Note: Descriptions are shown in the official language in which they were submitted.
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GENOTYPING USING PARTIALLY-COMPLEMENTARY PROBES
Technical Field
The present invention relates to detecting nucleic acid sequences and, in
particular,
relates to detecting distinct nucleic acid sequences with probes that are
complementary to
such distinct nucleic acid sequences, as well as complementary to each other.
Background of the Invention
to Studies designed to determine the genomic sequence of various organisms, as
well as
studies designed to compare genomic sequences, have elicited information
regarding
polymorphisms in various genomes ranging from the human genome to the HIV
genome. A
wide variety of polymorphisms in the genomes of such organisms have previously
been
described. The various types of genetic polymorphsims include single base
substitutions;
15 insertions or deletions; variable numbers of tandem repeats; deletions of
all or a large part of
a gene; gene amplifications; and chromosomal rearrangements. Generally,
polymorphisms
that involve a single nucleotide are called single nucleotide polymorphisms or
"SNPs". In
the event a genome contains a particular SNP, a sequence from the region
containing the SNP
may exist in one of two or more forms. If one of the forms can be identified
as occurring in
20 the majority of the population, or if it can be associated with full
functionality of a protein
product, it is referred to as the "wild-type" sequence. The other forms) of
the SNP would be
referred to as "mutant".
Some variations in certain genes are responsible for various disease states.
For
example, venous thrombosis is a fairly common disorder that annually affects
approximately
25 1 in 1000 people. In some cases, thrombosis can be traced to SNPs in genes
encoding
proteins that participate in the cascade of events responsible for blood
clotting. For example,
the so-called "factor V Leiden" mutation, a mutation in the gene encoding
factor V, has been
reported to be a factor responsible for thrombosis. Additionally, a variation
in the 3'
untranslated region of the prothrombin gene also has been reported to be
responsible for
30 thrombosis. Persons having either, or both, of the above gene variations
have been reported
to be at an even greater risk of thrombosis in the event that they are, for
example, undergoing
surgery, pregnant, or taking oral contraceptives.
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In certain cases, a mutant sequence and wild type sequence can be contained in
a
single genome. For example, such a case can arise where a sequence is derived
from an
organism that contains more than one set of chromosomes (variously referred to
as
"polyploidy"). Additionally, organisms containing a single chromosome may also
contain
variant forms of more than one gene in cases where the chromosome contains a
gene
amplification. While a genome may contain duplicate copies of either the wild-
type or
mutant version of the sequence in question (variously referred to as
"homozygous" for the
particular sequence), a further possibility is that the genome contains a copy
of each of the
wild-type and mutant sequence (variously referred to as "heterozygous" for the
particular
sequence). The existence of closely related sequences presents several
challenges when
attempting to detect these sequences in a test sample.
Previously, detecting related sequences in a test sample was performed using
RFLP
and gel electrophoresis. Dual and separate amplification reactions to
determine the presence
of a mutant and wild type sequence in a sample have also been performed to
detect the
presence of multiple related sequences in a test sample. For example, genetic
tests for the
factor V Leiden and the prothrombin mutations have been reported in the
literature. Many of
the previously reported genetic based assays are performed with the aid of an
amplification
. reaction such as the polymerase chain reaction (PCR) or the ligase chain
reaction (LCR).
After amplification of the regions suspected of containing the particular
mutation, the
amplification products are often times detected using gel electrophoresis.
Amplification
based assays for the above mutations are run individually and separately with
one reaction
designed to detect the wild type sequence and the other reaction designed to
detect the mutant
sequence. Accordingly, these assays can be cumbersome and expensive to
perform. Hence,
it would be useful to provide assays and assay reagents that allow an assay
for two related
sequences to be run in a single reaction vessel. Such reagents and methods
would therefore
avoid the use of dual and separate reactions and the use gel electrophoresis
for purposes of
detecting related sequences in a test sample.
Brief Description of the Invention
The present invention provides a method for detecting at least one of two
related
target nucleic acid sequences in a test sample. The method can be employed
with nucleic
acid amplification techniques that advantageously can be performed in a single
reaction
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vessel. Generally, the method comprises the steps of contacting a test sample
with a first and
second probe. The probes are designed such that (i) they are completely
complementary to
the each other except for at least one mismatch, (ii) the first probe is more
complementary to
a sense strand of the target sequence than to the second probe, (iii) the
second probe is more
complementary to an anti-sense strand of a second target sequence than to the
first probe, and
(iv) the probes hybridize to each other at an ambient temperature. A target
sequence/probe
hybrid is formed by hybridizing at least the first or second probe to the
first or second target
sequence. The so-formed hybrid is detected as an indication of the presence of
one of the
related target sequences in the test sample. Reagents for performing the above
method are
also provided.
Detailed Description of the Invention
The present invention provides methods for detecting two distinct target
sequences in
a test sample using a pair of probes that hybridize to one another but are not
completely
complementary to each other. Generally; the method comprises, if necessary,
amplifying
target sequences using any of the well known amplification techniques and
detecting
amplified target sequences, if any, with the probes. Advantageously, the
distinct target
sequences often times can be amplified using common amplification reagents.
Under certain
circumstances, such as when the target sequence is present in sufficient
concentration, the
target sequence may not require amplification before being detected with the
probes. The
probes generally are designed such that they are completely complementary to
their
respective target sequences and are sufficiently complementary to each other
to hybridize.
Typically, the probes have at least one mis-matched base pair preferably
located between the
5' and 3' terminal nucleotides of the respective probes. The use of probes
that are more
complementary to their respective target sequences than to each other has been
found to
enable the detection of distinct, but related, target sequences in a single
test sample without
the need to introduce separate probes to the products of separate
amplification reactions. The
invention is particularly suited for detecting the presence of variant forms
of a nucleic acid
sequence in a test sample.
A "target sequence" as used herein means a nucleic acid sequence that is
detected,
amplified, both amplified and detected, or otherwise is complementary to the
amplification
primers or one of the probes. Additionally, while the term target sequence is
often used
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herein to refer to a single strand of nucleic acid, those skilled in the art
will recognize that the
target sequence can also be double stranded. As previously mentioned, target
sequences that
can be detected using the method provided herein are related. Such target
sequences are
related insofar as one target sequence is usually a variation of the other
target sequence. In
particular, the target sequences that can be detected typically have the same
sequence except
that one of the target sequences contains, for example, a polymorphism that
makes its
sequence composition distinct from the other target sequence. Thus, for
example, a first
target sequence and second target sequence may be the same except that the
second target
sequence contains one or more nucleotide substitutions, deletions, and/or
insertions. Target
to sequences may be derived from any organism containing a nucleic acid
genome.
