Note: Descriptions are shown in the official language in which they were submitted.
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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
App'i_ Lion 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 secondset 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 targetstrand, 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 a/.
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 2 225 112 A and European
Patent Application EP-A-439 182.
One problem associated with ampliricaLion 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.
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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 associdLed 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 MgClz that span a relatively narrow range (e.g. 25 mM-35 mM). Concentrations of MgClz 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 MgC12, caution
must be exercised to avoid introducing either an excess or an insufficient quantity
of MgClz 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 MgC12.
SUMMARY OF THE INVENTION
The present invention provides an improved LCR method that minimizes
pre-amplification sample preparation and allows greater flexibility with respectto amplification reagent addition. These advantages are made available by addingan 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 targetand 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-
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ligatable to the other primary or secondary probe, respectfully, and;
(b) hybridizing the primary probes to the target and optionally,
hybridizing the secondaly probes to the target complement;
(c) correcting the modification in a target dependent manner to render the
~, 5 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 reiieve 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 concer,LI-dLion 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 LC~ amplification in the presence of final MgCI2
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.
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As a result, the target sequence is separated from conLar"i"ar,ts found in the
source material. After pUI iric~ion, the resulting test sample is contacted withthe 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 vspermidine 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 ril~l~Lion and
centrifugation; or which contain reagents such as, for example, acids, bases,
Iysing 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 Iysing agent.
It was also discovered that by using spermidine as taught herein, MgClz
concentrations that previously have been found to inhibit LCR ampl;ric~Lion by not
supporting LCR amplification, no longer inhibit and thereby support LCR
amplification. Thus, effective LCR amplification occurs in the presence of finalMgClz concentl~Lions less than 20 mM and greater than 35.5 mM. Typically,
final MgC12 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 probefrom 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 aprobe 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
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probe duplexes.
In accordance with modified LCR procedures, a group on the probe that is
obligatorily involved in the enzyme catalyzed step of the ~rll~Jliri~.dlion reaction is
masked or blocked with, for example, a chemical moiety. The enzymatic steps
5 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
"anll;liricaLion productn. 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
10 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
15 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
ZO 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
25 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
30 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
35 several parameters, including temperature, ionic strength, probe length and G:C
content of the probes. For example, lowering the temperature of the reaction
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promotes annealing. For any given set of probes, melt temperature, or Tm, can beestimated 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
5 temperature, since small cations tend to stabilize the formation of duplexes by
negating the negative charge on the phosphodiester backbone. Typical salt
concenL~Lions 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 fol~"aLion because G:C pairings
10 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 15 to 100 bases serve20 this purpose. Presently preferred are probes having a length of about 15 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;
25 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 1 ol 1 to about 1.2 x 1 ol Zmolecules of each probe; and around 1 x 1 o12 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
30 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 primary35 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
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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 co, ~ Lion of the modified end(s) in a target dependent manner.
Since enzymatic ligation is the preferred method of covalently attaching two
5 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 ~L~-d~ i"g two probes.
"Correction" refers to repair of the modiric;~lion that rendered the probe
ligation incompetent in the first place. Specific correction mechanisms relate
10 generally to one or more of: 1 ) creating or ~ Luri~g 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-dependentn, 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.
15 "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
el;."il,a~illg 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
20 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., themodification must be "corrected"). This can be accomplished using a polymerase
or a reverse transc~ tase and an excess of deoxyribonucleotide triphosphates
25 which are complementary to the target strand opposite the gap. Extension must be
terllli,l~ed 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
30 of double stranded target. Gap filling by extension is further described in W0
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.
35 "Upstream probe" refers to that probe partner whose 3' terminus points towardthe 5 terminus of the other probe partner regardless of whether the strand(s)
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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 UcorlecLed~ by removal of the non- ,
phosphate groups with resultant exposure of a 5' phosphate group. This is
5 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
10 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 thê
probe partners abut each other and can be ligated. A,l,pliric~Lion of target nucleic
acids using exonucleolytic activity is further described in W0 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 toexpose 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
182 and in more detail in PCT/US94/04113 the entire disclosure of which is
incorporated herein by reference.
