Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02922261 2016-02-23
Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013
ENRICHMENT AND QUANTIFICATION OF NUCLEIC ACID SEQUENCES
FIELD OF THE DISCLOSURE
This disclosure relates to detecting nucleic acid sequence alterations, or
mutations, in
the presence of highly similar wild-type sequences and to quantification of
the sequence
alteration or mutation.
BACKGROUND OF THE DISCLOSURE
Mutations in BRAF and KRAS are examples of genetic alterations that confer a
survival and growth advantage to cancer cells. Such genetic alterations can be
used for
selection of targeted therapies. But in a subject, the alterations are present
with a large excess
of non-altered, wild-type sequences.
Cell-free (cf) nucleic acids present in bodily fluids may be an aid in
identifying and
selecting individuals with cancer or other diseases associated with such
genetic alterations.
SUMMARY OF THE DISCLOSURE
The instant disclosure is based in part on the development of an assay using
cell-free
DNA (cfDNA) that enriches for the extremely low levels of altered, or mutant,
DNA to
provide high detection sensitivity. The disclosure utilizes a non-limiting
example of a KRAS
assay using cfDNA extracted from urine to illustrate aspects and principles of
the process.
In a first aspect, the disclosure provides a method for enriching a target
nucleic acid
sequence in an amplification reaction mixture. The method may comprise
a) preparing an amplification reaction mixture comprising a nucleic acid
sample comprising a reference (optionally wild-type) sequence and at least
suspected of
having one or more target (optionally mutant) sequences that are at least 50%
homologous to
the reference sequence and are also amplifiable by the same primer pair as the
reference
sequence, and an excess amount of reference blocking nucleic acid sequence
which is fully
complementary with at least a portion of the sequence of one of the strands of
the reference
sequence between its primer sites;
b) increasing the temperature of the reaction mixture to a first denaturing
temperature that is above the melting temperature (T.) of the reference
sequence and above
the melting temperature (T.) of the double stranded target sequence so as to
form denatured
reference strands and denatured target strands;
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c) reducing the temperature of the reaction mixture so as to permit formation
of duplexes of the reference blocking sequence and the complementary reference
strand and
heteroduplexes of the reference blocking sequence and target strands;
d) increasing the temperature of the reaction mixture to a critical
temperature
(Tc) sufficient to permit preferential denaturation of said heteroduplexes of
the reference
blocking sequence and target strands in preference to denaturation of the
duplexes of the
reference blocking sequence and reference strands;
e) reducing the temperature of the reaction mixture so as to permit the primer
pair to anneal to denatured target strands and any denatured reference strands
in the reaction
mixture;
t) increasing the temperature of the reaction mixture to a denaturing
temperature that is above the melting temperature (T.) of the reference
sequence and above
the melting temperature (T.) of the double stranded target sequence so as to
form denatured
reference strands and denatured target strands to extend the primers annealed
to the denatured
target strands and denatured reference strands in the reaction mixture; and
g) repeating c) through 1) for two or more cycles to enrich, in the reaction
mixture, the target sequence relative to the reference sequence.
The temperature increase in subparagraph f) is performed without maintenance
of any
one temperature as a "step" for extension of the annealed primers. Stated
differently, the
temperature of the reaction mixture is continually increased, after reaching
the temperature of
subparagraph e), until reaching the denaturing temperature in f). Optionally,
the denaturing
temperature of subparagraph f) is the same as that in b).
In some embodiments, the reference blocking sequence is complementary to a
portion
of the denatured target strand that is also complementary to at least a
portion of the 3' end of
one or both of the primers used. In other embodiments, the reference blocking
sequence may
include a 3'-end that is blocked to inhibit extension. In further embodiments,
the reference
blocking sequence strands may include a 5'-end comprising a nucleotide that
prevents 5' to 3'
exonucleolysis by Taq DNA polymerases. In yet additional embodiments, the
reference
blocking sequence may be a single-stranded nucleic acid reference blocking
sequence; a
double-stranded nucleic acid reference blocking sequence which denatures to
form single
strand reference blocking sequences in b) when the reaction mixture is heated
to the first
denaturing temperature; single stranded DNA, RNA, peptide nucleic acid or
locked nucleic
acid; or a chimera between single stranded DNA, RNA, peptide nucleic acid or
locked
nucleic acid or another modified nucleotide.