The term "test sample" as used herein, means anything suspected of containing
a
target sequence. The test sample can be derived from any biological source,
such as for
example, blood, bronchial alveolar lavage, saliva, throat swabs, ocular lens
fluid, cerebral
spinal fluid, sweat, sputa, urine, milk, ascites fluid, mucous, synovial
fluid, peritoneal fluid,
amniotic fluid, tissues such as heart tissue and the like, or fermentation
broths, cell cultures,
chemical reaction mixtures and the like. The test sample can be used (i)
directly as obtained
from the source or (ii) following a pre-treatment to modify the character of
the sample. Thus,
the test sample can be pre-treated prior to use by, for example, preparing
plasma from blood,
disrupting cells, preparing liquids from solid materials, diluting viscous
fluids, filtering
liquids, distilling liquids, concentrating liquids, inactivating interfering
components, adding
reagents, purifying nucleic acids, and the like.
A "primer sequence", "primer sequences" or "primer(s)" refers to reagents,
most
typically short strands of nucleic acid or "oligonucleotides", that prime
synthesis of multiple
copies of a target sequence. Oligonucleotides vary greatly in size but
typically are in the
range of between 10 and 1000 nucleotides in length, more typically in the
range of between
and 100 nucleotides in length. Amplification reactions that employ primer
sequences to
synthesize multiple copies of a target sequence (or amplify the target
sequence) are, by now,
well know in the art. LCR described in European Patent Number 320 308 and its
variations,
such as gap LCR described in U.S. Patent Number 5,792,607 (herein incorporated
by
reference), NASBA or similar reactions such as TMA described in U.S. Patent
Number
5,399, 491 (herein incorporated by reference), and preferably PCR, and
variations of PCR,
which are described in U.S. Patents Numbered 4,683,195, 4,683,202, and
5,310,652 (all of
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which are herein incorporated by reference) are examples of amplification
reactions that can
be employed according to the present invention.
The phrase "amplification reaction reagents" as used herein means reagents
which
are well known for their use in nucleic acid amplification reactions and may
include but are
not limited to: a single or multiple reagent, reagents, enzyme or enzymes
separately or
individually having reverse transcriptase, polymerase, and/or ligase activity;
enzyme
cofactors such as magnesium or manganese; salts; nicotinamide adenine
dinucleotide (NAD);
and deoxynucleoside triphosphates (dNTPs) such as, for example, deoxyadenosine
triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and
thymidine
triphosphate. The exact amplification reagents employed are largely a matter
of choice for
one skilled in the art based upon the particular amplification reaction
employed.
Probe sequences are similar to primer sequences insofar as they hybridize to
target
sequences, facilitate detection of target sequences, and typically are
oligonucleotides. Probes
generally are nucleic acid or they can be composed of nucleic acid analogs
such as, for
example, uncharged alkyl analogs such as methyl phosphonates, and
phosphotriesters,
peptide nucleic acids (as is disclosed in WO 93/25706), and morpholino analogs
(as disclosed
in U.S. Patents 5,142,047, 5,235,033, 5,166,315, 5,217,866 and 5,185,444).
Additionally, the
term probe is intended to mean that portion, or that group of consecutive
nucleotides, of a
nucleic acid sequence that is predetermined or designed to hybridize to its
target sequence, as
those skilled in the art will recognize that additional nucleotides can be
added to a probe for
other purposes. For example, such probe extensions, ar tails, are commonly
added for
purposes of assisting in detecting or purifying the probe.
Probes are provided in pairs and each probe of a particular pair hybridizes to
the other
member of the pair. The probes, however, are not perfectly complementary to
each other
which means that there is at least one mis-matched base pair on each probe. A
"mis-matched
base pair" (variously referred to as simply a "mismatch") means that one or
more nucleotides
on one probe does not hydrogen bond in the typical Watson-Crick fashion with
one or more
corresponding nucleotides on the other probe. Generally, probes are more
complementary to
their respective target sequences than they are to one another. Preferably,
however, the
probes are designed in such a manner so as to be perfectly complementary to
distinct target
sequences and therefore each nucleotide on a particular probe finds a
corresponding
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6
complementary nucleotide on its target sequence making the probe completely
complementary with its target.
There are numerous configurations that the probes may take and the exact probe
configuration is largely dependent upon particular target sequences selected.
Specifically,
mss-matches between the probes that make them partially non-complementary can
occur
anywhere and to any extent along the length of the probes. The probes,
however, should be
configured such that they hybridize to each other at ambient temperatures
(e.g. 15°C - 30°C)
in the absence of their respective target sequences but preferentially bind to
the respective
target sequences when they are present. Probes can be designed such that they
hybridize to
each other at ambient temperatures but preferentially bind to a target
sequence when it is
present (at any temperature where hybridization can occur) using well known
principles of
to hybridization or annealing kinetics. Hybridization is dependent in a rather
predictable
manner on several parameters, including temperature, ionic strength, sequence
length,
complementarity, and G:C content of the sequences. For example, lowering the
temperature
in the environment of complementary nucleic acid sequences promotes annealing.
For any
given set of sequences, melt temperature, or "Tm", can be estimated by any of
several known
methods. Ionic strength or "salt" concentration also impacts the melt
temperature, since
small rations 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 ration but are readily understood by those skilled in the art.
Similarly, high G:C
content and increased sequence length are also known to stabilize duplex
formation because
2o G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and
because longer
sequences have snore hydrogen bonds holding the sequences together. Thus, a
high G:C
content and longer sequence lengths impact the hybridization conditions by
elevating the Tm.
Once probes are selected for a given set of target sequences, the G:C content
and
length will be known and ran be accounted for in determining precisely what
the
hybridization conditions will encompass. Since ionic strength is typically
optimized for
enzymatic activity, the next most obvious parameter to vary is the
temperature. Generally,
the Tm of hybridized probes will be less than the Tm of the hybrid product
formed between
the probes and their respective target sequences. Thus, obtaining a suitable
probe
configuration is well within ordinary skill of one practicing this art.
Additionally,
determining whether a given probe configuration meets the above criteria can
be
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accomplished empirically. For example, the hybridization characteristics for a
particular set
of probes, and their respective target sequences, can be determined by raising
and lowering
the temperature in the environment of the probes and the probes and target.
Generally
speaking, given the fact that the probes are selected such that they are more
complementary
to their respective targets, complementary probes inherently will have a lower
Tm than the
Tm between the probes and their respective target sequences.
The exact size of the probes~is largely dependent upon the target sequences
and the
number of nucleotides involved in the variation between the two sequences.
Preferably,
however, the probes are between 10 and 1000 nucleotides long, more preferably
between 10
to and 100 nucleotides long, and most preferably between 10 and 50 nucleotides
long. The
probes hybridize to one another except in regions containing the variance
between the two
target sequences for which the probes are respectively specific. The region or
regions of the
probe that are not complementary also can be of any length as long as the
probes also have
regions that are sufficiently complementary to permit the probes to hybridize
at ambient
temperatures. Regions of the probes that are not complementary also can be
located at any
position in the probes as long as the probes hybridize to one another at
ambient temperatures.
