in a rather predictable manner on several parameters, including temperature, ionic strength, probe length and G:C content of the probes. For example, lowering the temperature of the reaction promotes annealing. For any given set of probes, melt temperature, or Tm, can be estimated by any of several known methods. Typically, diagnostic applications utilize hybridization temperatures which are slightly below the melt temperature. Ionic strength or "salt" concentration also impacts the melt temperature, since small cations tend to stabilize the formation of duplexes by negating the negative charge on the phosphodiester backbone.
  • Typical salt concentrations depend on the nature and valency of the cation but are readily understood by those skilled in the art.
  • high G:C content and increased probe length are also known to stabilize duplex formation because G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and because longer probes have more hydrogen bonds holding the probes together.
  • a high G:C content and longer probe lengths impact the "hybridization conditions" by elevating the melt temperature.
  • probe length, probe concentration and stringency of conditions affect the degree and rate at which hybridization will occur.
  • the probes are sufficiently long to provide the desired specificity; i.e., to avoid being hybridizable to nontarget sequences in the sample.
  • probes on the order of 1 5 to 100 bases serve this purpose.
  • Probes generally are added in approximately equimolar concentration since they are expected to react stoichiometrically. Each probe is generally present in a concentration ranging from about 5 nanomolar (nM) to about 90 nM; preferably from about 10 nM to about 35 nM. For a typical reaction volume of 200 ⁇ L, this is equivalent to adding from about 3 x 10 1 ⁇ to about 1 .2 x 10 1 2 molecules of each probe; and around 1 x 10 1 2 molecules per 200 ⁇ L has been a good starting point.
  • the optimum quantity of probe used for each reaction also varies depending on the number of cycles which must be performed and the reaction volume. Probe concentrations can readily be determined by one of ordinary skill in this art to provide optimum signal for a given number of cycles.
  • each corrected primary probe is ligated to its associated primary partner and each corrected secondary probe is ligated to its associated secondary partner.
  • An "adjacent" probe is either one of two probes hybridizable with the target in a contiguous orientation, one of which lies with its phosphorylated 5 ' end in abutment with the 3 ' hydroxyl end of the partner probe. "Adjacent" probes are created upon correction of the modified end(s) in a target dependent manner.
  • ligation Since enzymatic ligation is the preferred method of covalently attaching two adjacent probes, the term "ligation" will be used throughout the application.
  • ligation is a general term and is to be understood to include any method of covalently attaching two probes.
  • Correction refers to repair of the modification that rendered the probe ligation incompetent in the first place.
  • Specific correction mechanisms relate generally to one or more of: 1 ) creating or restoring a 3' hydroxyl; 2) creating or restoring a 5' phosphate or creating adjacency, either by cleaving an overhanging extension or by filling in a gap. It is important that correction be “target-dependent”, i.e. that it take place substantially only in the presence of target or target equivalent, and not in the presence of the other probes.
  • Template dependent is the same as “target dependent” in that the template is ligated probe product only, and not unligated probes.
  • modified ends are created by eliminating from one or more of the probes a short sequence of bases thereby leaving a recess or gap between the 5' end of one probe partner and the 3' end of the other probe partner when they are both hybridized to the target (or target complement, or polynucleotide generated therefrom).
  • the gaps between the probes must be filled in or extended (i.e., the modification must be "corrected”). This can be accomplished using a polymerase or a reverse transcriptase and an excess of deoxyribonucleotide triphosphates which are complementary to the target strand opposite the gap.
  • Extension must be terminated at the point of ligation so that the extended probe abuts the adjacent probe and can be ligated to it.
  • This method may be utilized in both single and double gap configurations wherein either one probe is extended (single gap) in the case of single stranded target or two probes are extended (double gap) in the case of double stranded target. Gap filling by extension is further described in WO 93/00447, the entire disclosure of which is incorporated herein by reference.
  • a non-phosphorylated 5' terminus is created which cannot be ligated to a 3' hydroxyl terminus of the upstream probe but which can be corrected in a target dependent manner to render it ligatable.
  • Upstream probe refers to that probe partner whose 3' terminus points toward the 5 terminus of the other probe partner regardless of whether the strand(s) possesses a "sense" direction for coding purposes.
  • the second probe partner is referred to as the "downstream” probe.