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Filing Date: October 20,2013
In cases of a peptide nucleic acid or locked nucleic acid, the position of the
peptide
nucleic acid or locked nucleic acid on the chimera sequence are selected to
match positions
where mutations are suspected to be present, thereby maximizing the difference
between the
temperature needed to denature heteroduplexes of the reference blocking
sequence and target
strands and the temperature needed to denature heteroduplexes of the reference
blocking
sequence and the complementary reference strand.
In other embodiments, the reference blocking sequence is fully complementary
with
one of the strands of the reference sequence between its primer binding sites,
or overlapping
at either end the primer binding sites. In further embodiments, the reference
blocking
sequence is equal to or shorter than the reference sequence. In yet additional
embodiments,
the reference blocking sequence is present in the reaction mixture at a
concentration of about
25 nM.
In some versions of the method, the temperature reducing in c) is less than
one
minute. In other versions, the melting temperature of the double-stranded
target sequence is
greater than or equal to the melting temperature of the double-stranded
reference sequence.
In additional versions, the T, is maintained for a period from 1 second to 60
seconds.
Including in the disclosure are methods wherein the reference and target
sequences
are first amplified by subjecting the reaction mixture to PCR and then
subjecting at least a
portion of the reaction mixture to the enrichment method described above.
In further embodiments, the target sequence may be that of a homozygous
mutation in
a subject, such as a human patient. In some cases, the reference and target
sequences are
KRAS sequences, optionally human KRAS sequences. In additional cases, the
reference and
target sequences comprise at least 25 base pairs. In further cases, the
reference and target
sequences are cell-free DNA (c1DNA), optionally obtained from a bodily fluid
such as urine,
blood, serum, or plasma.
In other embodiments, the disclosed method is performed in a real-time PCR
device,
optionally utilizing a labeled probe. In additional embodiments, the reaction
mixture in the
method contains a nucleic acid detection dye.
In a second aspect, the above enrichment method may be combined with a
detection
method for assessing one or more mutations post-enrichment. 'The disclosed
methods may
thus further include analyzing the reaction mixture with enriched target
sequence using one
or more methods selected from MALDI-TOF, HR-Melting, Di-deoxy-sequencing,
Single-
molecule sequencing, pyrosequencing, Second generation high-throughput
sequencing,
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SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR. These analytical
techniques
may be used to detect specific target (mutant) sequences as described herein.
In a third aspect, the assessment described above may be performed with
quantification of the detected target (mutant) sequences. The quantification
provides a means
for determining a calculated input percentage of the target sequence prior to
enrichment
based upon the output signal (optionally as a percentage) from the assessment.
This may be
performed by reference to a fitted curve like those illustrated in Figures 5
and 6. The actual
output from an assessment of a test sample is determined in combination with
one or more
control reactions containing a known quantity of target sequence DNA. The
outputs from the
test sample and the control(s) are compared to a fitted curve to interpolate
or extrapolate a
calculated input for the test sample. This permits a quantitative
determination of the amount
of a target sequence in a sample pre-enrichment based upon a post-enrichment
detection.
In some embodiments, a disclosed enrichment method is used as part of a method
for
determining the amount of a target sequence in a sample containing a reference
sequence is
provided. The method for determining may comprise performance of a disclosed
enrichment
method followed by a detection method, such as sequencing or massively
parallel sequences
ass non-limiting example, with a sample from a subject and one or more control
samples with
a known amount of the target sequence to measure the amount of the target
sequence; and
then calculating the amount of the target sequence and the one or more control
samples by
comparison to the measurement(s) of one or more known samples of target
sequence in the
sample. In some cases, the sample is urine, and the target sequence is cfDNA.