Typically, the probes are employed to hybridize to target sequences containing
"small
variations" or "small polymorphisms" such as, for example, base substitutions,
deletions, or
insertions involving ten or less nucleotides, more typically less than five
nucleotides,
preferably less than three nucleotides, and most preferably a single
nucleotide polymorphism.
Also, while the region or regions of the probes that are not complementary can
be
located at any region of the probes, it is preferable to have these regions
internal to the 5' and
3' terminal. nucleotides of the probes, except in cases where one or both
probes contain an
overhang which is discussed in detail below. The terminal nucleotides are
those that are the
last nucleotides on either end of the probe that are designed to hybridize to
the target
sequence. More preferably, the region or regions of the probes that are non-
complementary
are located between the terminal nucleotide and the center nucleotide or
nucleotides of the
probes. As used herein, the term "center nucleotide(s)" is intended to mean
the nucleotide or
nucleotides that are located in the middle of a particular probe. For example,
in the case of a
3o five nucleotide probe, the center nucleotide would be the third nucleotide
in from the 5'
terminal nucleotide. In the case of a six nucleotide probe, the center
nucleotides would be the
third and fourth nucleotides in from the 5' terminal nucleotide. Hence, it is
preferable to
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locate the region or regions of the probes that are not complementary off
center (e.g. closer to
the 5' end of one probe). Locating a mismatch off center is particularly
preferred in cases
where the mismatch is a result of the variation between the target sequences
and is the only
mismatch between the probes.
Given the above, when the probes axe hybridized to one another, several probe
pair
configurations are possible. Preferably, the probes are not co-extensive
insofar as the 3'ends
of the probes do not completely align with the 5' ends of the complementary
probe when they
are hybridized to one another. In other words one probe may be longer than the
other at one
end, which is commonly referred to in the art as an "overhang." Hence, an
overhang at the
to 3'end of one probe means that it extends by one or more nucleotides past
the 5' end of its
complementary probe. Preferably, one of the probes contains an overhang on the
3' end.
Alternatively, it is preferred to have a 3' overhang on both probes.
While probes generally are designed such that they are completely
complementary to
their respective target sequences, probes may have mis-matches with their
respective target
sequences as long as the number of mismatches between the probes is sufficient
to meet the
above criteria (i.e. the probes hybridize to each other at ambient
temperatures but
preferentially bind their respective targets). Probes also may have mis-
matches with their
respective target sequences when they are functionalized with, for example,
nucleic acid tails
that are not complementary to the tar get sequence. As previously mentioned,
tails are
typically used, for example, for purposes of detecting hybrids formed between
probes and
their respective target sequences.
As previously mentioned, the probes hybridize to distinct but related target
sequences.
Specifically, a first probe hybridizes to a first target sequence and a second
probe hybridizes
to a variant form of the first target sequence that is complementary to the
first target but for
the existence of a variation between the first target sequence and second
target sequence.
Accordingly, probes are designed such that they hybridize to opposite strands
of related target
sequences. Thus, for example, in the event the target sequences were double
stranded, a fixst
probe would be complementary to the sense strand of the first target sequence
and the second
probe would be complementary to the anti-sense strand of the second target
sequence.
Additionally, notwithstanding the fact that the target sequence, or its
variant form, may not be
present in a test sample, the probes nevertheless are pre-designed to
hybridize to both forms
of target sequence. Variant forms of a target sequence may include, for
example, a first
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target sequence and the same sequence that contains one or more nucleotide
substitutions,
deletions, and/or insertions. Hence, for example, a pair of probes may contain
a single
nucleotide mismatch when hybridized to one another when the target sequences
are the same
but for a single nucleotide polymorphism such as a single base pair
substitution.
Various target sequences and variations of such target sequences can be
detected
using the probes provided herein. For example, the respective target sequences
to which the
probes are complementary could be from a single stranded RNA viral genome and
a variant
or mutated strain of that particular viral genome. Specifically, the first
probe could be
complementar y to a region of the single stranded genome as it normally occurs
and the
second probe could be complementary to the opposite strand of the same genome
that
contains a mutation such as a single base pair substitution. In this
particular case, it will be
understood that the RNA may first be reverse transcribed into DNA and the
strand to which
the second probe hybridizes will be present as a result of amplification of
the cDNA strand
reverse transcribed from the RNA genome. Methods for reverse transcribing and
amplification have been described above. Additionally, according to this
embodiment, it is
possible that only one version of the virus is present in the test sample.
Accordingly, only
one probe will hybridize to its target.
Alternatively, the original target sequences may be derived from the human
genome.
In this case, a first probe could be complementary to the sense strand of a
gene (the "wild-
type sequence") and the second probe could be complementary to the antisense
strand of the
same gene containing two single base substitutions (the "mutant sequence").
Depending
upon the genotype of the individual sample, only the first probe would
hybridize to its target
if the genotype was homozygous for the wild-type sequence, both the first and
second probes
would hybridize to their targets if the genome was heterozygous, and only the
second probe
would hybridize if the genome were homozygous mutant.
Primer andlor probe sequences can be labeled for purposes of detecting hybrids
formed between the target sequences and probes using any of the well known
labels or
labeling chemistries and is largely a matter of choice for those skilled in
the art. Techniques
for labeling sequences are well known and include, for example, techniques
described in U.S.
Patent Numbers 4,762,,779; and 4,948,882, both of which are herein
incorporated by
reference. Typically, primer and probe sequences are labeled such that a
hybrid or hybrids
formed between the target sequences and complementary probes are
distinguishable. In other
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words, complementary probes are labeled so that a first probe/target sequence
hybrid can be
distinguished from a second probe/target sequence hybrid. Various
configurations for
making the above distinction are well known and also a matter of choice for
those skilled in
the art based upon the choice of detection apparatus.
The term "label" as used herein means a molecule or moiety having a property
or
characteristic which is capable of detection. A label can be directly
detectable, as with, for
example, radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal
particles,
fluorescent microparticles and the like; or a label may be indirectly
detectable, as with, for
example, specific binding members. It will be understood that directly
detectable labels may
l0 require additional components such as, for example, substrates, triggering
reagents, light, and
the like to enable detection of the label. When indirectly detectable labels
are used, they are
typically used in combination with a "conjugate". A conjugate is typically a
specific binding
member which has been attached or coupled to a directly detectable label.
Coupling
chemistries for synthesizing a conjugate are well known in the art and can
include, for
example, any chemical means and/or physical means that does not destroy the
specific
binding property of the specific binding member or the detectable property of
the label. As
used herein, "specific binding member" means a member of a binding pair, i.e.,
two different
molecules where one of the molecules through, for example, chemical or
physical means
specifically binds to the other molecule. In addition to antigen and antibody
specific binding
pairs, other specific binding pairs include, but are not intended to be
limited to, avidin and
biotin; haptens and antibodies specific for haptens; complementary nucleic
acid or nucleic
acid analog sequences; and the like.
Detection platforms that can be employed to detect the hybrids formed between
the
complementary probes and their respective target sequence include any of the
well known
homogeneous or heterogeneous techniques well known in the art. Examples of
homogeneous
detection platforms include the use of FRET labels attached to probes that
emit a signal in the
presence of the target sequence. So-called TaqMan assays described in U.S.
