EP0791075A2 - Use of spermidine to relieve inhibition of ligase chain reaction in a clinical test sample - Google Patents

Abstract

An improved method of amplifying target nucleic acids and particularly, to a method of alleviating the inhibitory effect of certain samples on ligase chain reaction amplifications. The relief of inhibition is accomplished by adding to the amplification mixture a polyamine reagent such as spermidine, in effective amounts, typically 1 to 4 nM. In other embodiments, the presence of spermidine allows for amplification to proceed in the presence of reduced concentrations of MgCl2 and ligase.

Description

USE OF SPERMIDINE TO RELIEVE INHIBITION OF LIGASE CHAIN REACTION IN A CLINICAL TEST SAMPLE This application is a continuation-in-part of co-pending U.S. Patent Application Serial number 08/331 ,391 , filed October 21 , 1994, the entirety of which is herein incorporated by reference.

BACKGROUND

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.

According to one LCR method, 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. In addition, 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. Of course, if the target is initially double stranded, the secondary probes in the first instance will also hybridize to the target complement. 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.

One problem associated with amplification of target nucleic acids in clinical samples is inhibition of amplification. While not completely understood, 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.

This results in the erroneous classification of the sample as negative for that agent and may lead to the improper diagnosis of a patient's condition.

Attempts have been made to overcome the problem of inhibition by purifying the target nucleic acid from endogenous and/or exogenous sample contaminants prior to LCR amplification. The literature describes various purification schemes well known to those skilled in the art, such as phenol extraction/ethanol precipitation, ion exchange chromatography, and purification gradients such as cesium chloride. These procedures add labor and time- consuming steps to the sample analysis.

Another problem associated with LCR amplification occurs through the improper addition of reaction reagents. This problem arises particularly when utilizing variant LCR technologies (mentioned above) which require the presence of high concentrations of MgCl2 that span a relatively narrow range (e.g. 25 mM- 35 mM). Concentrations of MgCl2 added to the amplification mixture that are either greater than or less than this optimal range may inhibit amplification. Consequently, in diagnostic assays which require the user to add MgCl2, caution must be exercised to avoid introducing either an excess or an insufficient quantity of MgCl2 into the reaction mixture. There is thus a need for an improved LCR method which requires fewer labor and time consuming pre-amplification purification procedures and which minimizes the potential for false results associated non-optimal addition of amplification reagents such as MgCtø-

SUMMARY OF THE INVENTION

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:

(a) providing a clinical test sample with an amount of spermidine effective to relieve amplification inhibition of and a 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, the two primary probes hybridizing at adjacent or near adjacent positions on the target, wherein at least one of the primary or secondary probes is modified at one end to render it non- -3- ligatable to the other primary or secondary probe, respectfully, and;

(b) hybridizing the primary probes to the target and optionally, hybridizing the secondary probes to the target complement;

(c) correcting the modification in a target dependent manner to render the primary probes ligatable to one another when hybridized to target and optionally, to render the secondary probes ligatable to one another when hybridized to target complement;

(d) ligating the primary probes and optionally, ligating the secondary probes to form a fused product; and (e) dissociating the fused product from the target and repeating the hybridization, correction and ligation steps to amplify the desired target sequence. In another embodiment, the improved method comprises the steps of:

(a) providing a clinical test sample with

(i) an amount of spermidine effective to relieve amplification inhibition and

(ii) a 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;

(b) hybridizing the primary probes to the target;

(c) ligating the primary probes to form a fused product; and

(d) dissociating the fused product from the target. 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. In addition to the unexpected reduction of inhibition afforded in LCR amplification in a clinical test sample, 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.

DETAILED DESCRIPTION OF THE INVENTION

As previously mentioned, 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".

For purposes of the present invention, the term "clinical test sample" or

"clinical sample" is a sample, taken from a mamalian 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. Thus, for example, a clinical test sample can be blood, serum, sputum or other mamalian body fluid which is centrifuged and resuspended in a buffer contining a lysing agent.

It was also discovered that by using spermidine as taught herein, MgU concentrations that previously have been found to inhibit LCR amplification by not supporting LCR amplification, no longer inhibit and thereby support LCR amplification. Thus, effective 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, as used herein, 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.