  • the 5' terminus is "corrected” by removal of the non- phosphate groups with resultant exposure of a 5' phosphate group. This is effected by removal of the entire nucleotide bearing the 5' non-phosphate group, using an agent having exonucleolytic activity which leaves a 5' phosphate terminus exposed on the next adjacent nucleotide.
  • an incompetent 5' end is created by a mismatched base(s) with respect to target within the downstream probe.
  • correction occurs in the presence of a polymerase, a ligase, and a dNTP pool, wherein the mismatched base is removed from the downstream probe and the upstream probe is extended until the probe partners abut each other and can be ligated.
  • Amplification of target nucleic acids using exonucleolytic activity is further described in WO 94/03636, the disclosure of which is incorporated herein by reference.
  • a modified end is created by adding a blocking moiety such as an abasic site or additional bases to the 3' hydroxyl end of at least one upstream probe, beyond the point of intended ligation.
  • the abasic site or the additional bases comprise an "overhang" and are the reason blunt-end ligation is not possible.
  • the overhang may be cleaved by a correcting reagent to expose a ligatable 3' terminus.
  • An example, of such a correcting agent is the enzyme endonuclease IV. Endonuclease IV correction is described in EP-A-439 1 82 and in more detail in PCT/US94/041 1 3 the entire disclosure of which is incorporated herein by reference.
  • Ligating reagents useful in the present invention include T4 ligase, and prokaryotic ligases such as E. coll DNA ligase, available from Molecular Biology Resources (Catalog No. 107001 , Milwaukee, Wl), Thermus aquaticus DNA ligase available from New England Biolabs (Catalog No. 208, Beverly, MA) and Pyrococc ⁇ s furious DNA ligase available from Stratagene, (Catalog No. 6001 91 , LaJolla, CA).
  • E. coll DNA ligase available from Molecular Biology Resources (Catalog No. 107001 , Milwaukee, Wl), Thermus aquaticus DNA ligase available from New England Biolabs (Catalog No. 208, Beverly, MA) and Pyrococc ⁇ s furious DNA ligase available from Stratagene, (Catalog No. 6001 91 , LaJolla, CA).
  • thermostable ligase is presently preferred for its ability to maintain activity during the thermal cycling of LCR. Absent a thermally stable ligase, the ligase must be added again each time the cycle is repeated. Also useful are eukaryotic ligases, including DNA ligase of Drosophila, reported by Rabin, et al., J. Biol. Chem. 261 :10637- 10647 (1986). Polymerizing reagents useful in the present invention include a polymerase isolated from Thermus flavus (T. fl) also available from Molecular
  • the fused (reorganized) probe is dissociated (e.g. melted) from the target and, as with conventional LCR, the process is repeated for several cycles.
  • the number of repeat cycles may vary from 1 to about 100, although from about 25 to about 50 are preferred presently.
  • spermidine reagent refers to the compound having the formula NH2-(CH2)3- H2-(CH2)3-NH2 which can be solubilized prior to its addition to the reaction mixture in an appropriate buffer.
  • inhibitor refers to the prevention of amplification of a target nucleic acid sequence where target is actually present. Inhibition may arise from the presence of inhibitory substances in the reaction mixture. For example, inhibition may occur from the presence of reagents that sequester required cofactors, and/or reagents that block active regulatory enzymatic sites. Although one or several mechanisms may be operating to cause inhibition of amplification in LCR, the actual mechanism by which inhibition occurs is not completely understood.
  • relieving inhibition means reducing the inhibitory effect of inhibitors on the amplification of a target nucleic acid sequence such that in the presence of a spermidine reagent, a measurable amount of target specific amplification product is produced in excess of that amount produced in the absence of the spermidine reagent. It is not possible to explicitly define the extent to which inhibition is relieved since the amount of inhibitor(s) present in clinical samples vary among patient samples. Thus, relief of inhibition is best defined in comparative terms.
  • spermidine for example, concentrations of spermidine effective at relieving inhibition are from 0.5 mM and about 4 mM, more typically, between about 1 mM and about 3 mM.
  • inhibitor refers to any substance that causes inhibition or prevents amplification of a target nucleic acid sequence(s) present in a clinical test sample.
