Taq DNA polymerase mutants for probe qPCR

Abstract

Disclosed are Taq DNA polymerase mutants which exhibit enhanced efficiency in qPCR compared to the wild type Taq DNA polymerase. Such mutants include: V62S, V64S, A70F, F73A, A77F, P253G, E255K, D257R, A259F, A271F, L288S, E289K, S357I, L361S, L376S, P382G, T385I, G418P, R419D, E421K, L461S, A472F, E497K, L498S, E524K, D551R, R556D, S679I, L789S, E189K/E507K/E742K (See Sequence Listing Guide for the mutants' amino acid and protein sequences).

Claims

1. A mutant Taq DNA polymerase comprising an amino acid sequence substantially identical to wild type Taq DNA polymerase but having one of the following mutations and amino acid sequences, and wherein said substantially identical amino acid sequence does not include the 6-membered histidine tag at the C-terminus and the six immediately preceding Glycine and Serine amino acids included in each of the following amino acid sequences, and thus differs from wild type Taq DNA polymerase only at the single indicated mutation site: V62S (SEQ ID NO: 4), V64S (SEQ ID NO: 6), A70F (SEQ ID NO: 8), F73A (SEQ ID NO: 10), P253G (SEQ ID NO: 14), E255K (SEQ ID NO: 16), D257R (SEQ ID NO: 18), A259F (SEQ ID NO: 20), A271F (SEQ ID NO: 22), L288S (SEQ ID NO: 24), E289K (SEQ ID NO: 26), S357I (SEQ ID NO: 28), L361S (SEQ ID NO: 30), L376S (SEQ ID NO: 32), P382G (SEQ ID NO: 34), T385I (SEQ ID NO: 36), G418P (SEQ ID NO: 38), R419D (SEQ ID NO: 40), E421K (SEQ ID NO: 42), L461S (SEQ ID NO: 44), A472F (SEQ ID NO: 46), E497K (SEQ ID NO: 48), L498S (SEQ ID NO: 50), E524K (SEQ ID NO: 52), D551R (SEQ ID NO: 54), R556D (SEQ ID NO: 56), S679I (SEQ ID NO: 58), L789S (SEQ ID NO: 60), E189K/E507K/E742K (SEQ ID NO: 62).

2. The mutant Taq DNA polymerase of claim 1 wherein the mutant Taq polymerase is capable of extending a primer where the extension conditions are limited to one second.

3. The mutant Taq DNA polymerase of claim 1 wherein when the extension temperature is 60° C. for 1 second, detectable fluorescent signal is generated in qPCR.

4. A kit for use in qPCR for a sample, comprising an amino acid sequence substantially identical to wild type Taq DNA polymerase but having one of the following mutations and amino acid sequences, and wherein said substantially identical amino acid sequence does not include the 6-membered histidine tag at the C-terminus and the six immediately preceding Glycine and Serine amino acids included in each of the following amino acid sequences, and thus differs from wild type Taq DNA polymerase only at the single indicated mutation site: V62S (SEQ ID NO: 4), V64S (SEQ ID NO: 6), A70F (SEQ ID NO: 8), F73A (SEQ ID NO: 10), P253G (SEQ ID NO: 14), E255K (SEQ ID NO: 16), D257R (SEQ ID NO: 18), A259F (SEQ ID NO: 20), A271F (SEQ ID NO: 22), L288S (SEQ ID NO: 24), E289K (SEQ ID NO: 26), S357I (SEQ ID NO: 28), L361S (SEQ ID NO: 30), L376S (SEQ ID NO: 32), P382G (SEQ ID NO: 34), T385I (SEQ ID NO: 36), G418P (SEQ ID NO: 38), R419D (SEQ ID NO: 40), E421K (SEQ ID NO: 42), L461S (SEQ ID NO: 44), A472F (SEQ ID NO: 46), E497K (SEQ ID NO: 48), L498S (SEQ ID NO: 50), E524K (SEQ ID NO: 52), D551R (SEQ ID NO: 54), R556D (SEQ ID NO: 56), S679I (SEQ ID NO: 58), L789S (SEQ ID NO: 60), E189K/E507K/E742K (SEQ ID NO: 62).

5. The kit of claim 4 further including an intercalating dye.

6. The kit of claim 5 wherein the intercalating dye is SYBR Green or EvaGreen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A to 1II are each amplification plots showing probe qPCR signal comparisons of Taq DNA wild type (FIGS. 1E, 1M, 1U, 1CC, 1GG, each labeled “WT”) and a number of the Taq DNA polymerase mutants, as identified below the X axis in each figure. The X axis in each figure is the cycle number and the Y axis is the change in fluorescent signal detected (ΔR), representing the indicated mutant's the change in fluorescence throughout the cycling protocol, with dots indicating specific measured signal value per cycle. All detection threshold lines are fixed at 0.02. The qPCR protocol for all mutants was run in duplicate, as indicated in figures where two distinct sets of dots and lines can be seen.