In accordance with modified LCR procedures, 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

"initiator") and the ligated or extended product is referred to as an

"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. In another aspect, 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.

In general, 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. Similarly, 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. Thus a high G:C content and longer probe lengths impact the "hybridization conditions" by elevating the melt temperature.

Hybridization of probes to target (and optionally to target complement) is widely known in the art and is illustrated in EP-A-320 308. Probe length, probe concentration and stringency of conditions affect the degree and rate at which hybridization will occur. Preferably, the probes are sufficiently long to provide the desired specificity; i.e., to avoid being hybridizable to nontarget sequences in the sample. Typically, probes on the order of 1 5 to 100 bases serve this purpose. Presently preferred are probes having a length of about 1 5 to about 40 bases.

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 μL, this is equivalent to adding from about 3 x 10¹ ^ to about 1 .2 x 10^{1 2} molecules of each probe; and around 1 x 10^{1 2} molecules per 200 μL 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.

Following addition of the probes, the next step in modified LCR methods is the specific correction step followed by the ligation of one probe to its adjacent partner. Thus, 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.

Since enzymatic ligation is the preferred method of covalently attaching two adjacent probes, the term "ligation" will be used throughout the application.

However, "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.

In the gap filling method referred to above, 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). In order for LCR to amplify the target, 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. In a second method of correction, 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. While the ligation incompetent probe is hybridized to target, 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. In another variation, an incompetent 5' end is created by a mismatched base(s) with respect to target within the downstream probe. In this situation, 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. In a third method of correction, 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.

The conditions and reagents which make possible the preferred enzymatic ligation step are generally known to those of ordinary skill in the art and are disclosed in the references mentioned in the background. 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 Pyrococcυs furious DNA ligase available from Stratagene, (Catalog No. 6001 91 , LaJolla, CA). A 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

Biology Resources or Taq DNA polymerase isolated from Thermus aquaticus

(available from several commercial sources including Strategene, Promega and

Perkin Elmer). Once ligated, 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.

During the course of amplification, inhibitors may be present within the sample that prevent the amplification of a target nucleic acid sequence(s). As provided in the present invention, a spermidine reagent is be added to the amplification reaction mixture in order to relieve this inhibitory effect. As used herein, spermidine or 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.

As used herein, "inhibition" 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.

As used herein, "relieving inhibition" or "relief of 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. For example, relief of inhibition is achieved when, in the amplification of target DNA from a patient sample (or in the presence of patient sample), a measurable amount of target specific amplification product is produced in the presence of spermidine in excess of that amount produced in the absence of spermidine, where equivalent amounts of patient sample were amplified under both conditions. Generally, 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.

As used herein, "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. For example, 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.

As used herein, "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.

In another embodiment of the present invention, the spermidine reagent allows LCR amplification under conditions of non-optimal gCl2- For example, reduced MgCtø 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.

In yet another embodiment of the present invention, 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. For example, 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 μL reaction volume (i.e. 68 to 100 units/μL). Reduced ligase concentrations according to the present invention refer to ligase concentrations of between about 1 ,000 units/200 μL and about 1 2,000 units/200 μL of reaction volume (i.e. 5- 60 units/μL), more typically between about 10 units/μL and about 50 units/μL of reaction volume.

Following amplification, 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.

