U.S. patent application number 14/044731 was filed with the patent office on 2014-05-22 for fast pcr for str genotyping.
This patent application is currently assigned to Life Technologies Corporation. The applicant listed for this patent is Life Technologies Corporation. Invention is credited to Lori Hennessy, Dennis Wang.
Application Number | 20140141422 14/044731 |
Document ID | / |
Family ID | 44505476 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140141422 |
Kind Code |
A1 |
Wang; Dennis ; et
al. |
May 22, 2014 |
Fast PCR for STR Genotyping
Abstract
Disclosed is a method of amplifying a nucleic acid sequence,
wherein the method comprises subjecting a reaction mixture to at
least one amplification cycle, wherein the reaction mixture
comprises a double-stranded nucleic acid and at least two primers
capable of annealing to complementary strands of the
double-stranded nucleic acid and amplifying at least one short
tandem repeat (STR) using a Family A DNA polymerase in a Fast PCR
protocol having a two-step amplification cycle in 25 seconds or
less. Also disclosed are real-time PCR methods using the two-step
protocol and kits for STR profiling using the Fast PCR
protocol.
Inventors: |
Wang; Dennis; (Dublin,
CA) ; Hennessy; Lori; (San Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Life Technologies Corporation |
Carlsbad |
CA |
US |
|
|
Assignee: |
Life Technologies
Corporation
Carlsbad
CA
|
Family ID: |
44505476 |
Appl. No.: |
14/044731 |
Filed: |
October 2, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13035849 |
Feb 25, 2011 |
8580505 |
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14044731 |
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61308862 |
Feb 26, 2010 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6858 20130101;
C12Q 1/686 20130101; C12Q 1/686 20130101; C12Q 2525/151 20130101;
C12Q 2527/107 20130101; C12Q 2527/113 20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-43. (canceled)
44. A composition comprising a Pol A enzyme and an amplified short
tandem repeat (STR) locus, wherein the enzymatic activity of the
Pol A enzyme present is such that a doubling of enzymatic activity
increases the presence of an amplified STR locus with a split
peak.
45. The composition of claim 44, wherein the Pol A enzyme is
derived from Thermus aquaticus.
46. The composition of claim 45, wherein the enzymatic activity is
7.5 Units.
47. The composition of claim 45, wherein the enzymatic activity is
from 7.5 Units to 10 Units.
48. The composition of claim 44, wherein the amplified STR locus is
D5S818.
49. The composition of claim 44, wherein the amplified STR locus is
D8S1179.
Description
FIELD
[0001] In general, the present teachings relate to the
amplification of a nucleic acid sample for the purposes of
obtaining an STR profile in less than 45 minutes.
BACKGROUND
[0002] Since the PCR process depends greatly on the performance of
the DNA polymerase used, various DNA polymerases have been searched
for in nature or re-engineered in vitro. Two key properties of a
DNA polymerase play important roles in determining the overall
reaction time required for the PCR amplification. The first
property is "elongation rate" (or "extension rate"), which is
defined as the number of nucleotides polymerized per second per
molecule of DNA polymerase. The second property is "processivity",
which is defined as the average number of nucleotides added by a
DNA polymerase in a single binding event. Both "elongation rate"
and "processivity" depends on the components of the reaction media
and on the DNA template sequence.
[0003] Rapid and accurate detection of DNA profiles is a key aspect
of forensic sample analysis and the technique of polymerase chain
reaction (PCR) plays an integral part in this process. Methods to
decrease the PCR time will save on technician labor. There is an
unmet need to decrease PCR time without compromising sensitivity,
specificity and accuracy of results.
SUMMARY OF SOME EMBODIMENTS OF THE INVENTION
[0004] In some embodiments, disclosed is a method for amplifying a
nucleic acid sequence wherein the method involves subjecting a
reaction mixture to at least one amplification cycle, wherein the
reaction mixture comprises a double-stranded nucleic acid, at least
two primers capable of annealing to complementary strands of the
double-stranded nucleic acid and amplifying at least one short
tandem repeat (STR), and a Family A DNA polymerase, and wherein the
at least one amplification cycle comprises denaturing the
double-stranded nucleic acid in the reaction mixture; and annealing
the at least two primers to complementary strands of the denatured
double-stranded nucleic acid and extending the at least two
primers; and wherein the time to complete one amplification cycle
is 25 seconds, 20 seconds, 15 seconds, 10 seconds or less.
[0005] In some embodiments the annealing temperature in the
amplification cycle is at least about 5.degree. C. greater,
10.degree. C. greater, or 15.degree. C. greater than the predicted
Tm of at least one of the at least two primers while the annealing
temperature ranges from about 55.degree. C. to about 75.degree. C.
and the annealing and extending occur at the same temperature. In
some embodiments the reaction mixture is held at the annealing
temperature for 1 second, 2 seconds, 3 seconds or up to 20 seconds
or more. In some embodiments the denaturing temperature is from 4
to 8 seconds and the annealing temperature in conjunction with the
extending temperature is from 5 seconds to 25 seconds.
[0006] In some embodiments, the DNA polymerase used in the Fast PCR
protocol herein is a Family A (Pol A) DNA polymerase from either a
natural or recombinant source including fragments and variants
thereof.
[0007] In some embodiments, the Fast PCR protocol method as taught
herein can be used for the amplification of a nucleic acid sample
to obtain an STR profile in 45 minutes or less. In other
embodiments a multiplex of at least 15 STR loci, at least 20 STR
loci or at least 40 STR loci plus the amelogenin loci are amplified
by the Fast protocol method. In some embodiments, the Fast PCR
protocol method is used for a real-time PCR reaction.