Patent Number
5,210,015 (herein incorporated by reference) are examples of techniques that
can be
employed to homogeneously detect nucleic acid sequences. Briefly, fox example,
according
to a TaqMan format one probe could be labeled with a first fluorophore that
emits a signal at
a first wavelength and an appropriate quenching entity that is capable of
quenching the signal
from the first fluorophore. The second probe could be labeled with a second
fluorophore that
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emits a signal at a second wavelength and an appropriate quenching entity that
is capable of
quenching the signal from the second fluorophore. According to the principles
of TaqMan-
type assays the probes would be cleaved in the presence of their respective
targets during the
course of an amplification reaction using an enzyme having 5' to 3'
exonucleolytic activity.
As a result, as many as two distinct signals could be detected or as few as no
signals could be
detected depending upon the presence of the target(s).
Heterogeneous formats typically employ a capture reagent to separate amplified
sequences from other materials employed to amplify and/or detect a target
sequence. Capture
reagents typically are a solid support material that is coated with one or
more specific binding
to members specific for the same or different binding members. A "solid
support material", as
used herein, refers to any material which is insoluble, or can be made
insoluble by a
subsequent reaction. Solid support materials thus can be a latex, plastic,
derivatized plastic,
magnetic or non-magnetic metal, glass or silicon surface or surfaces of test
tubes, microtiter
wells, sheets, beads, microparticles, chips, and other configurations known to
those of
ordinary skill in the art. An exemplary capture reagent includes an array
which generally
comprises oligonucleotides or polynucleotides immobilized to a solid support
material in a
spatially defined manner.
For example, a heterogeneous format that could be employed to detect probes
hybridized to their respective target sequences could include first and second
probes that are
labeled with first and second binding members. The primers employed to amplify
the
respective target sequences could be labeled with a third binding member. Once
the probes
were hybridized to the target sequences, if present, to form target
sequence/probe hybrids, the
hybrids could be immobilized to a solid support using the third binding member
attached to
the primer sequences. If desired, the solid support could be washed to remove
excess
reagents, and the hybrids, if any, could be detected using conjugates having
distinct directly
detectable labels. Signals, if any, associated with the solid phase could then
be detected to
indicate the presence of target sequences in the test sample.
According to a preferred embodiment, "oligonucleotide hybridization PCR"
(variably
referred to herein as "OH PCR") as described in U.S. Patent Application Serial
No.
08/514,704, filed August 14, 1995, that is herein incorporated by reference,
is employed.
Briefly, the reagents employed in the preferred method comprise primers,
complementary
probes, as well as amplification reagents for performing an amplification
reaction. The
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primers are employed to prime extension of a copy of a target sequence (or its
complement),
and are labeled with either a capture label or a detection label. The probe
sequences are used
to hybridize with the sequences generated by the primers, and typically
hybridize with a
sequence that does not include the primers. Similarly to the primers, the
probes are also
labeled with either a capture label or a detection label with the caveat that
when the primer is
labeled with a capture label the probe is labeled with a detection label and
vice versa.
Detection labels have the same definition as "labels" previously defined and
"capture labels"
are typically used to separate extension products, and probes associated with
any such
products, from other amplification reactants. Specific binding members (as
previously
defined) are well suited for this purpose. Also, probes used according to this
method are
preferably blocked at their 3' ends so that they are not extended under
hybridization
conditions. Methods for preventing extension of a probe are well known and are
a matter of
choice for one skilled in the art. Typically, adding a phosphate group to the
3' end of the
probe will suffice for purposes of blocking extension of the probe.
According to the above preferred embodiment, the probes initially are part of
the
reaction mixture. Additionally, it is preferred to select primers, probes and
amplification
conditions such that the probe sequence has a lower melt temperature than the
primer
sequences so that upon placing the reaction mixture under amplification
conditions copies of
the tar get sequence or its complement are produced at temperature above the
Tm of the
2o probe. After such copies are synthesized, they are denatured and the
mixture is cooled to
enable the formation of hybrids between the probes and any copies of the
target or its
complement. The rate of temperature reduction from the denaturation
temperature down to a
temperature at which the probes will bind to single stranded copies is
preferably quite rapid
(for example S to 15 minutes) and particularly through the temperature range
in which an
enzyme having polymerase activity is active for primer extension. Such a rapid
cooling
favors copy sequence/probe hybridization rather that primer/copy sequence
hybridization and
extension. .
As indicated above, various target sequences and variant forms of the target
sequence
can be detected in a single reaction vessel. For example, the portion of the
gene encoding
3o factor V where the factor V Leiden mutation occurs and the factor V Leiden
mutation could
be employed as the target sequence and the variant form of the target sequence
respectively.
Alternatively, the 3' untranslated region of the prothrombin gene susceptible
to containing
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13
the mutation causitive of thrombosis and the same region that contains the
mutation could be
employed as the target sequence and the variant form of the target sequence.
Based upon the
sequence of these genes and the known mutations, probes completely
complementary to, for
example, the sense strand of the wild type sequence and anti-sense strand of
the variant or
mutant sequence could be synthesized with or without overhangs as mentioned
above. Given
the nature of the above mutatations (i.e. single base substitutions) such
probes at least would
have a mismatch where the mutation occurs and nevertheless be sufficiently
complementary
to each other to hybridize at ambient temperatures while preferentially
hybridizing to their
respective target sequences. Exemplary, probe pairs meeting the above criteria
for the factor
1o V gene and factor V Leiden mutation include SEQ. m. NO. 5 and any one of
SEQ, m. NO.
6, SEQ. m. NO. 7, SEQ. )D. NO. 8, SEQ. B?. NO. 9, SEQ. m. NO. 10, or SEQ. ID.
NO.
11. Exemplary probe pairs meeting the above criteria for the 3' untranslated
region of the
prothrombin gene and mutation to the 3' untranslated region of the prothrombin
gene include
SEQ. )D. NO. 16 and any one of SEQ. B7. NO. 17, SEQ. m. NO. 18, or SEQ. )D.
NO. 19.
Probe pairs according to the invention, such as those exemplified above or
otherwise
meet the criteria stated above can be employed in assays to detect a target
sequence and its
variant (e.g. the factor V gene, or a part thereof which may contain the
factor V Leiden
mutation, and the portion that actually contains the factor V Leiden
mutation). In particular,
a test sample suspected of containing the target sequence and a variant form
of the target
2o sequence can be contacted with the probe pair in a single reaction vessel.
Under suitable
conditions known to those skilled in the art, the probes hybridize to their
respective target
sequences, if any, to form probe target sequence hybrids. Any hybrids formed
between the
probes and target sequences can then be detected as an indication of the
presence of the any
target sequences in the test sample.