In a particularly preferred configuration, haptens, or "hooks" (a subset of the generic terms "reporter" or "label"), 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. Typically, the hook(s) at one end of the fused duplex product (e.g. the 5^' end of A and the 3 ^' end of A') comprises an antigen or hapten capable of being immobilized by a specific binding reagent (such as antibody or avidin) coated onto a solid phase. The hook(s) at the other end (e.g. the 3^' end of B and the 5^' end of B^' ) contains a different antigen or hapten capable of being recognized by a label or a label system such as an antibody-enzyme conjugate. 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. 5,424,414 and carbazole and dibenzofuran derivatives as described in co-owned, co-pending U.S. Patent Application Serial No. 08/084,495 filed July 1 , 1993 both of which derive priority from applications filed December 17, 1 991 and both of which are incorporated herein by reference. A method for adding a hapten to the 3 '-end of an oligonucleotide is disclosed in co-owned U.S. Patent Number 5,290,925 filed December 20, 1990. Other methods (e.g. Amino Modifier II, Clontech, Palo Alto, California) are known and commercially available for labeling 3 ^' and 5' ends. The method for adding a hapten to the 5^' end is through the use of a phosphoramidite reagent as described in Thuong, N.T. et al., Tet. Letters, 29(46): 6905-5908 (1 988), or Cohen, J.S. et al., U.S. Patent Application Serial Number 07/246,688, abandoned (NTIS order no. Pat-Appl-7-246,688 (1 988). Thus, 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 . In brief, the protocol is as follows. 100 μL of the reaction mixture which has been amplified by LCR is pipetted into the sample well. 50 μL 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. After the incubation, the mixture is pipetted onto the glass fiber capture matrix of the IMx® reaction cell, and antiadamantane antibodies conjugated to alkaline phosphatase are added. This leads to a microparticle- oligonucleotide-enzyme complex which will stay near the surface of the glass fiber capture matrix. After the removal of excess reagent in a wash step (throughout this protocol, the blotter beneath the glass fiber capture matrix absorbs reagent solutions which would otherwise overflow the glass fiber capture matrix), the glass-fiber capture matrix is treated with 4-methylumbelliferyl phosphate (MUP). The surface-bound enzyme converts the nonfluorogenic MUP to 4-methylumbelliferone (MU), whose fluorescence can be measured at 448 nm. The numerical values given in the following examples are the rate reads of this process, expressed in counts/sec/sec (c/s/s). The amount of ligated probes is related to this rate. This concept of MEIA readout of labeled oligonucleotides is described in European Patent Application, publication No. 357,01 1 , published March 7, 1 990, "Detection and Amplification of Target Nucleic Acid Sequences," to Laffler, T.G., et al.; and elsewhere.

EXAMPLES

The invention will now be described further by way of examples. The examples are illustrative of the invention and are not intended to limit the invention in any way. Throughout the examples the following abbreviations have the meanings given. BSA refers to 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)

In the illustrative examples which follow, probe pairs are labeled with a "carbazole" hapten and an adamantaneacetic acid ("adamantane") hapten. Typically, "adamantane" and "carbazole" are used together in accordance with the description above, although any combination of virtually any haptens would be possible. Preferably, each member of a probe partner has a different label.

In all of the examples, 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). It should be noted that for purposes of the following examples, 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.

For the purposes of the following examples line diluent (LD) 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.

Example 1 Oliαonucleotide Synthesis and Haptenation

The following oligonucleotides (see Table 1 ) were synthesized following established procedures using β-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 β-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).

Table 1

Sequence ID

No SEQUENCE

2. CZ-GACTTTGCAACTCTTGGTGGTAGA

3. ACCACCAAGAGTTGCAAAGTC-CZ

4. GG I CAI AA I GGAC I I I I G I I G-AU

5. AD-CAACAAAAGTCCATTATGACCAAG

7. CZ-AACCTGTGGGGTCCGGCCTTT

8. GGCCGGACCCCACAGGTT-CZ

9. AD-GAGAGGTATCCGAACGTCAC

10. GTGACGTTCGGATACCTCTCGTG-AD

1 2. CZ-GCCATATTGTGTTGAAACACCGCCC

1 3. CGGTGTTTCAACACAATATGGC-CZ

14. AACCCGATATAATCCGCCCTT-AD

1 5. AD-AAGGGCGGATTATATCGGGTTCC

17 CZ-CCGACTGGGCAATTGGCTAAAGG

1 8 TTAGCCAATTGCCCAGTCGG-CZ

19 GCATCGGCGTCGGCACG-AD

20 AD-CGTGCCGACGCCGATGCGGG

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. 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.

All 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.

EXAMPLE 2 Effect of Spermidine on Relieving Inhibition of Target Directed DNA

Amplification bv LCR To determine the effect of spermidine on relieving inhibition of target directed DNA amplification during LCR, target nucleic acid (Chlamydia trachomatis (C. trachomatis) DNA ) was amplified in the presence of negative clinical sample and in either the presence or absence of 1 mM spermidine.