The conditions and reagents which make possible the preferred enzymatic
Z5 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. coli
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 Pyrococcus furious DNA ligase
available from Stratagene, (Catalog No. 600191, LaJolla, CA). A thermostable
ligase is presently preferred for its ability to Illaill-aill 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
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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 prèvent 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 inhibito~ effect. As used
herein, spermidine or spermidine reagent refers to the compound having the
formula NH2-(CH2)3-NH2-(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 mechani~"l 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 ofinhibitor(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
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- 1 0-
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
5 test sample. Inhibitors may be substances endogenous to a test sample or added to
the clinical test sample exogenously. Endogenous inhibitors are those substancesderived 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 prt:Lre~Lrnent steps. For example,
10 exogenous inhibitors may be introduced through the addition of detergents which
cause Iysis of cellular membranes and concurrent release of nucleic acids and/orthe 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
15 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 MgClz. For example,
20 reduced MgCI2 refers to concentrations of MgCI2 below those typically used in LCR
reactions. Reduced MgCI2 concentrations typically are final MgClz concentrationsbetween 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
25 reduced ligase conditions. Reduced ligase refers to concentrations of ligase below
those typically used in LCR reactions. For example, W0 93/00447 and W0
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 ~JL
reaction volume (i.e. 68 to 100 units/~L). Reduced ligase concentrations
30 according to the present invention refer to ligase concenL~Lions of between about
1,000 units/200 ~L and about 12,000 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.
Following amplification, the amplified sequences can be detected by a
35 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
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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 Ureporter" or Ulabel"), 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 dirrer~n~ 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 adal"a"Lane 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 17,1991 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 ll, 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 (1988), or Cohen, J.S. et
al., U.S. Patent Application Serial Number 07/246,688, abandoned (NTIS order
no. Pat-Appl-7-246,688 (1988). 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 l"ic,upa, licle enzyme immunoassay (MEIA) technology. The assay protocol is
similar to that used in the commercially available alpha-fetoprotein assay, with
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the following adapt;~lions: (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 ~Ik~line phosphataseare replaced with the conjugates of anti-3-phenyl-1-adamantaneacetic acid
5 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 ,uL 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
10 incubation well, the an~icalbazole antibody coated n~ upal ~icles 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
15 conjugated to alkaline phosphaLase 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 capture20 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
25 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, 1990, "Detection and Amplification of Target Nucleic Acid Sequences,"
to Laffler, T.G., et al.; and elsewhere.
E)CAMPLES
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-
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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, dl~P, 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 adar"antaneacetic acid ("adamantane") hapten.
Typically, "adamantane" and "carbazole" are used together in accordance with thedescription 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(l~) 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., 34/9:1726-
1732 (1988). It should be noted that for purposes of the following examples, a
modified IMx(~) instrument was used, which employs a stainless steel rather thanteflon coated steel pipetting probe. The IMx(l~) instrument typically generates
"machine" noise or background in the range of 5-12 counts/sec/sec. Other
equally suitable rnethods 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 hyl,ridi,a~ion 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 totalnucleotide into acid-insoluble ",~Leli~l in 30 min at 70DC. 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 1 o8 units. While this is not precisely
standardized and may vary by as much as 20%, optimization is within the skill ofthe routine practitioner.
Target sequences and probes were selected so as to include a "stop base" as
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taught in EP 439 182 by K. Backman et a/. published July 21, 1991 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
5 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
10 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
Oliqonucleotide Synthesis and Haptenation
The following oligonucleotides (see Table 1 ) were synthesized following
established procedures using 13-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'-
20 ends of oligonucleotides were conjugated with haptens, adamantane and carbazole.The conjugation of these haptens followed standard l~-cyanoethylphosphoramidite
chemistry, and is described in the aforementioned hapten applications. A similarprocedure is described for fluorescent label conjugates in published U.S.
application NTIS ORDER No. PAT-APPL-7-246,688) (Cohen, et al., 1989).