The number of control samples may be two or more, optionally selected to
include a
first control with an amount higher than that expected in the test sample and
a second control
with an amount that is lower than that expected in the test sample. Without
limiting the
disclosure, the control samples may be considered "markers" that are measured
at the same
time as the test sample.
Of course the disclosure further provides computer readable media comprising
program instructions for performing any disclosed method.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the two-step assay design for a 28-30 bp footprint in the
target
gene sequence.
Figure 2 illustrates the establishment of detection cutoffs using MAD scores.
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Attorney Docket No. 032035-052600US
Filing Date: October 20, 2013
Figure 3 illustrates that a cell line spike at 0.2% mutant indicates a highly
specific
assay.
Figure 4 illustrates KRAS detection in the urinary cfDNA of cancer patients.
Figure 5 shows a curve fit of KRAS enrichment data from known amounts of
mutant
sequence relative to wildtype sequence.
Figure 6 shows a log curve fit with 95% confidence bands and calculated input
mutation level of a cancer patient
DETAILED DESCRIPTION OF MODES OF PRACTICING THE DISCLOSUFtE
The disclosure provides a sensitive, easy and inexpensive test for the routine
clinical
detection of a gene alteration in cell-free nucleic acids from a sample of a
subject. In some
embodiments, the test is based on KRAS mutant detection. In other embodiments,
the test
may be for the BRAF V600E mutation.
The disclosed method for enriching a target nucleic acid sequence in an
amplification
reaction mixture was developed in part to achieve a high sensitivity, such as
the ability to
detect a target (mutant) sequence at a concentration between 0.01 to 0.05% or
higher in the
presence of excess reference (wildtype) sequence. In some embodiments, this
was achieved
in combination with a short DNA footprint of about 28 basepairs to about 50
basepairs. In
the non-limiting description of the KRAS assay herein, the footprint was about
30 basepairs.
The KRAS assay detects at least 7 different KRAS mutations in codons 12 and 13
of
human KRAS. A list of nucleotide substitutions for each of the KRAS mutations
tested in the
assay are shown in the following Table. Seven mutations were validated using
mutation
containing cell line DNA.
Table 1
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Filing Date: October 20, 2013
todbn 12 Cexion 13
. . . . . . . . . . .
WI GGTGGC
GI2A. .
.G C I ...... G. .
.
= === .===== ==
.................... .. . ...
............. .......... . ........ . . ..........
..............
. . ..
.
= = . = =
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. ..
.. . .
These mutations may be assessed by use of the disclosed enrichment method
followed
by next generation sequencing as a non-limiting example. This assessment, or
assay,
provides the ability to detect multiple mutations in a single run. Nine (9)
KRAS mutations
that were assessed in a single assay were Gl2A, G12C, G12D, G12F, G12R, G12S,
G12V,
G13C, and G13D.
In addition to KRAS mutations, the disclosure provides for the use of the
disclosed
methods for any cellular or mitochondrial mutation associated with a disease
or disorder in
the presence of wildtype sequences. In many embodiments, the disclosed methods
may be
performed in cases of cancer, including primary cancer or cancer that has
metastasized. In
other cases, the methods may be used in cases of a malignant, or non-
malignant, tumor.