Reactions were run in 50 mM EPPS buffer adjusted to pH 7.8 with KOH (EPPS- KOH buffer), 0.5 mM EDTA, 1 0 μM NAD, 3 to 6 μL of an approximate 10⁸ fold dilution of C. trachomatis infected McCoy cells (this amount was empirically chosen to yield IMx® signals in the range of about 500-1 300 c/s/s), 4.8-5 x 10^{1 1} molecules each of SEQ ID Nos. 2, 3, 4, and 5, 100 μL of clinical sample, 1 .7 μM each of dCTP and dTTP, 18,000 units Thermus thermophilus (T. th) DNA ligase and 2 units of Thermus flavus (T. fl) polymerase (Molecular Biology Resources, Milwaukee, Wl, cat. no 1070.01 ) in a final reaction volume of 200 μL. Probes were labeled with carbazole and adamantane as per Example 1. Control reactions were performed in buffer alone (i.e. in the absence of clinical sample) with either human placental (HP) DNA (Sigma) as a negative control or McCoy cell lysate as a positive control. Cycling was performed on a Perkin Elmer model 480 thermocycler at the following settings: 97°C, 1 second; 55°C, 1 second; 62°C, 50 seconds for 40 cycles. LCR amplification products were detected via a sandwich immunoassay performed using the Abbott IMx® automated immunoassay system. The results are shown in Table 2a.

Experiments were also performed as above in total reaction volumes of 100 μL. Probe and enzyme concentrations and clinical sample volumes were decreased proportionally (i.e. 2.4 x 10^{1 1} molecules of probes, 9,000 units ligase, 1 unit polymerase and 50 μL of clinical sample) in these reactions. The results are shown in Table 2b.

As seen in Tables 2a and 2b, amplification of target DNA (i.e. C. trachomatis) performed in the presence of clinical sample but in the absence of spermidine, showed little or no LCR amplification relative to a positive control. 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,

45, 60 and 68 in Table 2b).

TABLE 2a

MEIA Rate (c/s/s)

Sample 0 mM Spermidine 1 mM Spermidine

UI 3 1 2 1088 Ul 1 2 103 1009

McCoy lysate 1231 1 196 HP DNA 226 38

TABLE 2b

MEIA Rate (c/s/s)

Sample 0 mM Spermidine 1 mM Spermidine

Ul 1 2 39 555

UI 23 1 1 101 6

Ul 24 1 1 1045

UI 27 130 975

UI 34 43 247

UI 45 10 638

UI 60 277 914

UI 68 14 31 9

McCoy lysate 969 N.D.*

HP DNA 165 N.D.* N.D. refers to not performed

EXAMPLE 3 Gel Filtration Chromatooraohv

Experiments were performed in the presence of negative clinical samples (described above) using the experimental conditions of Example 1 for 100 μL 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. As shown in Table 3, 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 however, was effective at relieving inhibition of amplification in the presence of these partially purified samples.

TABLE 3

MEIA Rate (c/s/s)

Sample 0 mM Spermidine 1 mM Spermidine

NY 65 21 1 57

NY 77 1 8 758

NY 83 36 570

Ul 6944 14 51 0

Ul 6980 1 7 51 9

McCoy lysate 652 N.D.

HP DNA 14 N.D.

EXAMPLE 4

Effect of Spermidine on Relieving Inhibition of Target Directed DNA Amplification in the Presence of Varying Amounts of Inhibitor Experiments were performed to determine the effect of spermidine on relieving inhibition of target directed DNA amplification in the presence of a range of inhibitor concentrations. In this example, amplification of Mycobacterium tuberculosis (M. tb) DNA was performed in the presence of increasing amounts of negative clinical samples (described in Example 1 ). Reactions were performed in 200 μL total reaction volume containing 50 mM

EPPS-KOH buffer, 20 mM KCI, 30 mM MgCl2, 1 .7 μM each of dCTP and dATP, 10 μM 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. B 92: 209-21 1 , (1984)) and the DNA concentration of the sample preparation as determined either by OD26O or by DABA (diamino benzoic acid) reaction. Amplification reactions were performed both in the presence and absence of 1 mM spermidine. The volumes of clinical sample used per reaction were as indicated below. In control reactions, M. tb DNA and HP DNA, (positive and negative controls respectively), were amplified in the absence of clinical sample. Cycling was performed at 93°C, for 1 second; 65^βC, for 1 second; 68°C, for 1 minute, 1 5 seconds for 40 cycles.