  • Inhibitors may be substances endogenous to a test sample or added to the clinical test sample exogenously. Endogenous inhibitors are those substances derived naturally from the patient's system and which are therefore inherent in patient samples. Exogenous inhibitors may be any substances added to a clinical test sample for any purpose, such as during pretreatment steps.
  • exogenous inhibitors may be introduced through the addition of detergents which cause lysis of cellular membranes and concurrent release of nucleic acids and/or the addition of nuclease inhibitors which prevent the degradative action of endonucleases and exonucleases on nucleic acids.
  • reaction mixture or "amplification mixture” refers to any combination of test sample and reagents required to effect an amplification reaction.
  • Standard LCR reagents have been described in the literature and are also described in the examples.
  • the spermidine reagent allows LCR amplification under conditions of non-optimal gCl2-
  • reduced MgCt ⁇ refers to concentrations of MgU below those typically used in LCR reactions.
  • Reduced MgCl2 concentrations typically are final MgCl2 concentrations between 0.5 mM and less than 20 mM or greater than 35.5 mM.
  • the spermadine reagent is useful in a reaction mixture when amplification is performed under reduced ligase conditions.
  • Reduced ligase refers to concentrations of ligase below those typically used in LCR reactions.
  • WO 93/00447 and WO 94/03636 both to Carrino et al., describe LCR amplifications in which the amount of ligase used is 3,400 units or 5,000 units respectively, per 50 ⁇ L reaction volume (i.e. 68 to 100 units/ ⁇ L).
  • Reduced ligase concentrations refer to ligase concentrations of between about 1 ,000 units/200 ⁇ L and about 1 2,000 units/200 ⁇ L of reaction volume (i.e. 5- 60 units/ ⁇ L), more typically between about 10 units/ ⁇ L and about 50 units/ ⁇ L of reaction volume.
  • the amplified sequences can be detected by a number of conventional ways known in the art. Typically, detection is performed after separation, by determining the amount of label in the separated fraction. Of course, label in the separated fraction can also be determined subtractively by knowing the total amount of label added to the system and measuring the amount present in the unseparated fraction. Separation may be accomplished by electrophoresis, by chromatography or by affinity as in the preferred method described below.
  • haptens are attached at the available outside ends of at least two probes (opposite ends of fused product), and preferably to the outside ends of all four probes.
  • a "hook” is any moiety having an affinity to a specific binding partner.
  • the hook(s) at one end of the fused duplex product e.g. the 5 ' end of A and the 3 ' end of A'
  • the hook(s) at the other end e.g.
  • Exemplary hooks include but are not limited to biotin, fluorescein, digoxin, theophylline, phencyclidine, dansyl, 2-4-dinitrophenol, modified nucleotides such as bromouracil and others, complementary nucleotides, lectin/carbohydrate pairs, enzymes and their co-factors, and others known in the art.
  • Other exemplary hooks include adamantane acetic acid as described in U.S. Patent No.
  • exemplary ligated oligonucleotides may have a carbazole at one end and an adamantane at the other end for the detection by the IMx® instrument (Abbott Laboratories, Abbott Park, IL) using the microparticle enzyme immunoassay (MEIA) technology.
  • the assay protocol is similar to that used in the commercially available alpha-fetoprotein assay, with the following adaptations: (1 ) the anti-alpha-fetoprotein antibody coated microparticles are replaced with anti-carbazole antibody coated microparticles; and (2) the conjugates of anti-alpha-fetoprotein antibodies:alkaline phosphatase are replaced with the conjugates of anti-3 -phenyl- 1 -adamantaneacetic acid antibodies:alkaline phosphatase.
  • the protocol for the IMx® MEIA assays is further described in K. Backman et al., EP-A-439,182 published July 31 , 1991 .
  • the protocol is as follows. 100 ⁇ L of the reaction mixture which has been amplified by LCR is pipetted into the sample well. 50 ⁇ L of this sample is then pipetted into the incubation well, the anticarbazole antibody coated microparticles are added to the well. An appropriate period of incubation follows which allows the formation of a complex consisting of anticarbazole antibodies and nucleic acid sequences with the carbazole ends.
  • the mixture is pipetted onto the glass fiber capture matrix of the IMx® reaction cell, and antiadamantane antibodies conjugated to alkaline phosphatase are added.
  • the glass-fiber capture matrix is treated with 4-methylumbelliferyl phosphate (MUP).