In certain cases, such as when the target sequences are not in sufficient
quantity for
detection, primers and amplification reagents may be added to the test sample
prior to or at
the same time the probes are contacted with the test sample. Due to the nature
of the probes
and the target sequences with which they hybridize, often times a single set
of amplification
primers can be selected to amplify both target sequences. Specifically,
primers can be
3o selected such that they amplify the target sequence in such a fashion that
the variation in the
second target sequence will also be included in the amplification product if
it is present. For
example, primers capable of amplifying the factor V gene and the sequence
containing the
CA 02426346 2003-04-17
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14
factor V Leiden mutation include SEQ. ID. NO. 3 and SEQ. ID. NO. 4.
Additionally,
primers capable of amplifying the 3' untranslated region of the prothrombin
gene and
mutation to the 3' untranslated region of the prothrombin gene include SEQ.
ID. NO. 14 and
SEQ. ID. NO. 15.
Examples
Example 1
Se uences
A. Leiden Factor V Se uences : The following examples demonstrate detection of
wild type
and mutant Leiden Factor V using the DNA oligomer primers and probes herein
provided.
These DNA primers and probes are identified as SEQ. ID NO. 3, SEQ. ID NO. 4,
SEQ. ID
NO. 5, SEQ. ID NO. 6, SEQ. ll~ NO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9, SEQ. ID
NO. 10
and SEQ. ID NO. 11. A portion of a representative sequence of wild type Leiden
Factor V is
designated herein as SEQ. ID NO. l, and a portion of the similar
representative sequence of
mutant Leiden Factor V is designated herein as SEQ. ID NO. 2. SEQ. ID NO. 2
differs from
SEQ. ID NO. 1 at only one base, which is the point mutation that distinguishes
mutant Leiden
Factor V from wild type. SEQ. ID NO. 3 and SEQ. ID NO. 4 are specific for
regions found
2o in both wild type and mutant Leiden Factor V. SEQ. ID NO. 5 is specific for
a region found
in wild type Leiden Factor V only. SEQ. ID NO. 6, SEQ. ID NO. 7, SEQ. ID NO.
8, SEQ.
ID NO. 9, SEQ. ID NO. 10 and SEQ. ID NO. 11 are specific for a region found in
mutant
Leiden Factor V only.
In the following examples, SEQ. ID NO. 3 and SEQ. ID NO. 4 are used as
amplification primers for both wild type and mutant Leiden Factor V. SEQ. ID
NO. 5 is used
as an internal hybridization probe fox the wild type Leiden Factor V
amplification product.
SEQ. ID NO. 6, SEQ. ID NO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9, SEQ. ID NO. 10 and
SEQ.
m NO. 11 are used as internal hybridization probes for the mutant Leiden
Factor V
amplification product.
B. Prothrombin Sequences: The following examples demonstrate detection of the
wild type
and mutant prothrombin genes using the DNA oligomer primers and probes herein
provided.
These DNA primers and probes are identified as SEQ. ID NO. 12, SEQ. m NO. 13,
SEQ. ID
NO. 14, SEQ. ID NO. 15, SEQ. ID NO. 16, SEQ. ID NO. 17, SEQ. ID NO. 18, SEQ.
ID NO.
19 and SEQ. ID NO. 20. A portion of a representative sequence of the wild type
prothrombin
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gene is designated herein as SEQ. ID NO. 12, and a portion of the similar
representative
sequence of the mutant prothrombin gene is designated herein as SEQ. ID NO.
13. SEQ. 1D
NO. 13 differs from SEQ. ID NO. 12 at only one base, which is the point
mutation that
distinguishes the mutant prothrombin gene from the wild type. SEQ. ID NO. 14
and SEQ. ID
5 NO. 15 are specific for regions found in both the wild type and mutant
prothrombin genes.
SEQ. ID NO. 16 is specific for a region found in the mutant prothrombin gene
only. SEQ. ID
NO. 17, SEQ. ID NO. 18, SEQ. ll~ NO. 19 and SEQ. ID NO. 20 are specific for a
region
found in the wild type prothrombin gene only.
In the following examples, SEQ. ID NO. 14 and SEQ. ID NO. 15 are used as
10 amplification primers for both the wild type and mutant prothrombin genes.
SEQ. ID NO. 16
is used as an internal hybridization probe for the mutant prothrombin gene
amplification
product. SEQ. ID NO. 17, SEQ. ID NO. 18, SEQ. ID NO. 19 and SEQ. ID NO. 20 are
used
as internal hybridization probes for the wild type prothrombin gene
amplification product.
Example 2
Preparation of Primers and Probes
A. wild type and mutant Leiden Factor V Primers Primers were designed to
detect the target
2o sequence of both wild type and mutant Leiden Factor V by oligonucleotide
hybridization
PCR. These primers were SEQ. ID NO. 3 and SEQ. ID NO. 4. Primer sequences were
synthesized using standard oligonucleotide synthesis methodology. The SEQ. ID
NO. 4
primer was haptenated with carbazole at the 5' end using standard cyanoethyl
phosphoramidite coupling chemistry as described in U.S. Patent No. 5,424,414
incorporated
herein by reference.
B. wild type and mutant Leiden Factor V Probes Probes were designed to
hybridize with the
amplified target sequence of wild type or mutant Leiden Factor V by
oligonucleotide
hybridization. These probes were SEQ. ID NO. 5 for wild type Leiden Factor V
and SEQ. ID
NO. 6, SEQ. ID NO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9, SEQ. ID NO. 10 and SEQ. ID
NO.
11 for mutant Leiden Factor V. Probe sequences were synthesized using standard
oligonucleotide synthesis methodology. The SEQ. ID NO. 5 wild type probe was
haptenated
with adamantane at the 5'end, followed by 10 thymidines and blocked with
phosphate at the
3' end. The SEQ. ID NO. 6, SEQ. ID NO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9, SEQ.
ID NO.
10 and SEQ. ID NO. 11 mutant probes were haptenated with dansyl at the 5 '
end, followed
by 10 thymidines and blocked with phosphate at the 3' end. All syntheses used
standard
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16
cyanoethyl phosphoramidite coupling chemistry as described in U.S. Patent No.
5,464,746
(herein incorporated by reference).
C, wild type and mutant Prothrombin Primers Primers were designed to detect
the target
sequence of both the wild type and mutant prothrombin gene by oligonucleotide
hybridization PCR. These primers were SEQ. ID NO. 14 and SEQ. )D NO. 15.
Primer
sequences were synthesized using standard oligonucleotide synthesis
methodology. The SEQ.
ID NO. 14 primer was haptenated with either carbazole or adamantane at the 5'
end using
standard cyanoethyl phosphoramidite coupling chemistry as described in U.S.
Patent No.
l0 5,424,414 incorporated herein by reference. The SEQ. ID NO. 15 primer was
haptenated
with adamantane at the 5'end by the same method.