As shown in Table 4, the addition of increasing amounts of sample volume to reaction mixtures resulted in the inhibition of amplification of M. tb DNA.

Spermidine, at 1 mM concentration, was effective at relieving inhibition resulting from the presence of increased amounts of inhibitor(s).

TABLE 4

MEIA Rate (c/s/s/) Vol. of Sample

Sample (in (μl)) 0 mM Spermidine 1 mM Spermidine

M181 3 952 1 818

M181 10 699 1088

M181 30 1 7 910

M181 90 23 1 5

M.tb DNA 0 1813 1766

HP DNA 0 24 22

Ul 176 3 2365 2021

Ul 176 10 2067 2057

Ul 176 30 1 31 2 21 86

Ul 176 100 24 1 538

Ul 183 3 2330 1765

Ul 1 83 10 21 78 2020

Ul 1 83 30 1 900 1918

Ul 183 100 80 1989

Ul 199 3 2256 2001

Ul 199 10 2322 1 995

Ul 199 30 24 1776

Ul 199 100 37 1 341

M.tb DNA 0 2053 1709

HP DNA 0 29 1 9 EXAMPLE 5 Optimal Rangp nf Spermidine Concentrations Effective at Relieving Inhibition of Amnlification in the Presence of Clinical Samples Experiments were performed to determine the optimal range of spermidine concentrations that would effectively relieve inhibition of amplification of target nucleic acid in the presence of a varying amounts of clinical samples. Experimental conditions are described in Example 4. Sample volumes used per reaction were as indicated below.

The results in Table 5 are indicative of MEIA rates for LCR reactions performed in the presence of 1 mM and 3 mM spermidine. As shown, 1 mM spermidine was generally at least as effective or better than 3 mM spermidine in relieving inhibition of amplification.

TABLE 5

MEIA Rate (c/s/s)

Sample

UI194 10 1936 2264 2096

UI194 30 28 2349 2319

UI199 10 2304 2532 2116

UI199 30 31 2050 2015

UI199 100 34 696 109

M181 10 1462 2157 2003

M181 30 33 1660 583

M181 100 25 23 22

M249 3 1829 2797 2796

M249 10 25 2400 2793

M249 30 38 48 2064

M. tb DNA - 2193 2361 2024

HP DNA - 27 37 37

EXAMPLE 6 Magnesium Ion Conentration in the Presence of Spermidine

Experiments were performed to determine the effect of spermidine on the concentration of MgU2 required to effect LCR amplification. Specifically, experiments were performed to determine whether amplification could be effected at MgCl2 concentrations lower than those typically used in modified LCR reactions

(i.e. 25 to 35 mM MgCl2)- For this experiment, 25 genomes of M. tb DNA was amplified in 50 mM EPPS-KOH buffer, 20 mM KCI, 1 .7 μM each of dCTP and dATP, 10 μM NAD, approximately 1 x 10¹² molecules each of SEQ ID Nos. 7, 8, 9 and 10, 2 units of 7. fl polymerase, 18,000 units of 7. th DNA ligase and varying concentrations of gCl2 (indicated below). Total reaction volumes were 200 μL.

Cycling conditions were 93"C for 1 second, 63^βC for 1 second and 66°C for 40 second for a total of 40 cycles. As Table 6 shows, in the presence of 1 mM spermidine LCR amplification could be achieved with concentrations of MgCl2 as low as 5 and 10 mM.

TABLE 6

MEIA Rate (c/s/s)

Sample MgCl (mM) 0.0 mM Spermidine S.D.* 1 .0 mM Spermidine S.D.*

5.0 6 - 1 88 1 5

10.0 139 1 5 949 144

M. tb DNA 20.0 1 262 78 1 383 1 32

30.0 1 210 46 1 317 39

40.0 96 78 352 1 63

M. tb DNA 30.0 1 210 46 N.D. N.D.

HP DNA 30.0 6 0 N.D. N.D.