SEQUENCE LISTING GUIDE

(2) SEQ ID NO: 1 is the DNA sequence of Wild Type Taq DNA polymerase with a C terminal Histag.

(3) SEQ ID NO: 2 is the protein sequence of Wild Type Taq DNA polymerase with a C terminal Histag.

(4) SEQ ID NO: 3 is the DNA sequence of mutant (V62S) Taq DNA polymerase with a C terminal Histag.

(5) SEQ ID NO: 4 is the protein sequence of mutant (V62S) Taq DNA polymerase with a C terminal Histag.

(6) SEQ ID NO: 5 is the DNA sequence of mutant (V64S) Taq DNA polymerase with a C terminal Histag.

(7) SEQ ID NO: 6 is the protein sequence of mutant (V64S) Taq DNA polymerase with a C terminal Histag.

(8) SEQ ID NO: 7 is the DNA sequence of mutant (A70F) Taq DNA polymerase with a C terminal Histag.

(9) SEQ ID NO: 8 is the protein sequence of mutant (A70F) Taq DNA polymerase with a C terminal Histag.

(10) SEQ ID NO: 9 is the DNA sequence of mutant (F73A) Taq DNA polymerase with a C terminal Histag.

(11) SEQ ID NO: 10 is the protein sequence of mutant (F73A) Taq DNA polymerase with a C terminal Histag.

(12) SEQ ID NO: 11 is the DNA sequence of mutant (A77F) Taq DNA polymerase with a C terminal Histag.

(13) SEQ ID NO: 12 is the protein sequence of mutant (A77F) Taq DNA polymerase with a C terminal Histag.

(14) SEQ ID NO: 13 is the DNA sequence of mutant (P253G) Taq DNA polymerase with a C terminal Histag.

(15) SEQ ID NO: 14 is the protein sequence of mutant (P253G) Taq DNA polymerase with a C terminal Histag.

(16) SEQ ID NO: 15 is the DNA sequence of mutant (E255K) Taq DNA polymerase with a C terminal Histag.

(17) SEQ ID NO: 16 is the protein sequence of mutant (E255K) Taq DNA polymerase with a C terminal Histag.

(18) SEQ ID NO: 17 is the DNA sequence of mutant (D257R) Taq DNA polymerase with a C terminal Histag.

(19) SEQ ID NO: 18 is the protein sequence of mutant (D257R) Taq DNA polymerase with a C terminal Histag.

(20) SEQ ID NO: 19 is the DNA sequence of mutant (A259F) Taq DNA polymerase with a C terminal Histag.

(21) SEQ ID NO: 20 is the protein sequence of mutant (A259F) Taq DNA polymerase with a C terminal Histag.

(22) SEQ ID NO: 21 is the DNA sequence of mutant (A271F) Taq DNA polymerase with a C terminal Histag.

(23) SEQ ID NO: 22 is the protein sequence of mutant (A271F) Taq DNA polymerase with a C terminal Histag.

(24) SEQ ID NO: 23 is the DNA sequence of mutant (L288S) Taq DNA polymerase with a C terminal Histag.

(25) SEQ ID NO: 24 is the protein sequence of mutant (L288S) Taq DNA polymerase with a C terminal Histag.

(26) SEQ ID NO: 25 is the DNA sequence of mutant (E289K) Taq DNA polymerase with a C terminal Histag.

(27) SEQ ID NO: 26 is the protein sequence of mutant (E289K) Taq DNA polymerase with a C terminal Histag.

(28) SEQ ID NO: 27 is the DNA sequence of mutant (S357I) Taq DNA polymerase with a C terminal Histag.

(29) SEQ ID NO: 28 is the protein sequence of mutant (S357I) Taq DNA polymerase with a C terminal Histag.

(30) SEQ ID NO: 29 is the DNA sequence of mutant (L361S) Taq DNA polymerase with a C terminal Histag.

(31) SEQ ID NO: 30 is the protein sequence of mutant (L361S) Taq DNA polymerase with a C terminal Histag.

(32) SEQ ID NO: 31 is the DNA sequence of mutant (L376S) Taq DNA polymerase with a C terminal Histag.

(33) SEQ ID NO: 32 is the protein sequence of mutant (L376S) Taq DNA polymerase with a C terminal Histag.

(34) SEQ ID NO: 33 is the DNA sequence of mutant (P382G) Taq DNA polymerase with a C terminal Histag.