[0008] In other embodiments, kits are taught in varying
configurations for use in human identification comprising at least
one primer pair for the amplification of an STR loci in an
amplification cycle of 25 seconds or less.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1A-1E are schematics depicting results in replicate of
an enzyme titration experiment using Platinum.RTM. Taq and the Fast
PCR protocol.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0010] For the purposes of interpreting of this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
In the event that any definition set forth below conflicts with the
usage of that word in any other document, including any document
incorporated herein by reference, the definition set forth below
shall always control for purposes of interpreting this
specification and its associated claims unless a contrary meaning
is clearly intended (for example in the document where the term is
originally used). It is noted that, as used in this specification
and the appended claims, the singular forms "a," "an," and "the,"
include plural referents unless expressly and unequivocally limited
to one referent. The use of "or" means "and/or" unless stated
otherwise. For illustration purposes, but not as a limitation, "X
and/or Y" can mean "X" or "Y" or "X and Y". The use of "comprise,"
"comprises," "comprising," "include," "includes," and "including"
are interchangeable and not intended to be limiting. Furthermore,
where the description of one or more embodiments uses the term
"comprising," those skilled in the art would understand that, in
some specific instances, the embodiment or embodiments can be
alternatively described using the language "consisting essentially
of" and/or "consisting of". The term "and/or" means one or all of
the listed elements or a combination of any two or more of the
listed element.
[0011] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the described
subject matter in any way. All literature cited in this
specification, including but not limited to, patents, patent
applications, articles, books, and treatises are expressly
incorporated by reference in their entirety for any purpose. In the
event that any of the incorporated literature contradicts any term
defined herein, this specification controls. While the present
teachings are described in conjunction with various embodiments, it
is not intended that the present teachings be limited to such
embodiments. On the contrary, the present teachings encompass
various alternatives, modifications, and equivalents, as will be
appreciated by those of skill in the art.
[0012] The practice of the present invention may employ
conventional techniques and descriptions of organic chemistry,
polymer technology, molecular biology (including recombinant
techniques), cell biology, biochemistry, and immunology, which are
within the skill of the art. Such conventional techniques include
oligonucleotide synthesis, hybridization, extension reaction, and
detection of hybridization using a label. Specific illustrations of
suitable techniques can be had by reference to the example herein
below. However, other equivalent conventional procedures can, of
course, also be used. Such conventional techniques and descriptions
can be found in standard laboratory manuals such as Genome
Analysis: A Laboratory Manual Series (Vols. I-IV), PCR Primer: A
Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all
from Cold Spring Harbor Laboratory Press, 1989), Gait,
"Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press,
London, Nelson and Cox (2000), Lehninger, Principles of
Biochemistry 3.sup.rd Ed., W. H. Freeman Pub., New York, N.Y. and
Berg et al. (2002) Biochemistry, 5.sup.th Ed., W. H. Freeman Pub.,
New York, N.Y. all of which are herein incorporated in their
entirety by reference for all purposes.
[0013] As used herein, "amplify" refers to the process of
enzymatically increasing the amount of a specific nucleotide
sequence. This amplification is not limited to but is generally
accomplished by PCR. As used herein, "denaturation" refers to the
separation of two complementary nucleotide strands from an annealed
state. Denaturation can be induced by a number of factors, such as,
for example, ionic strength of the buffer, temperature, or
chemicals that disrupt base pairing interactions.
[0014] As used herein, the term "amplifying" refers to a process
whereby a portion of a nucleic acid is replicated using, for
example, any of a broad range of primer extension reactions.
Exemplary primer extension reactions include, but are not limited
to, PCR. Unless specifically stated, "amplifying" refers to a
single replication or to an arithmetic, logarithmic, or exponential
amplification.
[0015] As used herein, "annealing" refers to the specific
interaction between strands of nucleotides wherein the strands bind
to one another substantially based on complementarity between the
strands as determined by Watson-Crick base pairing. It is not
necessary that complementarity be 100% for annealing to occur.
[0016] The terms "amplification cycle" and "PCR cycle" are used
interchangeably herein and as used herein refers to the denaturing
of a double-stranded polynucleotide sequence followed by annealing
of a primer sequence to its complementary sequence and extension of
the primer sequence.
[0017] The terms "amplicon," "amplification product" and "amplified
sequence" are used interchangeably herein and refer to a broad
range of techniques for increasing polynucleotide sequences, either
linearly or exponentially and can be the product of an
amplification reaction. An amplicon can be double-stranded or
single-stranded, and can include the separated component strands
obtained by denaturing a double-stranded amplification product. In
certain embodiments, the amplicon of one amplification cycle can
serve as a template in a subsequent amplification cycle. Exemplary
amplification techniques include, but are not limited to, PCR or
any other method employing a primer extension step. Other
nonlimiting examples of amplification include, but are not limited
to, ligase detection reaction (LDR) and ligase chain reaction
(LCR). Amplification methods can comprise thermal-cycling or can be
performed isothermally. In various embodiments, the term
"amplification product" and "amplified sequence" includes products
from any number of cycles of amplification reactions.
[0018] As used herein, the terms "amplification primer" and
"oligonucleotide primer" are used interchangeably and refer to an
oligonucleotide, capable of annealing to an RNA or DNA region. The
region annealed to can be adjacent a target sequence, including but
not limited to a SNP, a STR or mutation region, and serving as an
initiation primer for DNA synthesis under suitable conditions well
known in the art. Typically, a PCR reaction employs an
"amplification primer pair" also referred to as an "oligonucleotide
primer pair" including an "upstream" or "forward" primer and a
"downstream" or "reverse" primer, which delimit a region of the RNA
or DNA to be amplified. A first primer and a second primer may be
either a forward or reverse primer respectively, and are used
interchangeably herein and are not to be limiting.
[0019] As used herein, "extension" refers to the amplification
cycle after the primer oligonucleotide and target nucleic acid have
annealed to one another, wherein the polymerase enzyme catalyzes
primer extension, thereby enabling amplification, using the target
nucleic acid as a replication template.
[0020] As used herein, the terms "polynucleotide",
"oligonucleotide", and "nucleic acid" are used interchangeably
herein and refer to single-stranded and double-stranded polymers of
nucleotide monomers, including without limitation
2`-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by
internucleotide phosphodiester bond linkages, or internucleotide
analogs, and associated counter ions, e.g., H.sup.+,
NH.sub.4.sup.+, trialkylammonium, Mg.sup.2.sup.+, Na.sup.+, and the
like. A polynucleotide may be composed entirely of
deoxyribonucleotides, entirely of ribonucleotides, or chimeric
mixtures thereof and can include nucleotide analogs. The nucleotide
monomer units may comprise any nucleotide or nucleotide analog.