*S.D. refers to standard deviation

EXAMPLE 7 Range of Magnesium Ion Concentrations that Effect LCR Amplification in the Presence of Spermidine

Experiments were performed to determine the range of magnesium ion concentrations at which amplification could be effected in the presence of spermidine. In this case target DNA was from either M. tb or C. trachomatis . M. tb reactions were performed in 200 μL total reaction volume in 50 mM EPPS- KOH buffer, 50 mM KCI, 2 mM spermidine, 1.7 μM each dCTP and dTTP, 10 μg/mL BSA, 10 μM 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 μg 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. After mixing all reagents, including the enzymes, 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.

As shown in Table 7a, 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.

TABLE 7a

Target MgCl2 Cone. (mM) MEIA Rate (c/s/s) S.D.

0.0 1 1 1

0.5 710 50

M. tb DNA 1 .0 1443 14

1 .5 1463 28

2.0 1 513 4

2.5 1 406 1 33

0.5 14 3

1 .0 10 1

HP DNA 1 .5 1 5 6

2.0 9 1

2.5 1 5 6

Standard Contro s

M. tb DNA 20 1 634 50 HP DNA 20 480 480

Essentially identical experiments were performed using C. trachomatis DNA as target DNA. C. trachomatis reactions were performed in 50 mM EPPS-

KOH buffer, 50 mM KCI, 2.5 mM spermidine, 1.7 μM each dCTP and dTTP, 10 μg/mL BSA, 10 μM NAD, 4.5 x 10^{1 ]} molecules each of SEQ ID Nos. 2, 3, 4 and 5,

1 .5 units of 7. fl polymerase, 1 ,800 units of 7. th DNA ligase, 25 molecules of

SEQ ID No. 1 (i.e. synthetic target) and MgCl2 as indicated below. Final reaction volumes were again 200 μL. Cycling was performed at 97^βC 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.

TABLE 7b

Target MgCl2 Cone. (mM) MEIA Rate (c/s/s) S.D.

0.0 1 3.0 0.0

0.5 1714.0 48.0

1.0 1 863.0 48.0

C. trachomatis 1 .5 1 940.0 8.0

2.0 1 955.0 45.0

2.5 1 957.0 1 6.0

3.0 2005.0 24.0

Standard Control

C. trachomatis 20.0 1 206.0 98.0

EXAMPLE 8 Optimal Range of Spermidine Concentrations that Effect Amplification Under Reduced oU£ Conditions Experiments were performed to determine the optimal range of spermidine concentrations that would effect LCR amplification in the presence of a reduced concentration of MgCl2 (i.e. 2 mM). Reactions were performed in 200 μL total volume and contained the following reagents: 50 mM EPPS-KOH buffer, 50 mM KCI, 2 mM MgCl2, 1 .7 μM dGTP, 10 μM NAD, 1 .4 x 10^{1 1} molecules each of SEQ ID Nos. 1 2, 13, 14 and 1 5, 10 μM BSA, 18,000 units 7. th DNA ligase, 2.0 units 7. fl DNA polymerase and 254 genomes of Neisseria gonorrhoeae (N. 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. In addition, in the negative control, target DNA was replaced with 1 50 ng of salmon sperm DNA (Sigma). Sample aliquots were counted on an IMx® instrument as described.

As shown in Table 8a, the concentration of MgCl2 typically used to effect LCR amplification of N. gonorrhoeae DNA is 30 mM. (See positive control, N. gonorrhoeae DNA). When the MgCl2 concentration was reduced to 2 mM, amplification was not accomplished in the absence of spermidine. (See sample 1 , MEIA rate = 1 8.0). However, as shown in samples 6 and 7, amplification of N. gonorrhoeae DNA was accomplished to nearly the same extent as that of the positive control in the presence of spermidine at concentrations ranging from 2.5 to 3 mM.

Table 8a Sample Spermidine (mM) Avg. MEIA Rate (c/s/s) S.D.

0.0 18.0 6.0

0.5 10.0 2.0

1.0 13.0 1 .0

N. gonorrhoeae DNA 1.5 63.0 6.0

2.0 202.0 1 2.0

2.5 659.0 3.0

3.0 637.0 93.0

Standard controls

N. gonorrhoeae DNA 0.0 81 2.0 10.0

Salmon sperm DNA 0.0 7.0 0.0

Experiments were performed under identical conditions to those in Example 8a with the following substitutions of deoxynucleotides, probes, and target DNA : 1.7 μM each of dCTP and dTTP, 4.5 x 10^{1 λ} 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.