D. wild type and mutant Prothrombin Probes Probes were designed to hybridize
with the
amplified target sequence of the Wild type or mutant prothrombin gene by
oligonucleotide
hybridization. These probes were SEQ. ID NO. 16 for the mutant prothrombin
gene and
SEQ. ID NO. 17, SEQ. ID NO. 18, SEQ. ID NO. 19 and SEQ. ID NO. 20 for the wild
type
prothrombin gene. Probe sequences were synthesized using standard
oligonucleotide
synthesis methodology. The SEQ. ID NO. 16 mutant probe was either haptenated
with 2
carbazoles at the 3' end, or haptenated with 2 dansyls at the 5'end, followed
by 10 thymidines
and blocked with phosphate at the 3' end. The SEQ. ID NO. 17, SEQ. ID NO. 18
and SEQ.
ID NO. 19 wild type probes were haptenated with 2 adamantanes at the 5 ' end,
followed by
10 thymidines and blocked with phosphate at the 3' end. Separately, both the
SEQ. ID NO.
17 and SEQ. ID NO. 20 wild type probes were haptenated with 2 carbazoles at
the 3' end.
All syntheses used standard cyanoethyl phosphoramidite coupling chemistry as
described in
U.S. Patent No. 5,464,746 (herein incorporated by reference).
Example 3
Sample Preparation
Wild type, heterozygous and homozygous mutant Leiden Factor V and Prothrombin
DNA was purified from 200 ~.1 of each whole blood sample using the QIAamp~
Blood Mini
Kit (QIAgen, Inc., Valencia, CA) nucleic acid extraction procedure and column
methodology
as described by the manufacturer. The DNA samples were simultaneously wild
type,
heterozygous or homozygous mutant for both genes. The genotype of all samples
was
verified by sequencing. Purified DNA was quantitated by taking the absorbance
reading at
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17
260 nm using a spectrophotometer. The final concentration of the purified DNA
was in the
range of 50 to 25 ng/~,1. '
Example 4
Detection of Mutant vs. Wild Type Leiden Factor V using Offset Probes
The probes used to detect the single base pair mutation distinguishing mutant
from
wild type Leiden Factor V were a sense strand probe and an antisense strand
probe,
respectively, and so would be exact compliments of one another except at the
single base pair
mismatch. Thus they could also hybridize with one another rather than with
their specific
target amplified DNA. It was hypothesized that offsetting the probes slightly
from one
another might help minimize any hybridization between them. This was tested by
deleting 2
bases from the 5' end of each probe.
Duplicates of each of the purified wild type, heterozygous and homozygous
mutant
Leiden Factor V/Prothrombin DNA samples were PCR amplified and detected using
SEQ. m
NO. 3 and SEQ. m NO. 4 primers with the SEQ. ID NO. 5 wild type probe and the
SEQ. ID
NO. 6 mutant probe. PCR was performed in buffer containing final
concentrations of 50 mM
Bicine (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.1, 150 mM potassium, 8% w/v
glycerol,
0.001 % bovine serum albumin (BSA), 0.1 mM EDTA and 0.02% sodium azide.
Recombinant Theru~us tl2ermophilus polymerase was used at a concentration of 5
units/reaction, with dNTPs (dATP, dGTP, dTTP and dCTP) present at a final
concentration
of 150 ~.M each. The SEQ. TD NO. 3 primer was used at a concentration of 120
nM, while
the SEQ. ID NO. 4 primer (labeled with carbazole) was used at a concentration
of 60 nM.
The SEQ. ID NO. 5 wild type probe was present at a concentration of 40 nM, and
the SEQ.
ID NO. 6 mutant probe was present at a concentration of 30 nM. A final
concentration of
3.25 mM Manganese chloride was used in a total reaction volume of 0.2 ml, with
sample
volume of 20 p1.
Reaction mixtures were amplified in an LCx° Thermal Cycler. Reaction
mixtures
were first incubated at 97°C for 4 minutes, followed by 45 cycles of
PCR amplification at
94°C for 30 seconds then 55°C for 45 seconds. After the reaction
mixtures were thermal
cycled, the mixtures were maintained at 97°C for 5 minutes and probe
oligo hybridization
was accomplished by lowering the temperature to 15°C. Samples were held
at 12°C for a
minimum of 5 minutes, and thereafter until reaction products were analyzed and
detected.
Reaction products were detected on the Abbott LCx° system (available
from Abbott
Laboratories, Abbott Park, IL). A suspension of anti-carbazole coated
microparticles, an
anti-adamantane antibody/alkaline phosphatase conjugate and an anti-dansyl
antibody/~3-
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18
galactosidase conjugate (all available from Abbott Laboratories, Abbott Park,
IL) were used
in conjunction with the LCxn to capture and detect the reaction products. The
enzyme
substrates used were 4-methyl-umbelliferyl phosphate (MUP) and 7-(3-D-
galactopyranosyloxy coumarin-4-acetic acid-(2-hydroxyethyl) amide (AUG) with
the rate of
conversion of substrate to product measured and reported as
counts/second/second (c/s/s).
The results in Table 1 show that while the wild type probe was able to detect
both the
homozygous wild type and the heterozygous DNA, and not the homozygous mutant
DNA (all
as expected), the mutant probe was unable to detect either the homozygous
mutant or the
heterozygous DNA.
to
TABLE 1
Wild type probe Mutant probe
Purified DNA Genoty LCxO rate (av . c/s/s)LCx~ rate (av . c/s/s)
a
Wild t a 667 27
Heterozy ous 475 32
Homozy ous Mutant 78 39
Modifications were made to the mutant probe, replacing the 2 removed bases at
the 5'
end and adding additional bases to the 3'end either with (SEQ. lD NO. 7, A) or
without
(SEQ. )D NO. 11, E) a mismatch G for destabilization, or still deleting the 2
bases at the 5'
end but adding 3, 4 or 5 bases to the 3' end (SEQ. ID NO. 8 (B), 9 (C) or 10
(D),
respectively). The experimental procedure described above was repeated using
each of these
mutant probes instead of SEQ. ID NO. 6 in separate reactions with the SEQ. TD
NO. 5 wild
2o type probe. The results are shown in Table 2 below.
TABLE 2
Purified Wild
DNA Type
/
Mutant
Probe
Pairs:
LCx
rate
(avg.
c/s/s)
Genotype ~WT Mut WT Mut WT Mut WT Mut WT Mut
A B C D E
WT 317 53 396 40 404 62 431 258 368 60
HT 240 219 244 298 244 378 259 530 284 210
HM ~ ~ 593 ~ 528 ~ 648 ~ 728 ~ 492
30 ~ 32 ~ 36 ~ 40 ~ 48
~ ~ ~ ~ ~
(WT = wild type; Mut = mutant; HT = heterozygous; HM = homozygous mutant)
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All mutant probes performed better than the previous SEQ. ID NO. 6 mutant
probe,
with the ability to detect both the heterozygous and homozygous mutant DNA.