Polynucleotides typically range in size from a few monomeric units,
e.g. 5-40 when they are sometimes referred to in the art as
oligonucleotides, to several thousands of monomeric nucleotide
units. Unless denoted otherwise, whenever a polynucleotide sequence
is represented, it will be understood that the nucleotides are in
5' to 3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytosine, "G" denotes
deoxyguanosine, "T" denotes thymidine, and "U" denotes
deoxyuridine, unless otherwise noted. The letters A, C, G, and T
can be used to refer to the bases themselves, to nucleosides, or to
nucleotides comprising the bases, as is standard in the art. In
naturally occurring polynucleotides, the inter-nucleoside linkage
is typically a phosphodiester bond, and the subunits are referred
to as "nucleotides."
[0021] When two different, non-overlapping oligonucleotides anneal
to different regions of the same linear complementary nucleic acid
sequence, the 3' end of one oligonucleotide points toward the 5'
end of the other; the former may be called the "upstream"
oligonucleotide and the latter the "downstream"
oligonucleotide.
[0022] The terms "polymerase" and "nucleic acid polymerase" are
used interchangeably and as used herein refer to any polypeptide
that catalyzes the synthesis or sequencing of a polynucleotide
using an existing polynucleotide as a template.
[0023] As used herein, "DNA polymerase" refers to a nucleic acid
polymerase that catalyzes the synthesis or sequencing of DNA using
an existing polynucleotide as a template.
[0024] As used herein, "Thermostable DNA polymerase" refers to a
DNA polymerase that, at a temperature higher than 37.degree. C.,
retains its ability to add at least one nucleotide onto the 3' end
of a primer or primer extension product that is annealed to a
target nucleic acid sequence. In certain embodiments, a
thermostable DNA polymerase remains active after exposure to a
temperature greater than about 37.degree. C. In certain
embodiments, a thermostable DNA polymerase remains active at a
temperature greater than about 42.degree. C. In certain
embodiments, a thermostable DNA polymerase remains active at a
temperature greater than about 50.degree. C. In certain
embodiments, a thermostable DNA polymerase remains active at a
temperature greater than about 60.degree. C. In certain
embodiments, a thermostable DNA polymerase remains active at a
temperature greater than about 70.degree. C. In certain
embodiments, a thermostable DNA polymerase remains active at a
temperature greater than about 80.degree. C. In certain
embodiments, a thermostable polymerase remains active at a
temperature greater than about 90.degree. C.
[0025] As used herein, "nucleoside" includes the natural
nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g. as
described in Komberg and Baker, DNA Replication, 2nd Ed. (Freeman,
San Francisco, 1992). "Analogs" in reference to nucleosides
includes synthetic nucleosides having modified base moieties and/or
modified sugar moieties, e.g. described by Scheit, Nucleotide
Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical
Reviews, 90: 543-584 (1990); or the like. Such analogs include
synthetic nucleosides designed to enhance binding properties,
reduce degeneracy, increase specificity, and the like.
[0026] The terms "elongation rate" and "extension rate" are used
interchangeably herein and as used herein refer to the number of
nucleotides polymerized per second per molecule of DNA
polymerase.
[0027] The term "processivity" as used herein refers to the average
number of nucleotides added by a DNA polymerase in a single binding
event.
[0028] The term "terminal transferase activity" as used herein
refers to the non-templated addition of a single nucleotide, mainly
adenosine, to the 3' end of the amplified DNA strand.
[0029] As defined herein, "5'.fwdarw.3' nuclease activity" or "5'
to 3' nuclease activity" refers to that activity of a
template-specific nucleic acid polymerase including either a
5'.fwdarw.3' exonuclease activity traditionally associated with
some DNA polymerases whereby nucleotides are removed from the 5'
end of an oligonucleotide in a sequential manner, (i.e., E. coli
DNA polymerase I has this activity whereas the Klenow fragment does
not), or a 5'.fwdarw.3' endonuclease activity wherein cleavage
occurs more than one nucleotide from the 5' end, or both.
[0030] As used herein, the phrase "thermostable" and "thermally
stable" are interchangeable.
[0031] As used herein, the term "thermostable nucleic acid
polymerase" refers to an enzyme which is relatively stable to heat
when compared, for example, to nucleotide polymerases from E. coli
and which catalyzes the polymerization of nucleosides. Generally,
the enzyme will initiate synthesis at the 3'-end of the primer
annealed to the target sequence, will proceed in the 5'-direction
along the template and if possessing a 5' to 3' nuclease activity,
hydrolyzing intervening, annealed probe to release both labeled and
unlabeled probe fragments, until synthesis terminates. A
representative thermostable enzyme isolated from Thermus aquaticus
(Taq) is described in U.S. Pat. No. 4,889,818 and a method for
using it in conventional PCR are described in Saiki et al., (1988),
Science 239:487.
[0032] Exemplary bacteria from which the DNA Pol A polymerase can
be isolated include but are not limited to Thermus aquaticus,
Thermus thermophilus, Thermatoga maritime, Bacillus caldotenax,
Carboxydothermus hydrogenformans, Thermoanaerobacter
thermohydrosulfuricus, Thermus brokianus, Thermus caldophilus GK24,
Thermus flavus, Thermus rubens, or a mutants of any of the
aforementioned thereof.
[0033] As used herein "hot-start" refers to the thermal exposure of
a reaction solution, often a PCR reaction mix, to a temperature
sufficient to restore enzymatic activity, i.e., thermal
reactivation of a DNA polymerase which had been inactivated by, for
example, chemical or antibody means.
[0034] As used herein, the "polymerase chain reaction" or PCR is a
an amplification of nucleic acid consisting of an initial
denaturation step which separates the strands of a double stranded
nucleic acid sample, followed by repetition of (i) an annealing
step, which allows amplification primers to anneal specifically to
positions flanking a target sequence; (ii) an extension step which
extends the primers in a 5' to 3' direction thereby forming an
amplicon polynucleotide complementary to the target sequence, and
(iii) a denaturation step which causes the separation of the
amplicon from the target sequence (Mullis et al., eds, The
Polymerase Chain Reaction, BirkHauser, Boston, Mass. (1994)). Each
of the above steps may be conducted at a different temperature,
preferably using an automated thermocycler (Applied Biosystems LLC,
a division of Life Technologies Corporation, Foster City, Calif.).