TABLE 8b

Sample Spermidine (mM) Avq. MEIA Rate (c/s/s) S.D.

0.0 31 .0 7.0

0.5 17.0 2.0

1.0 213.0 4.0

C. trachomatis 1 .5 1 372.0 1 2.0

2.0 1 891.0 26.0

2.5 2070.0 34.0

3.0 21 26.0 1 6.0

Standard controls

C. trachomatis - 2046.0 89.0

Salmon sperm - 69.0 58.0

DNA

EXAMPLE 9 Ligase Reguirement in Presence of Spermidine

Experiments were performed to determine the optimal range of ligase concentrations required to effect amplification of C. trachomatis in the presence of spermidine. 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:

Reagents Standard Conditions Modified Conditions

EPPS-KOH buffer 50.0 mM 50.0 mM

KCI 20.0 mM 50.0 mM

MgCl2 30.0 mM 4.0 mM

EDTA 0.5 mM 0.0 mM

Spermidine 0.0 mM 2.5 mM

BSA 1 0.0 μg/mL 10.0 μg/mL

NAD 10.0 μM 10.0 μM dCTP, dTTP (each) 1 .7 μM 1 .7 μM

7. fl polymerase 2.0 units 1 .5 units

SEQ ID Nos. 2, 3, 4, and 5 4.5 xl O^{1 1} 4.5 xl O^{1 1}

C. trachomatis DNA 10.0 genomes 10.0 genomes

Total reaction volumes were 200 μL. Final concentrations of T. thermophilus ligase were as indicated below.

The results in Table 9 show that in the presence of spermidine, a 10-fold less concentration of ligase could be used to effect amplification.

TABLE 9

MEIA Rate (c/s/s)

Ligase (units/μL) Std. Cond. S.D. Mod. Cond. S.D.

0.0 11.0 1.0 17.0 2.0

9.0 425.0 53.0 1494.0 24.0

22.5 1066.0 69.0 1638.0 44.0

45.0 1243.0 39.0 1661.0 48.0

67.5 1297.0 70.0 1634.0 49.0

90.0 1306.0 56.0 1631.0 7.0

While the invention has been described in detail and with reference to specific embodiments, it will be apparent to one skilled in the art that various changes and modifications may be made to such embodiments without departing from the spirit and scope of the invention. Additionally, all patents and publications mentioned above are herein incorporated by reference. SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: Davis, A.

Lee, E Cao, J.

(ii) TITLE OF INVENTION: Use of Spermidine to Relieve Inhibition of Ligase Chain Reaction in a Clinical

Sample

(iii) NUMBER OF SEQUENCES: 20 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Abbott Laboratories

(B) STREET: 100 Abbott Park Road

(C) CITY: Abbott Park

(D) STATE: Illinois (E) COUNTRY: USA

(F) ZIP: 60064-3500

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Macintosh

(C) OPERATING SYSTEM: System 7.0.1

(D) SOFTWARE: Microsoft Word 5.1a

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Paul D. Yasger

(B) REGISTRATION NUMBER: 37,477

(C) REFERENCE/DOCKET NUMBER: 5616.US.PI

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 708/938-3508

(B) TELEFAX: 708/938-2623

(C) TELEX: 186900006

(2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (extrachromosomal DNA) (vi) ORIGINAL SOURCE:

(A) ORGANISM: Chla ydia trachomatis (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GACTTTGCAA CTCTTGGTGG TAGACTTGGT CATAATGGAC TTTTGTTG 48

(2) INFORMATION FOR SEQ ID NO:2

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

GACTTTGCAA CTCTTGGTGG TAGA 24

(2) INFORMATION FOR SEQ ID NO:3 (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

ACCACCAAGA GTTGCAAAGT C 21 (2) INFORMATION FOR SEQ ID NO:4

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

GGTCATAATG GACTTTTGTT G 21

(2) INFORMATION FOR SEQ ID NO:5

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(i ) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 5 CAACAAAAGT CCATTATGAC CAAG 24

(2) INFORMATION FOR SEQ ID NO: 6 : (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE: (A) ORGANISM: Mycobacterium tuberculosis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