Non-limiting examples of cancer include, but are not limited to, adrenal
cortical
cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain or a
nervous system
cancer, breast cancer, cervical cancer, colon cancer, rectral cancer,
colorectal cancer,
endometrial cancer, esophageal cancer, Ewing family of tumor, eye cancer,
gallbladder
cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal cancer,
Hodgkin Disease,
intestinal cancer, Kaposi Sarcoma, kidney cancer, large intestine cancer,
laryngeal cancer,
hypopharyngeal cancer, laryngeal and hypopharyngeal cancer, leukemia, acute
lymphocytic
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leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia
(CLL),
chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), non-
HCL
lymphoid malignancy (hairy cell variant, splenic marginal zone lymphoma
(SMZL), splenic
diffuse red pulp small B-cell lymphoma (SDRPSBCL), chronic lymphocytic
leukemia (CLL),
prolymphocytic leukemia, low grade lymphoma, systemic mastocytosis, or splenic
lymphoma/leukemia unclassifiable (SLLU)), liver cancer, lung cancer, non-small
cell lung
cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, lymphoma of
the skin,
malignant mesothelioma, multiple myeloma, nasal cavity cancer, paranasal sinus
cancer,
nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,
non-Hodgkin
lymphoma, oral cavity cancer, oropharyngeal cancer, oral cavity and
oropharyngeal cancer,
osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary
tumor, prostate
cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma,
adult soft tissue
sarcoma, skin cancer, basal cell skin cancer, squamous cell skin cancer, basal
and squamous
cell skin cancer, melanoma, stomach cancer, small intestine cancer, testicular
cancer, thymus
cancer, thyroid cancer, uterine sarcoma, uterine cancer, vaginal cancer,
vulvar cancer,
Waldenstrom Macroglobulinemia, and Wilms Tumor.
Non-limiting examples of non-HCL lymphoid malignancy include, but are not
limited
to, hairy cell variant (HCL-v), splenic marginal zone lymphoma (SMZL), splenic
diffuse red
pulp small B-cell lymphoma (SDRPSBCL), splenic leukemia/lymphoma
unclassifiable
(SLLU), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, low grade
lymphoma, systemic mastocytosis, and splenic lymphoma/leukemia unclassifiable
(SLLU).
In additional embodiments, the disclosed methods are used in human subjects,
such as
those undergoing therapy or treatment for a disease or disorder associated
with a gene
alteration as described herein. Subjects may be of any age, including, but not
limited to
infants, toddlers, children, minors, adults, seniors, and elderly individuals.
In many eases, the sample containing a target sequence is a bodily fluid. Non-
limiting
examples of a bodily fluid include, but are not limited to, peripheral blood,
serum, plasma,
urine, lymph fluid, amniotic fluid, and spinal fluid. .
The disclosure demonstrates that massively parallel sequencing can be an
effective
tool to monitor mutation status of the KRAS gene in urinary cfDNA. The assay
is selective
and highly specific for all seven KRAS mutations within KRAS codons 12 and 13.
Preliminary results show that mutated KRAS could be detected in the urine of 8
out of 9
patients whose tumor tissue contained a KRAS mutation. The discrepancy of the
called
nucleotide in 4 of the 8 detectable tumor samples may highlight discrepancies
in patient
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Filing Date: October 20, 2013
tumor heterogeneity of these samples. Using massively parallel DNA sequencing
to detect
mutations from cell-free urinary DNA non-invasively monitors metastatic
patients for
response, non-response and the emergence of resistance mechanisms of
molecularly targeted
therapies.
The present disclosure also provides, in part, a kit for performing the
disclosed
methods. The kit may include a specific binding agent that selectively binds
to a BRAF
mutation, and instructions for carrying out the method as described herein.
As used herein the term "sample" refers to anything which may contain an
analyte for
which an analyte assay is desired. In many cases, the analyte is a cf nucleic
acid molecule,
such as a DNA or cDNA molecule encoding all or part of BRAF. The sample may be
a
biological sample, such as a biological fluid or a biological tissue. Examples
of biological
fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum,
cerebral spinal fluid,
tears, mucus, amniotic fluid or the like. Biological tissues are aggregate of
cells, usually of a
particular kind together with their intercellular substance that form one of
the structural
materials of a human, animal, plant, bacterial, fungal or viral structure,
including connective,
epithelium, muscle and nerve tissues. Examples of biological tissues also
include organs,
tumors, lymph nodes, arteries and individual cell(s).