If desired, RNA samples can be converted to DNA/RNA heteroduplexes
or to duplex cDNA by methods known to one of skill in the art. The
PCR method also includes reverse transcriptase-PCR and other
reactions that follow principles of PCR.
[0035] The term "primer" refers to a polynucleotide
(oligonucleotide) and analogs thereof that are capable of
selectively hybridizing to a target nucleic acid or "template", a
target region flanking sequence or to a corresponding
primer-binding site of an amplification product; and allows the
synthesis of a sequence complementary to the corresponding
polynucleotide template, flanking sequence or amplification product
from the primer's 3' end. Typically a primer can be between about
10 to 100 nucleotides in length and can provide a point of
initiation for template-directed synthesis of a polynucleotide
complementary to the template, which can take place in the presence
of appropriate enzyme(s), cofactors, substrates such as nucleotides
(dNTPs) and the like.
[0036] The term "primer extension" as used herein refers to both to
the synthesis of DNA resulting from the polymerization of
individual nucleoside triphosphates using a primer as a point of
initiation, and to the joining of additional oligonucleotides to
the primer to extend the primer. As used herein, the term "primer
extension" is intended to encompass the ligation of two
oligonucleotides to form a longer product which can then serve as a
target in future amplification cycles. As used herein, the term
"primer" is intended to encompass the oligonucleotides used in
ligation-mediated amplification processes which are extended by the
ligation of a second oligonucleotide which hybridizes at an
adjacent position.
[0037] As used here, the term "primer extension reaction" refers to
a reaction in which a polymerase catalyzes the template-directed
synthesis of a nucleic acid from the 3' end of a primer. The term
"primer extension product" refers to the resultant nucleic acid. A
non-limiting exemplary primer extension reaction is the polymerase
chain reaction (PCR). The terms "extending" and "extension" refer
to the template-directed synthesis of a nucleic acid from the 3'
end of a primer, which is catalyzed by a polymerase.
[0038] The term "nucleic acid sequence" as used herein can refer to
the nucleic acid material itself and is not restricted to the
sequence information (i.e. the succession of letters chosen among
the five base letters A, C, G, T, or U) that biochemically
characterizes a specific nucleic acid, for example, a DNA or RNA
molecule. Nucleic acids shown herein are presented in a
5'.fwdarw.3' orientation unless otherwise indicated.
[0039] The term "Tm" as used herein refers to the melting
temperature at which half of the DNA strands in the double-stranded
state and half are in the single-stranded state.
[0040] The term "denaturing" as used herein refers to separation of
a double-stranded nucleic acid into single, complementary strands.
Most often, hydrogen bonds are broken to accomplish denaturing
using either heat or chemical methods such as urea.
[0041] The terms "annealing" and "hybridization" are used
interchangeably and mean the base-pairing interaction of one
nucleic acid with another nucleic acid that results in formation of
a duplex or other higher-ordered structure. The primary interaction
is base specific, i.e. A/T and G/C, by Watson/Crick and
Hoogsteen-type hydrogen bonding.
[0042] The terms "complement" and "complementary" as used herein
refer to the ability of two single stranded polynucleotides (for
instance, a primer and a target polynucleotide) to base pair with
each other, where an adenine on one strand of a polynucleotide will
base pair to a thymine or uracil on a strand of a second
polynucleotide and a cytosine on one strand of a polynucleotide
will base pair to a guanine on a strand of a second polynucleotide.
Two polynucleotides are complementary to each other when a
nucleotide sequence in one polynucleotide can base pair with a
nucleotide sequence in a second polynucleotide. For instance,
5'-ATGC and 5'-GCAT are complementary.
[0043] The term "extending" as used herein refers to increasing the
primer sequence length in a 5' to 3' direction thereby forming an
amplicon polynucleotide complementary to the target sequence
[0044] The terms "detecting" and "detection" are used in a broad
sense herein and encompass any technique by which one can determine
the presence of or identify a nucleic acid sequence. In some
embodiments, detecting comprises quantitating a detectable signal
from the nucleic acid, including without limitation, a real-time
detection method, such as quantitative PCR ("Q-PCR"). In some
embodiments, detecting comprises determining the sequence of a
sequencing product or a family of sequencing products generated
using an amplification product as the template; in some
embodiments, such detecting comprises obtaining the sequence of a
family of sequencing products. In other embodiments detecting can
be achieved through measuring the size of a nucleic acid
amplification product.
[0045] As used herein, "DNA" refers to deoxyribonucleic acid in its
various forms as understood in the art, such as genomic DNA, cDNA,
isolated nucleic acid molecules, vector DNA, and chromosomal DNA.
"Nucleic acid" refers to DNA or RNA in any form. Examples of
isolated nucleic acid molecules include, but are not limited to,
mRNA, siRNA, miRNA, shRNA, recombinant DNA molecules contained in a
vector, recombinant DNA molecules maintained in a heterologous host
cell, partially or substantially purified nucleic acid molecules,
and synthetic DNA molecules. Typically, an "isolated" nucleic acid
is free of sequences that naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, is generally substantially free of other cellular
material or culture medium when produced by recombinant techniques,
or free of chemical precursors or other chemicals when chemically
synthesized.
[0046] As used herein, the term "short tandem repeat (STR) loci"
refers to regions of a genome which contains short, repetitive
sequence elements of 2 to 7 base pairs in length. Each sequence
element is repeated at least once within an STR and is referred to
herein as a "repeat unit." The term STR also encompasses a region
of genomic DNA wherein more than a single repeat unit is repeated
in tandem or with intervening bases, provided that at least one of
the sequences is repeated at least two times in tandem. Examples of
STRs, include but are not limited to, a triplet repeat, e.g., ATC
in tandem; a 4-peat (tetra-repeat), e.g., GATA in tandem; and a
5-peat (penta-repeat), e.g., ATTGC in tandem and so on. Information
about specific STRs that can be used as genetic markers can be
found in, among other places, the STRbase at
www.cstl.nist.gov/strbase.