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Table 1
Sequence ID
No SEQUENCE
'. 2. CZ-GACIIIGCAACTCTTGGTGGTAGA
3. ACCACCAAGAGTTGCAAAGTC-CZ
4. GGTcATAATGGA[~llllGllG-AD
5. AD-CAACAAAAGTCCATTATGACCAAG
7. CZ-AACCTGTGGGGTCCGGCCIII
8. GGCCGGACCCCACAGGTT-CZ
9. AD-GAGAGGTATCCGAACGTCAC
1O. GTGACGl~CGGATACCTCTCGTG-AD
12. CZ-GCCATATTGTGTTGAAACACCGCCC
13. CG(il(~lll(;AACACAATATGGC-CZ
14. AACCCGATATMTCCGCCCTT-AD
15. AD-AAGGGCGGATTATATCGGGTTCC
17 CZ-CCGACTGGGCAATTGGCTAAAGG
18 TTAGCCAATTGCCCAGTCGG-CZ
19 GCATCGGCGTCGGCACG-AD
AD-CGTGCCGACGCCGATGCGGG
Oligonucleotides corresponding to SEQ ID Nos. 2, 3, 4 and 5 were selected
5 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. 16 (9):4053-4067
(1988)). 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
10 Mycobacterium tuberculosis. Oligonucleotides corresponding to SEQ ID Nos. 12,13,14 and 15 were selected to detect a target sequence (SEQ ID No. 11, 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 (1986)). Oligonucleotides
15 corresponding to SEQ ID Nos.17,18,19 and 20 were selected to detect an
unmapped genomic sequence (SEQ ID No. 16) from Mycobacterium tuberculosis.
All oligonucleotides were purified by reversed-phase HPLC or by PAGE
electrophoresis (Maniatis, T., et al., Molecular Cloning, Cold Spring Harbor
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Laboratory, 1972) to remove failure sequences and, in the case of haptenated
oligos, any urll1aptendLed species.
EXAMPLE 2
Effect of SDermidine on Relieving Inhibition of Target Directed DNA
Amplification by 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
10 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, 10 ~M NAD, 3 to 6 uL of an approximate 1 o8 fold
dilution of C. trachomatis infected McCoy cells (this amount was er~pi~icc.'ly
chosen to yield IMx(g) signals in the range of about 500-1300 c/s/s), 4.8-5 x
15 1 ol 1 molecules each of SEQ ID Nos. 2, 3, 4, and 5, 100 ~L of clinical sample, 1.7
M each of dCTP and dTTP, 18,000 units Thermus thermophilus (T. th) DNA
ligase and 2 units of Thermus flavus (T. f/) polymerase (Molecular Biology
Resources, Milwaukee, Wl, cat. no 1070.01 ) in a final reaction volume of 200
,uL. Probes were labeled with carbazole and adamantane as per Example 1.
20 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 Iysate as a positive control. Cycling was performed on a Perkin Elmermodel 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 detected25 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 ,uL. Probe and enzyme concentrations and clinical sample volumes were
decreased proportionally (i.e. 2.4 x 101 1 molecules of probes, 9,000 units
30 ligase, 1 unit polymerase and 50 ~L 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.
35 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
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and 12 in Table 2a and increased MEIA rates of samples Ul 12, 23, 24, 27, 34,
45 60 and 68 in Table 2b).
TABLE Za
MEIA Rate (c/s/s)
Sample0 mM Spermidine 1 mM Spermidine
Ul 3 1 2 1 088
Ul 1 2 1 03 1 009
McCoy Iysate 1 231 1 196
HP DNA 226 38
TABLE 2b
MEIA Rate (c/s/s)
Sample0 mM S~ermidine 1 mM Spermidine
Ul 12 39 555
Ul23 11 1016
Ul 24 11 1045
Ul 27 130 975
Ul 34 43 247
Ul 45 1 0 638
Ul 60 277 914
Ul68 14 319
McCoy Iysate 969 N.D.*
HP DNA 165 N.D.*
*N.D. refers to not performed
EXAMPLE 3
Gel Filtration Chromatography
Experi",enL~ were performed in the presence of negative clinical samples
(described above) using the experimental conditions of Example 1 for 100 uL
reactions with the modification that test samples were first subjected to gel
filtration chromatography prior to use in LCR reactions. This procedure is a
20 partial puriri~ lion 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
#P5194). 0.5 mL of sample was loaded per column. The nucleic acid was eluted
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with TE by placing the column within a collection tube and spinning at 1600 rpm
for 5 minutes at 15~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
5 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)
10Sample O mM Spermidine 1 mM S,cermidine
NY 65 21 157
NY 77 18 758
NY 83 36 570
Ul 6944 14 510
Ul 6980 17 519
McCoy Iysate 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. tbJ DNA was performed in the presence of
increasing amounts of negative clinical samples (described in Example 1).