  • MUP 4-methylumbelliferyl phosphate
  • the surface-bound enzyme converts the nonfluorogenic MUP to 4-methylumbelliferone (MU), whose fluorescence can be measured at 448 nm.
  • BSA bovine serum albumin
  • EPPS refers to a buffer of N-(2-hydroxyethyl)piperazine-N'-(3- propane-sulfonic acid).
  • EPPS-KOH refers to a buffer of EPPS adjusted to pH 7.8 with KOH
  • NAD refers to nicotinamide adenine denucleotide, an energy source for certain biological reactions.
  • EDTA refers to ethylenediaminetetraacetic acid.
  • dATP, dTTP, dCTP, and dGTP refer to deoxyribonucleotides adenosine triphosphate, thymidine triphosphate, cytosine triphosphate, and guanidine triphosphate respectively.
  • TRIS refers to Tris[hydroxylmethyl]aminomethane TE refers to a buffer of Tris-EDTA (10 mM Tris, 1 mM EDTA, pH 8.0)
  • probe pairs are labeled with a “carbazole” hapten and an adamantaneacetic acid (“adamantane”) hapten.
  • adamantane adamantaneacetic acid
  • adamantane adamantaneacetic acid
  • adamantane adamantaneacetic acid
  • adamantane adamantaneacetic acid
  • adamantane adamantaneacetic acid
  • each member of a probe partner has a different label.
  • results were read in an IMx® instrument.
  • This is commercially available from Abbott Laboratories (Abbott Park, Illinois) and is described in EP-A-288 793 and in Fiore, M. et al Clin. Chem., 34/9:1 726- 1732 (1 988).
  • a modified IMx® instrument was used, which employs a stainless steel rather than teflon coated steel pipetting probe.
  • the IMx® instrument typically generates "machine" noise or background in the range of 5-1 counts/sec/sec.
  • Other equally suitable methods of detection useful in the practice of the present invention include ELISA, EIA, and immunochromatography and nucleic acid hybridization techniques including southern blotting, dot blotting, slot blotting, solution hybridization and others well known in the art.
  • Quantities of polymerase are expressed in units, defined as follows by Molecular Biology Resources, the source of polymerase used herein: 1 unit of enzyme equals the amount of enzyme required to incorporate 10 nanomoles of total nucleotide into acid-insoluble material in 30 min at 70°C. Units of ligase enzyme are defined herein as: 1 mg of 95% purified Thermus thermophilus DNA ligase has a specific activity of about 1 x 10 * units. While this is not precisely standardized and may vary by as much as 20%, optimization is within the skill of the routine practitioner.
  • Target sequences and probes were selected so as to include a "stop base" as taught in EP 439 1 82 by K. Backman et al. published July 21 , 1 991 to terminate gap filling extension precisely at the point of ligation so that the extended probe abuts its probe partner and can be ligated to it.
  • line diluent is a buffer reagent consisting of 50 mM Tris acetate pH 7.5 used to detect machine noise in the absence of target DNA and probe. All data are expressed as IMx® rates of counts/second/second (c/s/s).
  • Respiratory samples were used throughout the examples. Unless otherwise noted, the respiratory samples were prepared using procedures routinely employed for preparing respitory specimens for culture determination of the presence of Mycobacteria. More specifically, samples were decontaminated using alkali conditons, neutralized, sedimented and resuspended.
  • oligonucleotides were synthesized following established procedures using ⁇ -cyanoethylphosphoramidites on a model 380A DNA synthesizer (Applied Biosystems, Foster City CA). A,C,G, and T have their usual meanings. Probes are written 5' to 3' from left to right. The 3' and 5'- ends of oligonucleotides were conjugated with haptens, adamantane and carbazole. The conjugation of these haptens followed standard ⁇ -cyanoethylphosphoramidite chemistry, and is described in the aforementioned hapten applications. A similar procedure is described for fluorescent label conjugates in published U.S. application NTIS ORDER No. PAT-APPL-7-246,688) (Cohen, et al., 1989).