[0047] The term "detectable signal" as used herein refers to a
signal that is capable of being detected under certain conditions.
In certain embodiments, a detectable signal is detected when it is
present in a sufficient quantity.
[0048] The term "signal moiety" as used herein refers to a moiety
that is capable of producing a detectable signal.
[0049] The term "indicator molecule "as used herein refers to a
molecule comprising a label that can be detected.
[0050] The term "probe" as used herein refers to a polynucleotide
that comprises a specific portion designed to hybridize in a
sequence-specific manner with a complementary region of a specific
nucleic acid sequence, e.g., a target nucleic acid sequence. In
certain embodiments, the specific portion of the probe may be
specific for a particular sequence, or alternatively, may be
degenerate, e.g., specific for a set of sequences. In certain
embodiments, the probe is labeled. The probe can be an
oligonucleotide that is complementary to at least a portion of an
amplification product formed using two primers.
[0051] The term "indicator probe" as used herein refers to a probe
comprising a label that can be detected.
[0052] The term "5'-nuclease probe" as used herein refers to a
probe that comprises a signal moiety linked to a quencher moiety or
a donor moiety through a short oligonucleotide link element. When
the 5'-nuclease probe is intact, the quencher moiety or the donor
moiety influences the detectable signal from the signal moiety.
According to certain embodiments, the 5'-nuclease probe selectively
hybridizes to a target nucleic acid sequence and is cleaved by a
polypeptide having 5' to 3' exonuclease activity, e.g., when the
probe is replaced by a newly polymerized strand during a primer
extension reaction, such as PCR.
[0053] As used herein "quencher moiety" refers to a moiety that
causes the detectable signal of a signal moiety to decrease when
the quencher moiety is sufficiently close to the signal moiety.
[0054] Also herein, the recitations of numerical ranges by
endpoints include all numbers subsumed within that range (e.g., 1
to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0055] Reference will now be made to various embodiments, examples
of which are illustrated in the accompanying drawings.
[0056] DNA polymerases are known to those skilled in the art. DNA
polymerases include DNA-dependent polymerases, which use DNA as a
template, or RNA-dependent polymerases, such as reverse
transcriptase, which use RNA as a template.
[0057] Based on sequence homology, bacterial DNA polymerases can be
subdivided into seven different families: A, B, C, D, X, Y, and RT.
DNA-dependent DNA polymerases fall into one of six families (A, B,
C, D, X, and Y), with most falling into one of three families (A,
B, and C). See, e.g., Ito et al. (1991) Nucleic Acids Res.
19:4045-4057; Braithwaite et al. (1993) Nucleic Acids Res.
21:787-802; Filee et al. (2002) J. Mol. Evol. 54:763-773; and Alba
(2001) Genome Biol. 2:3002.1-3002.4. Certain DNA polymerases may be
single-chain polypeptides (e.g., certain family A and B
polymerases) or multi-subunit enzymes (e.g., certain family C
polymerases) with one of the subunits having polymerase activity.
Id. A fusion protein may comprise a DNA polymerase selected from a
family A, B, C, D, X, or Y polymerase.
[0058] There are five known DNA polymerases in bacteria. All have
5'-3' polymerase activity and include Pol I, Pol II, Pol III, Pol
IV and Pol V. Pol IV and Pol V are Y-family DNA polymerases, known
to have weak fidelity on normal templates and can replicate through
damaged DNA. Pol I, Pol II and Pol III all have 3'-5' exonuclease
activity while Pol I is also implicated in DNA repair, having both
5'-3' polymerase and 3'-5' proofreading exonuclease activity.
[0059] Family A polymerases ("Pol A") include both replicative and
repair polymerases. Replicative members from this family include T7
DNA polymerase and the eukaryotic mitochondrial DNA Polymerase y.
Among the repair polymerases are E. coli DNA Pol I, Thermus
aquaticus Pol I (Taq DNA polymerase), and Bacillus
stearothermophilus Pol I. Excision repair and processing of Okazaki
fragments generated during lagging strand synthesis are performed
by the repair polymerases. Because most thermostable Pol A enzymes
do not possess the 3' to 5' exonuclease activity, they are
incapable of proofreading the newly synthesized nucleic acid strand
and consequently have high error rates.
[0060] Family B polymerases ("Pol B") are substantially replicative
polymerases including the major eukaryotic DNA polymerases .alpha.,
.delta., .epsilon., and also DNA polymerase .zeta.. Pol B
polymerases also include DNA polymerases encoded by some bacteria
and bacteriophages, of which the best characterized are from T4,
Phi29 and RB69 bacteriophages. Pol B enzymes are involved in both
leading and lagging strand synthesis and are noteworthy for their
remarkable accuracy during replication as many have strong 3'-5'
exonuclease activity the exceptions being DNA polymerase a and
which lack proofreading activity.
[0061] In certain embodiments, amplification methods comprise at
least one cycle of amplification, for example, but not limited to,
the sequential procedures of: hybridizing primers to
primer-specific portions of target sequence or amplification
products from any number of cycles of an amplification reaction;
synthesizing a strand of nucleotides in a template-dependent manner
using a polymerase; and denaturing the newly-formed nucleic acid
duplex to separate the strands. The cycle may or may not be
repeated.
[0062] PCR amplification time can be decreased significantly by
changing the enzyme used. Three key intrinsic properties of a DNA
polymerase play important roles in determining the overall reaction
time required for the PCR amplification. The first property is
"elongation rate" (or "extension rate"), which is defined as the
number of nucleotides polymerized per second per molecule of DNA
polymerase. The second property is "processivity", which is defined
as the average number of nucleotides added by a DNA polymerase in a
single binding event. Both "elongation rate" and "processivity"
depend on the components of the reaction media and on the DNA
template sequence. The third property is the presence or absence of
terminal transferase activity, which is the non-templated addition
of a single nucleotide, mainly adenosine, to the 3' end of the
amplified DNA strand.