20 Reactions were performed in 200 ~L total reaction volume containing 50 mM
EPPS-KOH buffer, 20 mM KCI, 30 mM MgClz,1.7 ~M each of dCTP and dATP,10
~M NAD, approximately 1 x 1 ol 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 pubiished
25 genome size of M. tb DNA (Baess, l., Acta Path. Microbiol. Immunol. Scand., Sect.
B 92: 209-211, (1984)) and the DNA concentration of the sample preparation
as determined either by ~D260 or by DABA (diamino benzoic acid) reaction.
Ampliric~Lion reactions were performed both in the presence and absence of 1 mM
spermidine. The volumes of clinical sample used per reaction were as indicated
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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~C, for 1 second; 68~C, for 1 minute,15
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 ar~ lirica~ion 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 (~/I)) ~ mM Spermidine1 mM S,cermidine
M181 3952 1818
M181 10 699 1088
M181 30 17 910
M181 90 23 15
M.tbDNA 0 1813 1766
HP DNA 0 24 22
Ul 176 3 2365 2021
Ul 176 10 2067 2057
Ul 176 30 1312 2186
Ul 176 100 24 1538
Ul 183 3 2330 1765
Ul 183 10 2178 2020
Ul 183 30 1900 1918
Ul 183 100 80 1989
Ul 193 3 2256 2001
Ul 199 10 2322 1995
Ul 199 30 24 1776
Ul 199 100 37 1341
M.tb DNA 0 2053 1709
HP DNA 0 29 19
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EXAMPLE 5
Optimal Range of S,cermidine Concentrations Effective at Relieving ll,hibilion of
Amplification in the Presence of Clinical Samples
Experiments were performed to determine the optimal range of
5 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
10 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 inrelieving inhibition of amplification.
TABLE 5
MEIA Rate (c/s/s)
Sample ~L sample O mM Spermidine 1 mM Spermidn~ 3 mM Spermidine
Ul194 10 1936 2264 2096
Ul194 30 28 2349 2319
Ul199 10 2304 2532 2116
Ul199 30 31 2050 2015
Ul199 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 lon Conentration in the Presence of Spermidine
Experiments were performed to determine the effect of spermidine on the
concentration of MgCI2 required to effect LCR amplification. Specifically,
experiments were performed to determine whether amplification could be effected
CA 02202990 1997-04-17
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at MgC12 concentrations lower than those typically used in modified LCR reactions
(i.e. 25 to 35 mM MgCI2). For this experiment, Z5 genomes of M. tb DNA was
amplified in 50 mM EPPS-KOH buffer, ZO mM KCI, 1.7 ~M each of dCTP and
dATP, 10 ~M NAD, approximately 1 x 1 o12 molecules each of SEQ ID Nos. 7, 8, 9
and 10, 2 units of T. fl polymerase, 18,000 units of T. th DNA ligase and varying
concer,L,dLions of MgCI2 (indicated below). Total reaction volumes were 200 ~L.
Cycling conditions were 93~C for 1 second, 63~C 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 con~:enlr~Lions of MgClz as
low as 5 and 10 mM.
TABLE 6
MEIA Rate (c/s/s)
15 Sample MgClz (mM) 0.0 mM Spermidine S.D.* 1.0 mM Spermid ne S.D.*
5.0 6 - 188 15
10.0 139 15 949 144
M. tbDNA20.0 1262 78 1383 132
30.0 1210 46 1317 39
40.0 96 78 352 1 63
M tbDNA 30.0 1210 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 lon 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 ,LrL 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 I~M NAD, 1 x 10 1 2 molecules each of SEQ ID Nos. 1 7, 1 8, 1 9 and
20, 2 units of T. fl polymerase, 18,000 units of T. th DNA ligase, 25 genomes ofM. tb DNA and MgCI2 as indicated below. It should be noted that for purposes of
this and all subsequent examples, the KCI concentration used was 50 mM.