  • Oligonucleotides corresponding to SEQ ID Nos. 2, 3, 4 and 5 were selected to detect a region (SEQ ID No. 1 , see Sequence Listing) of a cryptic plasmid found in Chlamydia trachomatis (Hatt, C, et al., Nucl. Acids Res. 1 6 (9):4053-4067 (1 988)). Oligonucleotides corresponding to SEQ ID Nos. 7, 8, 9 and 10 were selected to detect a target sequence (SEQ ID No. 6, see Sequence Listing) corresponding to nucleotides 347-390 of the protein antigen b (pab) gene in Mycobacterium tuberculosis.
  • pab protein antigen b
  • Oligonucleotides corresponding to SEQ ID Nos. 1 2, 1 3, 14 and 1 5 were selected to detect a target sequence (SEQ ID No. 1 1 , see Sequence Listing) of the Opa A gene of Neiserria gonorrhoeae and correspond to map positions 66.1 , 66.2, 66.3 and 66.4 respectively . (Stern, A., Brown, M., Nickel, P. and Meyer, T.F., Cell 47: 61 -71 (1 986)). Oligonucleotides corresponding to SEQ ID Nos. 1 7, 1 8, 1 9 and 20 were selected to detect an unmapped genomic sequence (SEQ ID No. 1 6) from Mycobacterium tuberculosis.
  • oligonucleotides were purified by reversed-phase HPLC or by PAGE electrophoresis (Maniatis, T., et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1972) to remove failure sequences and, in the case of haptenated oligos, any unhaptenated species.
  • target nucleic acid Chomydia trachomatis (C. trachomatis) DNA
  • C. trachomatis C. trachomatis
  • amplification of target DNA i.e. C. trachomatis
  • C. trachomatis target DNA
  • the presence of 1 mM spermidine was sufficient to relieve inhibition of C. trachomatis DNA amplification (as shown by increased MEIA rates of samples Ul 3 and 12 in Table 2a and increased MEIA rates of samples Ul 1 , 23, 24, 27, 34,
  • HP DNA 165 N.D.* N.D. refers to not performed
  • Example 2 Experiments were performed in the presence of negative clinical samples (described above) using the experimental conditions of Example 1 for 100 ⁇ L reactions with the modification that test samples were first subjected to gel filtration chromatography prior to use in LCR reactions. This procedure is a partial purification step that helps to remove LCR inhibitors from the sample.
  • Spun columns were prepared as 5 mL packed bed volumes of Sephadex G-50-80 (Sigma) in plastic screening columns purchased from Baxter S/P (Catalogue #P51 94). 0.5 mL of sample was loaded per column.
  • the nucleic acid was eluted with TE by placing the column within a collection tube and spinning at 1 600 rpm for 5 minutes at 1 5°C in a Beckman Instruments TY.JS 4.2 rotor in a J6B centrifuge.
  • spun column chromatography was ineffective at removing all inhibitors from patient samples prior to amplification by LCR.
  • the addition of spermidine at 1 mM concentration was effective at relieving inhibition of amplification in the presence of these partially purified samples.
  • EPPS-KOH buffer 20 mM KCI, 30 mM MgCl2, 1 .7 ⁇ M each of dCTP and dATP, 10 ⁇ M NAD, approximately 1 x 10 1 2 molecules each of SEQ ID Nos. 7, 8, 9 and 10, 25 genomes of M. tb DNA, 2 units of T. fl polymerase, and 18,000 units of T. th DNA ligase. 25 genomes of M. tb DNA was calculated based on the published genome size of M. tb DNA (Baess, I., Acta Path. Microbiol. Immunol. Scand., Sect.
  • target DNA was from either M. tb or C. trachomatis .
  • M. tb reactions were performed in 200 ⁇ L total reaction volume in 50 mM EPPS- KOH buffer, 50 mM KCI, 2 mM spermidine, 1.7 ⁇ M each dCTP and dTTP, 10 ⁇ g/mL BSA, 10 ⁇ M NAD, 1 x 10 1 2 molecules each of SEQ ID Nos. 17, 1 8, 1 9 and 20, 2 units of T. fl polymerase, 18,000 units of 7. th DNA ligase, 25 genomes of M.
  • tb DNA and MgCl2 as indicated below. It should be noted that for purposes of this and all subsequent examples, the KCI concentration used was 50 mM. (Preliminary experiments had shown that signal from background amplification was reduced in reaction mixtures containing 50 mM KCI while the signal from target amplification was unaffected (data not shown). Accordingly, this concentration was used to further reduce the background signal). Parallel controls were run under identical reaction conditions with 2 ⁇ g of HP DNA. Standard positive and negative control reactions were performed under the same set of conditions with the following modifications: 20 mM KCI, 20 mM MgCl2 and no spermidine.