With the
exception of mutant probe D, the mutant probes also showed good specificity
inasmuch as
they did not detect wild type DNA. The wild type probe continued to perform
well, detecting
both wild type and heterozygous, but not homozygous mutant, DNA. The best
signals were
achieved using mutant probes C (SEQ. 117 NO. 9) and D (SEQ. ID NO. 10). Thus,
the best
combination of probes for detecting either wild type or mutant Leiden Factor V
DNA was
using the wild type probe (SEQ. ID NO. 5), having a 2 base deletion at the 5'
end, with
mutant probe C (SEQ. ID NO. 9), also having a 2 base deletion at the 5' end
but also
containing 4 additional bases on the 3' end. This effectively offset the base
pair mismatch
between the wild type and mutant Leiden Factor V sequence, moving it from the
middle to
closer to the 5' end.
Thus, as a general matter, the offset probes were able to specifically
hybridize to their
appropriate amplified target sequences.
Example 5
Detection of Mutant vs. Wild Type Prothrombin
A. Using Sense Strand Probes Initial experiments to detect the wild type vs.
mutant
2o polymorphism on the prothrombin gene were performed using similar sense
strand probes,
one for the mutant gene (SEQ. ID NO. 16) and the other for the wild type gene
(SEQ. ID NO.
20), in separate reactions. In addition to the one base pair difference at the
wild type vs.
mutant locus, the wild type probe also contained 2 additional base pairs at
the 5' end.
Duplicates of the purified heterozygous and homozygous mutant Leiden Factor
V/Prothrombin DNA samples were diluted 1:100, 1:1000 and 1:10,000 in Molecular
Grade
Water (5'-3', Iilc., Boulder, CO), PCR amplified using SEQ. ID NO. 14 and SEQ.
ID NO. 15
primers labeled with adamantane and detected using either the carbazole
labeled SEQ. ID
NO. 16 mutant probe or the carbazole labeled SEQ. ID NO. 20 wild type probe,
in separate
reactions. PCR was performed in buffer containing final concentrations of 50
mM Bicine
(N,N,-bis[2-Hydroxyethyl]glycine), pH 8.1, 150 mM potassium, 8% w/v glycerol,
0.001%
bovine serum albumin (BSA), 0.1 mM EDTA and 0.02% sodium azide. Recombinant
Thermos theYmophilus polymerase was used at a concentration of 5
units/reaction, with
dNTPs (dATP, dGTP, dTTP and dCTP) present at a final concentration of 150 ~.M
each. The
primers were used at a concentration of 250 nM each, with the probes present
at a
concentration of 5 nM each. A final concentration of 3.25 mM Manganese
chloride was
used in a total reaction volume of 0.2 ml, with sample volume of 20 ~,1.
Duplicates of Poly-A
at 20 ng/[ul were run as a negative control.
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Reaction mixtures were amplified,in an LCx° Thermal Cycler. Reaction
mixtures
were first incubated at 95°C for 1 minute, then 56°C for 30
seconds, and 97°C for 2 minutes,
followed by 40 cycles of PCR amplification at 95°C for 40 seconds then
58°C for 60 seconds.
After the reaction mixtures were thermal cycled, the mixtures were maintained
at 72°C for 5
5 minutes, then 97°C for 5 minutes, with probe oligo hybridization
accomplished by lowering
the temperature to 15°C for 2 minutes. Samples were held at 4°C
thereafter until reaction
products were analyzed and detected.
Reaction products were detected on the Abbott LCx° system (available
from Abbott
Laboratories, Abbott Park,1L). A suspension of anti-carbazole coated
microparticles and an
to anti-adamantane antibody/alkaline phosphatase conjugate (all available from
Abbott
Laboratories, Abbott Park,1L) were used in conjunction with the LCx° to
capture and detect
the reaction products. The enzyme substrate used was 4-methyl-umbelliferyl
phosphate
(MUP) with the rate of conversion of substrate to product measured and
reported as
counts/second/second (c/s/s).
15 As can be seen in Table 3 below, while the mutant probe detected both the
heterozygous and the homozygous mutant samples, the wild type probe also
detected both
samples, though it should only react with the heterozygous sample and not the
homozygous
mutant. Thus, this probe combination, with both probes from the sense strand,
was not be
able to distinguish the wild type from the mutant polymorphism of the
prothrombin gene.
TABLE 3
Purified DNA Genotype Wild type sense probeMutant sense probe
(Sam 1e Dilution) LCx rate (av . c/s/s)LCx rate (av . c/s/s)
Heterozygous 850 553
1:100
1:1000 594 357
1:10,000 195 148
Homozygous Mutax~,t 731 642
1:100
1:1000 485 441
1:10,000 172 111
Poly A - Negative Control~ 24 ~ 17
~
B. Using Sense vs. Antisense Probes Since probes from the sense strand failed
to
differentiate the wild type from the mutant allele of the prothrombin gene,
the wild type
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21
probe was moved to the antisense strand to determine if this would facilitate
discrimination of
the 2 alleles.
This experiment was performed as described in Example S.A. above, except using
the
carbazole labeled SEQ. ID NO. 17 wild type probe instead of the SEQ. ID NO. 20
probe, and
testing duplicates of samples at the 1:100 dilution only. Thermal cycling
conditions were also
modified as follows: reaction mixtures were first incubated at 97°C for
2 minutes, followed
by 40 cycles of PCR amplification at 95°C for 40 seconds then
58°C for 60 seconds. After
the reaction mixtures were thermal cycled, the mixtures were maintained at
97°C for 5
minutes, with probe oligo hybridization accomplished by lowering the
temperature to 15°C
1o for 2 minutes. Samples were held at 15°C thereafter until reaction
products were analyzed
and detected.
TABLE 4
Purified DNA Genotype Wild type sense probe Mutant sense probe
(Sam 1e Dilution) LCx rate (av . c/s/s) LCx rate (av . c/s/s)
Heterozygous 586 792
1:100
Homozygous Mutar~,t 172 938
1:3.00
Poly A - Ne ative Control17 19
The results in Table 4 above, show that moving the wild type probe to the
antisense
strand allows discrimination between the wild type and mutant alleles to
occur, with the wild
type probe now showing a much higher detection signal with the heterozygous
sample,
containing one wild type allele, than with the homozygous mutant sample. The
mutant probe
2o also maintains its ability to detect both heterozygous and homozygous
mutant samples.
Additionally, the mutant probe, under these conditions, gives a slightly
higher signal with the
homozygous mutant sample, containing two mutant alleles, than with the
heterozygous
sample, with only one mutant allele, vs. what was seen in the experiment in
Example S.A.
C. Using Offset Probes Although discrimination of wild type and mutant
prothrombin was
achieved using one probe on the sense strand and the other on the antisense
strand, in
Example S.B. above, the ability to combine both probes in a single reaction
mixture, so that a
sample would only have to be tested once to determine the genotype, would be
advantageous.
This experiment also used the sense (mutant)/antisense (wild type) probes in
separate
reactions, requiring two tests to be run for each sample to determine the
sample's genotype. .