AACCTGTGGG GTCCGGCCTT TCACGAGAGG TATCCGAACG TCAC 44 (2) INFORMATION FOR SEQ ID NO:7 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : AACCTGTGGG GTCCGGCCTT T 21

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 :

GGCCGGACCC CACAGGTT 18

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9 :

GAGAGGTATC CGAACGTCAC 20

(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GTGACGTTCG GATACCTCTC GTG 23 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genoraic) (vi) ORIGINAL SOURCE:

(A) ORGANISM: Neisseria gonorrheae (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

GCCATATTGT GTTGAAACAC CGCCCGGAAC CCGATATAAT CCACCCTT 48

(2) INFORMATION FOR SEQ ID NO:12

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

GCCATATTGT GTTGAAACAC CGCCC 25 (2) INFORMATION FOR SEQ ID NO:13

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

CGGTGTTTCA ACACAATATG GC 22

(2) INFORMATION FOR SEQ ID NO: 14

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AACCCGATAT AATCCGCCCT T 21

(2) INFORMATION FOR SEQ ID NO: 15

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

AAGGGCGGAT TATATCGGGT TCC 23

(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:

(A) ORGANISM: Mycobacterium tuberculosis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CCGACTGGGC AATTGGCTAA AGGCCCGCAT CGGCGTCGGC ACG 43

(2) INFORMATION FOR SEQ ID NO: 17

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CCGACTGGGC AATTGGCTAA AGG 23

(2) INFORMATION FOR SEQ ID NO:18

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

TTAGCCAATT GCCCAGTCGG 20

(2) INFORMATION FOR SEQ ID NO:19

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

GCATCGGCGT CGGCACG 17

(2) INFORMATION FOR SEQ ID NO:20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: synthetic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

CGTGCCGACG CCGATGCGGG 20

Claims

We claim:

1 . A method for relieving amplification inhibition in a ligase chain reaction comprising the steps of:

(a) providing a clinical test sample with (i) an amount of spermidine effective to relieve amplification inhibition and (ii) a 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, the two primary probes hybridizing at adjacent or near adjacent positions on the target, wherein at least one of the primary or secondary probes is modified at one end to render it non-ligatable to the other primary or secondary probe to form a reaction mixture and;

(b) hybridizing the primary probes to the target;

(c) correcting the modification in a target dependent manner to render the primary probes ligatable to one another when hybridized to the target;

(d) ligating the primary probes to form a fused product; and (e) dissociating the fused product from the target.

2. The method of claim 1 wherein said amount of spermidine in said reaction mixture is at a final concentration of between about 0.5 mM and about 4 mM.

3. The method of claim 2 wherein said composition further comprises MgCl2 and the final concentration of MgCl2 in said reaction mixture is greater than 35.5 mM.

4. The method of claim 2 wherein said composition further comprises MgCl and the final concentration of MgCl2 in said reaction mixture is between 0.5 mM and less than 20 mM.

5. The method of claim 1 wherein said amount of spermidine in said reaction mixture is at a final concentration of between 2.0 mM and 3 mM and wherein said composition further comprises MgCl2 at a final concentration of MgU in said reaction mixture of less than 10 mM. -33-

6. A method for relieving amplification inhibition in a ligase chain reaction comprising the steps of:

(a) providing a clinical test sample with (i) an amount of spermidine effective to relieve amplification inhibition and (ii) a 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;

(b) hybridizing the primary probes to the target;

(c) ligating the primary probes to form a fused product; and (d) dissociating the fused product from the target.

7. The method of claim 6 wherein said amount of spermidine in said reaction mixture is at a final concentration of between about 0.5 mM and about 4 mM.

8. The method of claim 7 wherein said composition further comprises MgU and the final concentration of MgCl2 in said reaction mixture is greater than 35.5 mM.

9. The method of claim 7 wherein said composition further comprises MgCl2 and the final concentration of MgCl2 in said reaction mixture is between 0.5 mM and less than 20 mM.

10. The method of claim 6 wherein said amount of spermidine in said reaction mixture is at a final concentration of between 2.0 mM and 3 mM and wherein said composition further comprises MgCl2 at a final concentration of MgU2 in said reaction mixture of less than 10 mM.