[0063] Considerations to for reducing PCR time can start with
evaluation of the polymerase enzyme used. For example, the
mechanism for enzyme activation can decrease PCR time by about 10
minutes. Hot start enzymes with chemical modifications such as
AmpliTaq Gold.RTM. DNA polymerase (Applied Biosystems, Foster City,
Caif.), can require an eight to eleven minute heat activation step
while hot start enzymes having antibody (Platinum.RTM. Taq DNA
Polymerase, Invitrogen, Carlsbad, Calif., SpeedSTAR.TM. HS DNA
Polymerase, Takara, Madison, Wis.), oligonucleotide, or use of
single-stranded binding proteins to primers can reduce the hot
start mechanism to one to two minutes.
[0064] Improvements which decrease the denaturation, annealing and
extension times can further reduce PCR time by use of a more
processive DNA polymerase as well as utilization of a thermal
cycler with faster ramping rates or changing from a 3-step cycling
protocol to a 2-step protocol which removes one ramping time per
PCR cycle. Further, use of a Pol B DNA polymerase can eliminate the
need for a final extension step, saving up to 60 minutes. However,
use of a variety of Pol B family DNA polymerases has been shown to
produce higher stutter peak heights making STR profile
interpretation difficult (data not shown).
[0065] In some embodiments, envisioned are Pol A and Pol B DNA
polymerases for amplifying a target nucleic acid sequence in under
at least 50 minutes, under at least 45 minutes, under at least 40
minutes, under at least 35 minutes, under at least 30 minutes,
under at least 25 minutes and under at least 20 minutes. The
resulting amplification product can be detected. In some
embodiments the detection is selected from microfluidics,
electrophoresis, mass spectrometry and the like known to one of
skill in the art for detecting amplification products.
[0066] In some embodiments, PCR amplification products may be
detected by fluorescent dyes conjugated to the PCR amplification
primers, for example as described in PCT patent application WO
2009/059049. PCR amplification products can also be detected by
other techniques, including, but not limited to, the staining of
amplification products, e.g. silver staining and the like.
[0067] In some embodiments, detecting comprises an instrument,
i.e., using an automated or semi-automated detecting means that
can, but need not, comprise a computer algorithm. In some
embodiments, the instrument is portable, transportable or comprises
a portable component which can be inserted into a less mobile or
transportable component, e.g., residing in a laboratory, hospital
or other environment in which detection of amplification products
is conducted. In certain embodiments, the detecting step is
combined with or is a continuation of at least one amplification
step, one sequencing step, one isolation step, one separating step,
for example but not limited to a capillary electrophoresis
instrument comprising at least one fluorescent scanner and at least
one graphing, recording, or readout component; a chromatography
column coupled with an absorbance monitor or fluorescence scanner
and a graph recorder; a chromatography column coupled with a mass
spectrometer comprising a recording and/or a detection component; a
spectrophotometer instrument comprising at least one UV/visible
light scanner and at least one graphing, recording, or readout
component; or a microarray with a data recording device such as a
scanner or CCD camera. In certain embodiments, the detecting step
is combined with an amplifying step, for example but not limited
to, real-time analysis such as Q-PCR. Exemplary means for
performing a detecting step include the ABI PRISM.RTM. Genetic
Analyzer instrument series, the ABI PRISM.RTM. DNA Analyzer
instrument series, the ABI PRISM.RTM. Sequence Detection Systems
instrument series, and the Applied Biosystems Real-Time PCR
instrument series (all from Applied Biosystems); and microarrays
and related software such as the Applied Biosystems microarray and
Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and
other commercially available microarray and analysis systems
available from Affymetrix, Agilent, and Amersham Biosciences, among
others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De
Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al.,
Nat. Med. 9:140-45, including supplements, 2003) or bead array
platforms (Illumina, San Diego, Calif.). Exemplary software
includes GeneMapper.TM. Software, GeneScan.RTM. Analysis Software,
and Genotyper.RTM. software (all from Applied Biosystems).
[0068] In some embodiments, an amplification product can be
detected and quantified based on the mass-to-charge ratio of at
least a part of the amplicon (m/z). For example, in some
embodiments, a primer comprises a mass spectrometry-compatible
reporter group, including without limitation, mass tags, charge
tags, cleavable portions, or isotopes that are incorporated into an
amplification product and can be used for mass spectrometer
detection (see, e.g., Haff and Smirnov, Nucl. Acids Res.
25:3749-50, 1997; and Sauer et al., Nucl. Acids Res. 31:e63, 2003).
An amplification product can be detected by mass spectrometry. In
some embodiments, a primer comprises a restriction enzyme site, a
cleavable portion, or the like, to facilitate release of a part of
an amplification product for detection. In certain embodiments, a
multiplicity of amplification products are separated by liquid
chromatography or capillary electrophoresis, subjected to ESI or to
MALDI, and detected by mass spectrometry. Descriptions of mass
spectrometry can be found in, among other places, The Expanding
Role of Mass Spectrometry in Biotechnology, Gary Siuzdak, MCC
Press, 2003.
[0069] In some embodiments, detecting comprises a manual or visual
readout or evaluation, or combinations thereof. In some
embodiments, detecting comprises an automated or semi-automated
digital or analog readout. In some embodiments, detecting comprises
real-time or endpoint analysis. In some embodiments, detecting
comprises a microfluidic device, including without limitation, a
TaqMan.RTM. Low Density Array (Applied Biosystems). In some
embodiments, detecting comprises a real-time detection instrument.
Exemplary real-time instruments include, the ABI PRISM.RTM. 7000
Sequence Detection System, the ABI PRISM.RTM. 7700 Sequence
Detection System, the Applied Biosystems 7300 Real-Time PCR System,
the Applied Biosystems 7500 Real-Time PCR System, the Applied
Biosystems 7900 HT Fast Real-Time PCR System (all from Applied
Biosystems); the LightCycler.TM. System (Roche Molecular); the
Mx3000P.TM. Real-Time PCR System, the Mx3005P.TM. Real-Time PCR
System, and the Mx4000.RTM. Multiplex Quantitative PCR System
(Stratagene, La Jolla, Calif.); and the Smart Cycler System
(Cepheid, distributed by Fisher Scientific). Descriptions of
real-time instruments can be found in, among other places, their
respective manufacturer's users manuals; McPherson; DNA
Amplification: Current Technologies and Applications, Demidov and
Broude, eds., Horizon Bioscience, 2004; and U.S. Pat. No.