  • reaction mixtures were incubated at room temperature (approximately 22°C) for 2 hours. Cycling was then performed at 93°C for 1 second, 65°C for 1 second and 68°C for 1 minute and aliquots counted by IMx® as described.
  • MgCl2 concentrations could be reduced to 1 .0 mM and still effect LCR amplification of target DNA as efficiently as the positive control. Furthermore, non-target directed amplification was not apparent throughout the range tested since variations in MgCl2 concentrations did not effect amplification of HP DNA at any concentration tested.
  • SEQ ID No. 1 i.e. synthetic target
  • MgCl2 MgCl2 as indicated below.
  • Final reaction volumes were again 200 ⁇ L. Cycling was performed at 97 ⁇ C for 1 second, 55°C for 1 second and 62°C for 50 seconds. Aliquots of samples were counted on an
  • IMx® instrument as described. As shown in Table 7b, spermidine again substantially reduced the gCl2 concentration range at which amplification could be effected.
  • EXAMPLE 8 Optimal Range of Spermidine Concentrations that Effect Amplification Under Reduced oU£ Conditions
  • Reactions were performed in 200 ⁇ L total volume and contained the following reagents: 50 mM EPPS-KOH buffer, 50 mM KCI, 2 mM MgCl2, 1 .7 ⁇ M dGTP, 10 ⁇ M NAD, 1 .4 x 10 1 1 molecules each of SEQ ID Nos. 1 2, 13, 14 and 1 5, 10 ⁇ M BSA, 18,000 units 7. th DNA ligase, 2.0 units 7.
  • N. gonorrhoeae Neisseria gonorrhoeae DNA. 254 genomes was calculated from the DNA concentration of the sample preparation (as determined by OD26O) and the weight per genome of N. gonorrhoeae DNA (calculated as 2.3 femtograms DNA per genome). Spermidine was added to final concentrations ranging from 0 to 3 mM. Cycling conditions were established at 97°C, for 1 second, 55°C for 1 second and 62°C for 50 seconds.
  • Positive and negative control reactions were performed under essentially the same set of conditions with the following modifications: 20 mM KCI, 30 mM MgCl2 and no spermidine.
  • target DNA was replaced with 1 50 ng of salmon sperm DNA (Sigma). Sample aliquots were counted on an IMx® instrument as described.
  • Example 8a Experiments were performed under identical conditions to those in Example 8a with the following substitutions of deoxynucleotides, probes, and target DNA : 1.7 ⁇ M each of dCTP and dTTP, 4.5 x 10 1 ⁇ oligos of C. trachomatis probe set 6917, and 25 molecules of SEQ ID No. 1 (i.e. synthetic target). Cycling conditions and controls were as described. As shown in Table 8b, in the presence of spermidine at concentrations ranging from 2-3 mM, amplification of C. trachomatis DNA was accomplished to nearly the same extent as that of the positive control.
  • Target DNA was amplified under standard reaction conditions (i.e. in the absence of spermidine) and under modified reaction conditions (i.e. in the presence of spermidine) as follows:
  • T. thermophilus ligase were as indicated below.
  • ORGANISM Chla ydia trachomatis
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:1: GACTTTGCAA CTCTTGGTGG TAGACTTGGT CATAATGGAC TTTTGTTG 48
  • MOLECULE TYPE synthetic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:2 :
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE synthetic DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:9 :
  • MOLECULE TYPE DNA (genoraic) (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE synthetic DNA
  • MOLECULE TYPE DNA (genomic)
  • ORIGINAL SOURCE
  • ORGANISM Mycobacterium tuberculosis
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 16:
  • MOLECULE TYPE synthetic DNA
  • MOLECULE TYPE synthetic DNA

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Abstract

Procédé amélioré d'amplification d'acides nucléiques cibles et en particulier procédé d'atténuation des effets inhibiteurs de certains échantillons lors de réactions d'amplification en chaine par ligases. L'atténuation de l'inhibition s'obtient par adjonction au mélange d'amplification d'une quantité efficace d'un réactif de type polyamine tel que la spermidine, par exemple de 1 à 4 mM. Dans des variantes, la présence de la spermidine permet l'amplification en présence de concentrations réduites de MgCl2 et de ligase.