However, since the sense/antisense probes would be exact compliments of one
another,
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22
except at the single base pair mismatch, they could also hybridize with one
another rather
than with their specific target amplified DNA. Thermal cycling conditions were
modified to
help prevent this, and, additionally, the strategy that had been employed with
the Leiden
Factor V probes, offsetting the probes from one another around the base pair
mismatch, was
utilized.
The probe to detect the mutant allele of the prothrombin gene (SEQ. ID NO. 16)
was
the same as that used in Examples 5.A. and B. above, and contained the base
pair mismatch
in the center (base 6 of 13). Three wild type probes were tested with this
mutant probe: wild
type probe F (SEQ. ID NO. 19) was complementary to the mutant probe except for
the single
base pair mismatch; wild type probe G (SEQ. ID NO. 17) contained 2 additional
bases at the
3' end; and wild type probe H (SEQ. JD NO. 18) contained 2 additional bases at
the 3' end
and deleted 2 bases from the 5' end, creating a 2 base overhang on each
probe's 3' end.
Duplicates of each of the purified wild type, heterozygous and homozygous
mutant
Leiden Factor V/Prothrombin DNA samples were PCR amplified and detected as in
Example
4 except using SEQ. ID NO. 14 and SEQ. D7 NO. 15 primers, and the SEQ. H~ NO.
16
mutant probe with each of the 3 wild type probes described above in separate
reactions (i.e.
the mutant probe and one of each of the wild type probes were used together in
the same
reaction mixture). The reaction conditions used were those described in
Example 4 above,
except for the primer and probe concentrations which were as follows: the SEQ.
ID NO. 14
primer (labeled with carbazole) was used at a concentration of 200 nM, the
SEQ. ID NO. 15
primer was used at a concentration of 150 nM; the SEQ. ID NO. 16 mutant probe
was used at
a concentration of 70 nM, and either the SEQ. ID NO. 17 (G), 18 (H) or 19 (F)
wild type
probe was present in each reaction mixture at a concentration of 45 nM. The
results from this
experiment are shown in Table 5 below.
TABLE 5
Purified DNA Wild
T a
/ Mutant
Prolbe
Pairs:
LCxO
rate
(av
. c/s/s)
Genotype WT F Mut WT G Mut WT H Mut
Wild t a 277 67 302 59 365 54
Heterozygous 215 400 206 403 204 290
Homozy ous Mutant 59 720 88 703 59 444
(WT = wild type; Mut = mutant)
3o All wild type/mutant probe pairs showed specific detection of the
appropriate wild
type or mutant allele with the wild type probes detecting both homozygous wild
type and
heterozygous prothrombin samples but not detecting homozygous mutant
prothrombin DNA
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23
as positive. The mutant probe detected both homozygous mutant and heterozygous
prothrombin samples but did not detect homozygous wild type prothrombin
samples as
positive. As expected, all probes detected the heterozygous samples since they
contain one
wild type and one mutant allele, and these probes did so in a dose dependent
manner. Thus,
all probes showed excellent specificity.
The wild type probe (F) that was completely complementary to the mutant probe
(except at the base pair mismatch) worked, but gave the lowest signal with the
homozygous
wild type sample. The wild type probe that showed the best signal when
detecting the wild
type allele was mutant probe H (SEQ. B7 NO. 18), the probe combination giving
a 2 base
overhang at both ends. This probe combination also showed the best balance
(equivalency)
between mutant and wild type positive signals.
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.
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1/5
SEQUENCE LISTING
<110> Abbott Laboratories
Solomon, Natalie A.
Erickson, Dwight D.
Ziegler, Sharon R.
<120> Nucleic Acid Sequences Using Offset
Probes For Detecting Leiden Factor V And Prothrombin Wild
Type DNA
<130> 6746.US.01
<140> Not Yet Assigned
<141> 2000-10-27
<160> 20
<170> FastSEQ for Windows Version 4.0
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<213> Homo Sapiens
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gagagacatc gcctctgggc taataggact acttctaatc tgtaagagca gatccctgga 120
caggcgagga atacaggtat tttgtccttg aagtaacctt tcagaaattc tgagaatttc 180
ttctggctag aacatgttag gtctcctggc taaataatgg ggca 224
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<213> Homo Sapiens
<400> 2
acccacagaa aatgatgccc agtgcttaac~aagaccatac tacagtgacg tggacatcat 60
gagagacatc gcctctgggc taataggact acttctaatc tgtaagagca gatccctgga 120
caggcaagga atacaggtat tttgtccttg aagtaacctt tcagaaattc tgagaatttc 180
ttctggctag aacatgttag gtctcctggc taaataatgg ggca 224
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Amplification Primer
<400> 3
acccacagaa aatgatgccc ag 22
<210> 4
<211> 22
<212> DNA
CA 02426346 2003-04-17
WO 02/46476 PCT/USO1/50997
2/5
<213> Artificial Sequence
<220>
<223> Amplification Primer
<400> 4
tgccccatta tttagccagg ag 22
<210> 5
<211> 11
<212> DNA
<~13> Artificial Sequence
<220>
<223> Probe
<400> 5
cctcgcctgt c 1l
<210> 6
<211> 1l
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 6
caggcaagga a 11
<210> 7
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 7
gacaggcaag gaagac 16
<210> 8
<211> l4
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 8
caggcaagga atac
14
<210> ,~
<211> 15
<212> DNA
<213> Artificial Sequence
CA 02426346 2003-04-17
WO 02/46476 PCT/USO1/50997
3/5
<220>
<223> Probe
<400> 9
caggcaagga ataca 15
<210> 10
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 10
caggcaagga atacag 16
<210> 11
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 11
gacaggcaag gaata 15
<210> 12
<211> 127
<212> DNA
<213> Homo Sapiens
<400> 12
catattctgg gctectggaa ccaatcccgt gaaagaatta tttttgtgtt tctaaaacta 60
tggttcccaa taaaagtgac tctcagcgag cctcaatgct cccagtgcta ttcatgggca 120
gctctct 127
<210> 13
<211> 127
<212> DNA
<213> Homo Sapiens
<400> 13
catattetgg gctcetggaa ccaatccegt gaaagaatta tttttgtgtt tctaaaacta 60
tggttcccaa taaaagtgac tctcagcaag cctcaatgct cccagtgcta ttcatgggca 120
gctctet - 127
<2l0> Z4
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Amplification Primer
<400> 14
CA 02426346 2003-04-17
WO 02/46476 PCT/USO1/50997
4/5
catattctgg gctcctggaaic 21
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Amplification Primer
<400> 15
agagagctgc ccatgaatag cac 23
<210> 16
<211> 13
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 16
tcagcaagcc tca 13
<210> 17
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 17
tgaggctcgc tgaga 15
<210> 18
<211> 13
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 18
aggctcgctg aga 13
<210> 19
<211> 13
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 19
tgaggctcgc tga 13
CA 02426346 2003-04-17
WO 02/46476 PCT/USO1/50997
5/5
<210> 20
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Probe
<400> 20
tctcagcgag cctca 15