6,814,934.
[0070] Those in the art understand that the detection techniques
employed are generally not limiting. Rather, a wide variety of
detection means are within the scope of the disclosed methods and
kits, provided that they allow the presence or absence of a
microorganism in the sample to be determined.
[0071] In various embodiments of the present teachings it has been
discovered that use of a two-step amplification cycle in a PCR
cycling protocol can reduce the PCR time to under 45 min. while
obtaining a complete, interpretable STR profile using 1 ng of
control DNA 9947A. Examples of Fast PCR protocols with Pol A family
enzymes are illustrated in Table 1.
TABLE-US-00001 TABLE 1 Fast PCR Protocols with Pol A DNA
Polymerases STR Kit: Identifiler .RTM. Plus Identifiler .RTM. Plus
AmpliTaq Gold .RTM. Platinum .RTM. Taq Enzyme: DNA Polymerase DNA
Polymerase No. of No. of Amplification Amplification PCR Protocol
cycles cycles Template 95.degree. C./11 min. 95.degree. C./1 min.
Denaturation Amplification 94.degree. C./5 sec. 29X 94.degree. C./5
sec. 29X cycle 59.degree. C./20 sec. 59.degree. C./20 sec.
Nontemplate 72.degree. C./1 min. 72.degree. C./1 min. Adenylation
Total PCR Time: 45 min. 36.5 min.
[0072] In some embodiments the two-step amplification cycle is 30
seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds
or less or 10 seconds or less. In some embodiments the
amplification cycle is carried our at least 25 times, at least 26
times, at least 27 times, at least 28 times or at least 30
times.
[0073] In some embodiments, the predicted Tm of the primers is
5.degree. C. less than the annealing temperature, 10.degree. C.
less than the annealing temperature, or 15.degree. C. less than the
annealing temperature. Likewise, the annealing temperature in the
amplification cycle can be 5.degree. C. greater than the annealing
temperature, 10.degree. C. greater than the annealing temperature,
or 15.degree. C. greater than the annealing temperature. The Tm is
a reflection of the temperature that double-stranded DNA becomes
single-stranded.
[0074] In various embodiments of the present teachings the
annealing temperature and the elongation temperature are identical
and the extending of primer annealed to the template occurs at the
annealing temperature, for example, but not limited to, the
two-step amplification cycling protocol. In some embodiments the
annealing temperature is from about 55.degree. C. to about
75.degree. C., and from about 57.degree. C. to about 72.degree. C.,
from about 58.degree. C. to about 72.degree. C., from about
59.degree. C. to about 72.degree. C. and from about 60.degree. C.
to about 72.degree. C. In some embodiments the reaction mixture
undergoing PCR amplification is held at the annealing temperature
for 5 seconds or less, 4 seconds or less, 3 seconds or less, 2
seconds or less or 1 second or less.
[0075] In various embodiments of the present teaching the
denaturing temperature, the temperature at which the
double-stranded deoxyribonucleotide separates into single strands,
occurs at a denaturing temperature sufficient to denature the
double-stranded nucleic acid such as at from about 85.degree. C. to
about 100.degree. C. and the reaction mixture is held at the
denaturing temperature for 9 seconds or less, 8 seconds or less, 7
seconds or less, 6 seconds or less, 5 seconds or less, 4 seconds or
less, 3 seconds or less, 2 seconds or less or 1 second and the
annealing/elongation temperature is 30 seconds or less, 25 seconds
or less, 20 seconds or less, 15 seconds or less, 10 seconds or less
or 5 seconds or less.
[0076] In various embodiments the temperature of denaturing,
annealing/elongation and extension reflect the temperature of the
heating element or heat block within the thermal cycler or a
microfluidic device. In other embodiments the temperatures of
denaturing, annealing/elongation and extension reflect the
temperature of the reaction mixture. The reaction mixture, once it
reaches the denaturing and/or annealing/elongation temperature is
not required to be held at the denaturing and/or
annealing/elongation temperature once the denaturing and/or
annealing/elongation temperature is reached by the reaction
mixture.
[0077] In various embodiments of the present teachings the Pol A
enzyme can be an enzyme derived from Thermus aquaticus, Thermus
thermophilus HB-8 or HB-27, Thermus flavus, Thermus maritime; large
fragment, Thermotoga naepolitana, Thermococcus gorgonarius,
Thermococcus litoralis, Thermococcus aggregans, and Thermomicrobium
roseum. The Pol A family polymerase can be a fragment, variant or
recombinant form of the thermostable DNA polymerase. Such
fragments, variants or recombinant forms that retain their DNA
polymerase activity are well known to one of skill in the art.
[0078] In various embodiments of the present teachings it has been
discovered that high concentrations of a Pol A enzyme along with
high concentrations of primers unexpectedly and substantially
reduce PCR amplification times for obtaining complete,
interpretable STR profiles. Titrating the selected Pol A enzyme to
slowly increase the enzyme in the reaction mix was performed to
identify the optimal concentration (Example A). Contrary to the
studies of Butler et al. (Forensic Sci. Intl.: Gen. 3:42-45 2008)
and Tan et al. (J. Forensic Sci. 54(6):1287-1296, 2009), successful
amplification for the determination of an STR profile of a nucleic
acid sample is dependent not only on the level of Pol A enzyme, but
the level of primer concentration and required no added enzymes or
supplements.
[0079] Using the AmpF/STR.RTM. Identifiler Plus.RTM. PCR
Amplification Kit (Applied Biosystems, Foster City, Calif.), for
the primers and substituting Platinum.RTM. Taq in the Master Mix,
the concentration of the Pol A enzyme, Platinum.RTM. Taq, was
increased incrementally as shown in FIGS. 1A-1E. Amplification was
performed in an Applied Biosystems GeneAmp.RTM. 9700 PCR instrument
(Table 1). Unexpectedly, insufficient enzyme resulted in incomplete
STR profiles (FIG. 1A), e.g., D5 and enzyme in excess resulted in
split peak morphologies (FIGS. 1D and 1E, .about.140-160 bp),
particularly in the D5 and D8 loci. Surprisingly, a full,
interpretable STR profile was obtained using the Fast PCR protocol
and Platinum Taq enzyme (FIGS. 1B to 1C).