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(P~ ,aly experiments had shown that signal from background amplification
was reduced in reaction mixtures containing 50 mM KCI while the signal from
target ampliric~Lion was unaffected (data not shown). Accordingly, this
concentration was used to further reduce the background signal). Parallel
5 conL,uls were run under identical reaction conditions with Z ,ug 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 MgClz 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, MgCI2 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
15 throughout the range tested since variations in MgCI2 concentrations did not effect
amplification of HP DNA at any concentration tested.
TABLE 7a
Target MgC12 Conc. (mM)MEIA Rate (c/s/s)S.D.
o.O 11
0.5 71 0 50
M. tb DNA 1.0 1443 14
1 .5 1 463 28
2.0 1513 4
2.5 1 406 1 33
0.5 14 3
1.0 10
HP DNA 1.5 15 6
2.0 9
2.5 15 6
Standard Controls
M. tbDNA 20 1634 50
HP DNA 20 480 480
Essentially identical experiments were performed using C. trachomatis
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DNA as target DNA. C. trachomatis reactions were performed in 50 mM EPPS-
KOH buffer, 50 mM KCI, 2.5 mM spermidine,1.7,uM each dCTP and dTTP,10
/~g/mL BSA, 10 ~M NAD, 4.5 x 1011 molecules each of SEQ ID Nos. 2,3, 4 and 5,
1.5 units of T. fl polymerase,1,800 units of T. th DNA ligase, 25 molecules of
5 SEQ ID No.1 (i.e. synthetic target) and MgCI2 as indicated below. Final reaction
volumes were again ZOO ~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 MgClz concentration range at which amplification could10 be effected.
TABLE 7b
Target MgCI2 Conc. (mM) MEIA Rate (c/s/s) S.D.
0.0 13.0 0.0
0.5 1714.0 48.0
1.0 1863.0 48.0
C. trachomatis1.5 1940.0 8.0
2.0 1955.0 45.0
2.5 1957.0 16.0
3.0 2005.0 24.0
Standard Control
C. trachomatis ¦ 20.0 ¦1206.0 ¦ 98.0
EXAMPLE 8
Optimal Ran~e of Spermidine Concentrations that Effect Amplification
Under Reduced MaCI? Conditions
Experiments were performed to determine the optimal range of
20 spermidine concentrations that would effect LCR ar"~liri~ ion in the presence of a
reduced concenll~ion of MgCI2 (i.e. 2 mM). Reactions were performed in 200
~rL total volume and contained the following reagents: 50 mM EPPS-KOH buffer,
50 mM KCI, 2 mM MgCI2,1.7 ~M dGTP,10 ~M NAD,1.4 x 1 ol 1 molecules each
of SEQ ID Nos.12,13,14 and 15,10 ~M BSA,18,000 units T. th DNA ligase,
25 Z.O units T. 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 OD260) and the weight per genome of M
gonorrhoeae DNA (calculated as 2.3 femtograms DNA per genome). Spermidine
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was added to final concen~l;dLions 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
5 the same set of conditions with the following modiri- dLions: 20 mM KCI, 30 mMMgCI2 and no spermidine. In addition, in the negative control, target DNA was
replaced with 150 ng of salmon sperm DNA (Sigma). Sample aliquots were
counted on an IMx(~ instrument as described.
As shown in Table 8a, the concentration of MgCI2 typically used to effect
10 LCR amplification of N gonorrhoeae DNA is 30 mM. (See positive control, M
gonorrhoeae DNA). When the MgCI2 concentration was reduced to 2 mM,
amplification was not accomplished in the absence of spermidine. (See sample 1,
MEIA rate = 18.0). However, as shown in samples 6 and 7, amplification of N.
gonorrhoeae DNA was accomplished to nearly the same extent as that of the
15 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 1 8.0 6.0
0.5 1 0.0 2.0
1 .0 1 3.0 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 0.0 812.0 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 ,uM each of dCTP and dTTP, 4.5 x 1 ol 1 oligos of C. trachomatisprobe 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
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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
SampleSpermidine (mM)Av~. MEIA Rate (c/s/s) S.D.