As used herein, a "subject" includes a mammal. The mammal can be e.g., any
mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, home,
goat, camel,
sheep or a pig. In many cases, the mammal is a human being.
Cancer is a group of diseases that may cause almost any sign or symptom. The
signs
and symptoms will depend on where the cancer is, the size of the cancer, and
how much it
affects the nearby organs or structures. If a cancer spreads (metastasizes),
then symptoms may
appear in different parts of the body.
One skilled in the art may refer to general reference texts for detailed
descriptions of
known techniques discussed herein or equivalent techniques. These texts
include Ausubel et
al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005);
Sambrook et
al., Molecular Cloning, A Laboratory Manual (ri edition). Cold Spring Harbor
Press, Cold
Spring Harbor, New York (2000); Coligan et al., Current Protocols in
Immunology, John
Wiley & Sons, N.Y.; Enna et at., Current Protocols in Pharmacology, John Wiley
& Sons,
N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975),
Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990).
These texts
can, of course, also be referred to in making or using an aspect of the
disclosure.
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Filing Date: October 20, 2013
Other features and advantages of the present disclosure are apparent from the
different
examples. The provided examples illustrate different components and
methodology useful in
practicing the present disclosure. The examples do not limit the disclosure.
EXAMPLES
Example 1: Materials and Methods:
The following methods described herein were utilized in the examples that
follow.
Patient urine samples
A total of 9 patients with advanced cancers, who were previously tested for
mutations
in KRAS by a CLIA certified laboratory, were tested for codon 12 and 13 KRAS
mutations
along with 27 healthy controls. Cell free urinary DNA was collected from each
patient.
Two-step assay design
A two-step assay design was developed for a 28-30 basepair footprint in the
target
mutant gene sequence. Figure 1 summarizes the assay design, which includes a
first pre-
amplification step to increase the number of copies of a target mutant gene
sequence relative
to wild-type gene sequences that are present in the sample. The pre-
amplification is
conducted in the presence of a wild-type (non-mutant) suppressing "WT blocker"
oligonucleotide that is complementary to the wild-type sequence (but not the
mutant
sequence) to decrease amplification of wild-type DNA. The pre-amplification is
performed
with primers that include adapters (or "tags") at the 5' end to faciliatate
amplification in the
second step.
"lhe second step is additional amplification with primers complementary to the
tags on
the ends of the primers used in the first step and substitution of multiplex
sequencing for the
illustrated use of digital droplet PCR with a Taqman (reporter) probe
oligonucleotide
complementary to the mutant sequence,.
Assay development
Figure 2 illustrates the establishment of detection cutoffs using MAD scores.
Dotted
vertical lines represent z-score cutoffs of 2 sigma. z score density
distribution of KRAS
G12V target/non-target ratios observed in a healthy control (grey) with
mutation detection
results from colon cancer patient (h.), forward reads (gold point) and reverse
reads in (blue
point).
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Figure 3 illustrates that a cell line spike at 0.2% mutant indicates a highly
specific
assay. Figure 4 illustrates KRAS detection in the urinary cfDNA of cancer
patients.
Together, these figures show results from sample libraries that were combined
and sequenced
on an Illumina MiSeq instrument (mean sample sequence counts of 129k).
Following
demultiplexing, KRAS fragments were identified by matching left and right
flanking regions
(mean counts of 117k). The target mutant variants were quantified by computing
the
frequency of occurrence of each 5 bp sequence (Table 1) in the KRAS identified
samples. For
each targeted mutation, the frequency of non-target nucleotides (not including
wildtype) was
also computed. These values were used to normalize against variation in the
nucleotide
substitution rates inherent in the enrichment, library prep, and sequencing
steps of sample
processing. The ratio of target to non-target frequencies was used as a test
statistic for KRAS
mutation detection. Target to non-target ratios were standardized by
converting them to
robust z-scores. The robust z-score of a raw score x is:
robust z = (x ¨ m) /1.4826 MAD
where m is the median of the healthy control sample population and MAD is the
median
absolute deviation of the healthy control sample population. Using the median
and MAD of
the population produce a z-score that is more robust to outliers than z-scores
computed using
the mean and standard deviation.