[0080] Studies to compare the sensitivities of AmpliTaq Gold.RTM.,
Platinum.RTM. Taq and SpeedSTAR.TM. enzymes with the Fast PCR
protocol verse the Identifiler Plus kit and standard PCR protocol
showed that the standard protocol was able to detect a complete STR
profile with as little as 125 pg of input DNA while the Fast PCR
protocol achieved a complete STR profile down to 500 ng DNA with
AmpliTaq Gold and 250 pg DNA for Platinum and SpeedSTAR. As would
be expected, the average peak height was larger with the standard
protocol verses the Fast PCR protocol (data not shown).
[0081] Specificity studies indicated higher cross-species
reactivity with Platinum and SpeedSTAR enzymes with the Fast PCR
protocol verse AmpliTaq Gold enzyme. Additionally, when compared to
the standard protocol, inhibition of PCR when the Fast PCR protocol
was followed was present with as little as 100 uM Hematin and 25
ng/ul Humic acid for AmpliTaq Gold enzyme, 200 uM Hematin and 50
ng/ul Humic acid for Platinum enzyme and 200 uM Hematin and 75
ng/ul Humic acid for SpeedSTAR enzyme. These results can be
reversed by increasing the cycle number or optimization of the
master mix formulation.
[0082] In various embodiments of the present teachings the family A
polymerase can be a bacterial polymerase or a fragment or variant
of a bacterial family A polymerase having polymerase activity. The
thermostable polymerase can also be a recombinant polymerase, a
fragment or variant of a recombinant DNA polymerase that has
polymerase activity. In some embodiments the polymerase is a
variant of a Taq DNA polymerase with increased processivity
relative to naturally occurring Taq DNA polymerase.
[0083] In various embodiments the polymerase used in the Fast PCR
protocol PCR reaction mixture can further have an indicator
molecule to indicate the amount of nucleic acid in the reaction
mixture. In some embodiments the indicator molecule can be an
indicator probe capable of hybridizing to the double-stranded
nucleic acid such as in a 5' nuclease reaction as would be known to
one of skill in the art. The probe can be a 5' nuclease probe, a
molecular beacon, a PNA probe or other probe known to one of skill
in the art.
[0084] The present teachings are also directed to kits for
determining an STR profile that utilize the methods described
above. In some embodiments, a basic kit can comprise a container
having at least one pair of oligonucleotide primers capable of
amplifying an STR locus. A kit can also optionally comprise
instructions for use. A kit can also comprise other optional kit
components, such as, for example, one or more of an allelic ladder
directed to each of the loci amplified, a sufficient quantity of
enzyme for amplification, amplification buffer to facilitate the
amplification, divalent cation solution to facilitate enzyme
activity, dNTPs for strand extension during amplification, loading
solution for preparation of the amplified material for
electrophoresis, genomic DNA as a template control, a size marker
to insure that materials migrate as anticipated in the separation
medium, and a protocol and manual to educate the user and limit
error in use. The amounts of the various reagents in the kits also
can be varied depending upon a number of factors, such as the
optimum sensitivity of the process. It is within the scope of these
teachings to provide test kits for use in manual applications or
test kits for use with automated sample preparation, reaction
set-up, detectors or analyzers.
[0085] Those in the art understand that the detection techniques
employed are generally not limiting. Rather, a wide variety of
detection means are within the scope of the disclosed methods and
kits, provided that they allow the presence or absence of an
amplicon to be determined.
EXAMPLES
A. Enzyme Titration
[0086] A basic PCR buffer solution in bulk was prepared without
enzyme having 10-50 mM Tris-HCl, pH 8.0, 1-70 mM of KCl, and
MgCl.sub.2, 0.15-0.4 mM of each dNTP, 0.4-0.8% Tween 20, optionally
0.05%-1% Triton-x100, 700-3000 ng BSA, 1-8% Glycerol, 0.008-0.05%
Sodium azide, optionally 0.5%-2% DMSO, and 1 ng control DNA 9947A
for a 25 ul reaction. Aliquots of the PCR buffer were prepared with
varying amounts of Platinum Taq DNA polymerase from 1 to 15
units/25 ul PCR reaction, in duplicate, (FIGS. 1A-1E). The Fast PCR
protocol in Table 1 was followed in conjunction with the primers
used in the Identifiler Plus Kit being added to the reaction mix.
The amplification products were loaded on an Applied Biosystems
3130xl capillary electrophoresis instrument and analyzed using
GeneMapper.RTM. ID-X software. Optimal enzyme concentration was
determined based on the ability to obtain a complete STR profile
with interpretable peak heights overall.
[0087] Insufficient enzyme resulted in incomplete STR profiles
(FIG. 1A), e.g., D5 and enzyme in excess resulted in split peak
morphologies (FIGS. 1D and 1E, .about.140-160 bp), particularly in
the D5 and D8 loci. Surprisingly, a full, interpretable STR profile
was obtained using the Fast PCR protocol and Platinum Taq enzyme
(FIGS. 1B to 10).
B. Primer Titration
[0088] Primer concentration also appeared to impact the success of
the Fast PCR protocol for the ldentifiler.RTM. Direct and NGM.TM.
Kits (Applied Biosystems) (data not shown). Thus, all primer
concentrations were adjusted to 0.100 uM in a 25 ul reaction mix.
PCR was performed according to the Fast PCR protocol in Table 1 and
evaluated as described for Enzyme Titration.
[0089] While the principles of this invention have been described
in connection with specific embodiments, it should be understood
clearly that these descriptions are made only by way of example and
are not intended to limit the scope of the invention. What has been
disclosed herein has been provided for the purposes of illustration
and description. It is not intended to be exhaustive or to limit
what is disclosed to the precise forms described. Many
modifications and variations will be apparent to the practitioner
skilled in the art. What is disclosed was chosen and described in
order to best explain the principles and practical application of
the disclosed embodiments of the art described, thereby enabling
others skilled in the art to understand the various embodiments and
various modifications that are suited to the particular use
contemplated. It is intended that the scope of what is disclosed be
defined by the following claims and their equivalence.
* * * * *
References