0.0 31.0 7.0
0.5 1 7.0 2.0
1 .0 21 3.0 4.0
C. trachomatis 1.5 1372.0 12.0
2.0 1 891 .0 26.0
Z.5 2070.0 34.0
3.0 2126.0 16.0
Standard contr~ ls
C. trachomatis - 2046.0 89.0
Salmon sperm - 69.0 58.0
DNA
EXAMPLE 9
Ligase Reauirement in Presence of Spermidine
Experiments were performed to determine the optimal range of ligase
concentrations required to effect ampliricaLion of C. trachomatis in the presence of
spermidine. Target DNA was amplified under standard reaction conditions (i.e. inthe absence of spermidine) and under modified reaction conditions (i.e. in the
15 presence of spermidine) as follows:
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Rea~entsStandard Conditions Modified Conditions
EPPS-KOH buffer 50.0 mM 50.0 mM
KCI 20.0 mM 50.0 mM
MgCI2 30.0 mM 4.0 mM
EDTA 0.5 mM 0.0 mM
Spermidine 0.0 mM 2.5 mM
BSA 10.0,ug/mL 10.0,ug/mL
NAD 10.0 ~M 10.0 ,uM
dCTP, dTTP (each) 1.7 ,uM 1.7 ~M
T. flpolymerase2.0 units 1.5 units
SEQID Nos. 2, 3,4, and 5 4.5 xloll 4.5 xlo11
C. trachomatis DNA 10.0 genomes 10.0 genomes
Total reaction volumes were 200 ~L. Final concentrations of T. thermophilus
ligase were as indicated below.
The results in Table 9 show that in the presence of spermidine, a 1 O-fold
less concentration of ligase could be used to effect amplification.
TABLE 9
MEIA Rate (c/s/s)
1 0 Ligase
(units/LIL)Std. Cond. S.D. Mod. Cond. S.D.
0.0 1 1 .0 1 .0 1 7.0 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 various15 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.
=
CA 02202990 1997-04-17
W O96S12~24 PCTAUS9~12874
SEQUENCE LISTING
(l) GENERAL INFORMATION:
~ 5 (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: l00 Abbott.Park Road
(C) CITY: Abbott Park
(D) STATE: Illinois
Z0 (E) COUNTRY: USA
(F) ZIP: 60064-3500
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
Z5 (B) COMPUTER: Macintosh
(C) OPERATING SYSTEM: System 7Øl
(D) SOFTWARE: Microsoft Word 5.la
(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: 56l6.US.P1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 708/938-3508
(B) TELEFAX: 708/938-2623
(C) TELEX: 186900006
(2) INFORMATION FOR SEQ ID NO:l
(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: Chlamydia trachomatis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
' 55 GACTTTGCAA CTCTTGGTGG TAGACTTGGT CATAATGGAC TTTTGTTG 48
(2) INFORMATION FOR SEQ ID NO:2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
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W O96/12824 PCTrUS95/12874
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GACTTTGCAA CTCTTGGTGG TAGA 24
(2) INFORMATION FOR SEQ ID NO:3
(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:3:
ACCACCAAGA GTTGCAAAGT C 21
(2) INFORMATION FOR SEQ ID NO:4
(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:~:
GGTCATAATG GA~l~ G 21
(2) INFORMATION FOR SEQ ID NO:5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID No:5:
40 CAACAAAAGT CCATTATGAC CAAG 24
(2) INFORMATION FOR SEQ ID NO:6:
(i) 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:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02202990 l997-04-l7
WO 96112824 PCT/US95~12874
-29-
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
5 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:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Neisseria gonorrheae
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
GCCATATTGT GTTGAAACAC CGCCCGGAAC CCGATATAAT CCACCCTT ~8
(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
CA 02202990 l997-04-l7
W O96/12824 PCTrUS95112874
-30~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GCCATATTGT GTTGAAACAC CGCCC 25
5 (2) INFORMATION FOR SEQ ID NO:13
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid s
(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:
25 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:
_
CA 02202990 l997-04-l7
WO 96112'824 PCl'JlJS95~2874
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:l9
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
Z0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
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