Quantifation
The following table and Figure 5 illustrate curve fit and calculated input
mutation
level of a cancer patient containing the KRAS G12D mutation.
Table 2
Sample Actual% Calculated Up-per Lower
Output %Input
etiltiletitiFINISMOSEUINICASSIMPIPR.RIVA4SME041351(GIZD
11
MiiinigeningiginaMeMEM:EMONWSMIMMONN:
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Filing Date: October 20, 2013
The raw data plot (Figure 5) of the enriched reference data shows a best fit
to a
hyperbolic curve (also known as a saturation binding or dose response curve)
demonstrating a
strong nonlinear enrichment of low level mutant species. Mutant DNA input at
known
amounts of 0.2%, 0.05%, 0.01% and 0.0% of the total DNA returned observed
detection
levels of 18.25%, 4.45%, 1.84% and 0.54% respectively as a percentage of the
total sequence
reads. Using urinary DNA from a stage IV colorectal carcinoma patient with a
known
mutation at the 612D site it was found that the mutation accounted for 13.06%
of the total
sequence after enrichment, corresponding to an input amount of approximately
0.14%
mutational load in the patient's urine.
The following table and Figure 6 illustrate curve fit and calculated input
mutation
using a log transformed axis and showing the 95% confidence bands. Figure 6 is
a log
transformed plot of the data in Figure 5 with inclusion of the 95% confidence
bands based on
repeated assessment of known amounts of mutant sequence.
Table 3
Sample Actual %
Calculated
Output % Inpuõ t =
liC01005012A9 EiRIV803.1108"EN02261548EM
Patient signals whose z score is above the cutoff (Figure 2, >2.0, 95%
confidence) can
be quantified using the ratio of mutant to wildtype sequence counts for that
position. These
are converted to a percentile and plotted to a reference curve to interpolate
the input mutation
level of the original sample.
Example 2: KRAS mutations in cfDNA
The agreement between tumor tissue and cfKRAS is shown in the table below.
KRAS
mutations were detected in the urine of 8 out of 9 metastatic cancer patients
previously
detected in tumor tissue by a CLIA certified laboratory. A z score of 2.0 or
more indicates a
95% or greater confidence level of a true call compared to background.
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Table 4:
Detection or mutant ,
Urtriary tfDNA
1 Cance=rType Tumorl KRAS in Urinary z :score,
(WA) KRAS Mutation
cfONA
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LungAdenocarcinoma 612V
:V G12V: =
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id3.iglligg.Nt'it;,:.O.,.lgling4Uki,,oaIV:A.
C910.r.0;ta.V .;. .:.:=:=:
.=::GiI5 32
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'Me citation of documents herein is not to be construed as reflecting an
admission that
any is relevant prior art. Moreover, their citation is not an indication of a
search for relevant
disclosures. All statements regarding the date(s) or contents of the documents
is based on
available information and is not an admission as to their accuracy or
correctness.
All references cited herein, including patents, patent applications, and
publications,
are hereby incorporated by reference in their entireties, whether previously
specifically
incorporated or not.
Having now fully described the inventive subject matter, it will be
appreciated by
those skilled in the art that the same can be performed within a wide range of
equivalent
parameters, concentrations, and conditions without departing from the spirit
and scope of the
disclosure and without undue experimentation.
While this disclosure has been described in connection with specific
embodiments
thereof, it will be understood that it is capable of further modifications.
This application is
intended to cover any variations, uses, or adaptations of the disclosure
following, in general,
the principles of the disclosure and including such departures from the
present disclosure as
come within known or customary practice within the art to which the disclosure
pertains and
as may be applied to the essential features hereinbefore set forth.
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