PCT/US1995/012874 1994-10-21 1995-10-18 Emploi de spermidine pour attenuer les effets inhibiteurs de reactions en chaine de ligase dans un echantillon d'essai clinique WO1996012824A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP51396296A JP2002501361A (ja) 1994-10-21 1995-10-18 臨床検査試料におけるリガーゼ連鎖反応の阻害を軽減するためのスペルミジンの使用
EP95938175A EP0791075A2 (fr) 1994-10-21 1995-10-18 Emploi de spermidine pour attenuer les effets inhibiteurs de reactions en chaine de ligase dans un echantillon d'essai clinique

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US33139194A 1994-10-21 1994-10-21
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US08/532,212 1995-10-12

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136533A (en) * 1997-07-03 2000-10-24 Id Biomedical Additives for use in cycling probe reactions
WO2000071698A2 (fr) * 1999-04-30 2000-11-30 Connex Gesellschaft Zur Optimierung Von Forschung Und Entwicklung Mbh Melange pour neutraliser des inhibiteurs d'enzymes
US20150218608A1 (en) * 2014-01-31 2015-08-06 New England Biolabs, Inc. Compositions and Methods for Improvement of Ligation Yields
WO2017170376A1 (fr) 2016-03-30 2017-10-05 和光純薬工業株式会社 Jeu d'amorce pour la détection de chlamydia trachomatis, méthode de détection de chlamydia trachomatis utilisant ce dernier, et kit de réactifs associé

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WO1991017239A1 (fr) * 1990-05-03 1991-11-14 Cornell Research Foundation, Inc. Systeme d'amplification d'adn induit par ligase thermostable, servant a detecter des maladies genetiques.
EP0320308B1 (fr) * 1987-12-11 1993-11-03 Abbott Laboratories Procédé pour détecter une séquence cible d'acide nucléique

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JPS6236187A (ja) * 1985-08-09 1987-02-17 Takara Shuzo Co Ltd Ｄｎａリガ−ゼ組成物及びその使用方法
JP3416981B2 (ja) * 1993-03-30 2003-06-16 株式会社島津製作所 核酸合成法

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EP0320308B1 (fr) * 1987-12-11 1993-11-03 Abbott Laboratories Procédé pour détecter une séquence cible d'acide nucléique
WO1991017239A1 (fr) * 1990-05-03 1991-11-14 Cornell Research Foundation, Inc. Systeme d'amplification d'adn induit par ligase thermostable, servant a detecter des maladies genetiques.

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DATABASE WPI Week 8712 Derwent Publications Ltd., London, GB; AN 87-084125 XP002001117 "dna synthetase composition option temperature degree contain dna synthetase polyethylene glycol one mono di valence cation poly amine " & JP,A,62 036 187 (TAKARA SHUZO KK) , 17 February 1987 *
DATABASE WPI Week 9444 Derwent Publications Ltd., London, GB; AN 94-353745 XP002001116 "nucleic acid amplify presence poly amine efficiency remove inhibit cause impure biological sample" & JP,A,06 277 061 (SHIMADZU CORP.) , 4 October 1994 *
See also references of EP0791075A2 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136533A (en) * 1997-07-03 2000-10-24 Id Biomedical Additives for use in cycling probe reactions
WO2000071698A2 (fr) * 1999-04-30 2000-11-30 Connex Gesellschaft Zur Optimierung Von Forschung Und Entwicklung Mbh Melange pour neutraliser des inhibiteurs d'enzymes
WO2000071698A3 (fr) * 1999-04-30 2001-02-15 Connex Ges Zur Optimierung Von Melange pour neutraliser des inhibiteurs d'enzymes
US20150218608A1 (en) * 2014-01-31 2015-08-06 New England Biolabs, Inc. Compositions and Methods for Improvement of Ligation Yields
WO2017170376A1 (fr) 2016-03-30 2017-10-05 和光純薬工業株式会社 Jeu d'amorce pour la détection de chlamydia trachomatis, méthode de détection de chlamydia trachomatis utilisant ce dernier, et kit de réactifs associé

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