(35) SEQ ID NO: 34 is the protein sequence of mutant (P382G) Taq DNA polymerase with a C terminal Histag.

(36) SEQ ID NO: 35 is the DNA sequence of mutant (T385I) Taq DNA polymerase with a C terminal Histag.

(37) SEQ ID NO: 36 is the protein sequence of mutant (T385I) Taq DNA polymerase with a C terminal Histag.

(38) SEQ ID NO: 37 is the DNA sequence of mutant (G418P) Taq DNA polymerase with a C terminal Histag.

(39) SEQ ID NO: 38 is the protein sequence of mutant (G418P) Taq DNA polymerase with a C terminal Histag.

(40) SEQ ID NO: 39 is the DNA sequence of mutant (R419D) Taq DNA polymerase with a C terminal Histag.

(41) SEQ ID NO: 40 is the protein sequence of mutant (R419D) Taq DNA polymerase with a C terminal Histag.

(42) SEQ ID NO: 41 is the DNA sequence of mutant (E421K) Taq DNA polymerase with a C terminal Histag.

(43) SEQ ID NO: 42 is the protein sequence of mutant (E421K) Taq DNA polymerase with a C terminal Histag.

(44) SEQ ID NO: 43 is the DNA sequence of mutant (L461S) Taq DNA polymerase with a C terminal Histag.

(45) SEQ ID NO: 44 is the protein sequence of mutant (L461S) Taq DNA polymerase with a C terminal Histag.

(46) SEQ ID NO: 45 is the DNA sequence of mutant (A472F) Taq DNA polymerase with a C terminal Histag.

(47) SEQ ID NO: 46 is the protein sequence of mutant (A472F) Taq DNA polymerase with a C terminal Histag.

(48) SEQ ID NO: 47 is the DNA sequence of mutant (E497K) Taq DNA polymerase with a C terminal Histag.

(49) SEQ ID NO: 48 is the protein sequence of mutant (E497K) Taq DNA polymerase with a C terminal Histag.

(50) SEQ ID NO: 49 is the DNA sequence of mutant (L498S) Taq DNA polymerase with a C terminal Histag.

(51) SEQ ID NO: 50 is the protein sequence of mutant (L498S) Taq DNA polymerase with a C terminal Histag.

(52) SEQ ID NO: 51 is the DNA sequence of mutant (E524K) Taq DNA polymerase with a C terminal Histag.

(53) SEQ ID NO: 52 is the protein sequence of mutant (E524K) Taq DNA polymerase with a C terminal Histag.

(54) SEQ ID NO: 53 is the DNA sequence of mutant (D551R) Taq DNA polymerase with a C terminal Histag.

(55) SEQ ID NO: 54 is the protein sequence of mutant (D551R) Taq DNA polymerase with a C terminal Histag.

(56) SEQ ID NO: 55 is the DNA sequence of mutant (R556D) Taq DNA polymerase with a C terminal Histag.

(57) SEQ ID NO: 56 is the protein sequence of mutant (R556D) Taq DNA polymerase with a C terminal Histag.

(58) SEQ ID NO: 57 is the DNA sequence of mutant (S679I) Taq DNA polymerase with a C terminal Histag.

(59) SEQ ID NO: 58 is the protein sequence of mutant (S679I) Taq DNA polymerase with a C terminal Histag.

(60) SEQ ID NO: 59 is the DNA sequence of mutant (L789S) Taq DNA polymerase with a C terminal Histag.

(61) SEQ ID NO: 60 is the protein sequence of mutant (L789S) Taq DNA polymerase with a C terminal Histag.

(62) SEQ ID NO: 61 is the DNA sequence of mutant (E189K/E507K/E742K) Taq DNA polymerase with a C terminal Histag.

(63) SEQ ID NO: 62 is the protein sequence of mutant (E189K/E507K/E742K) Taq DNA polymerase with a C terminal Histag.

(64) SEQ ID NO: 63 is the DNA sequence of the 2019-nCoV_N2 Forward Primer.

(65) SEQ ID NO: 64 is the DNA sequence of the 2019-nCoV_N2 Reverse Primer.

(66) SEQ ID NO: 65 is the DNA sequence of the 2019-nCoV_N2 Probe.

(67) The respective DNA and protein sequences of wild type Taq DNA polymerase and each mutant above, with a C terminal Histag, is shown in the sequence listing attached.

DETAILED DESCRIPTION

(68) As used herein, “about,” “approximately,” and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within ±10% of the indicated value, whichever is greater.

(69) The term “labeled probe” refers to a labeling probe used in an amplification reaction, typically for quantitative or qPCR analysis, as well as end-point analysis. Such labeling probes may be used to monitor the amplification of the target polynucleotide, and are suitable for monitoring the amount of amplicon(s) produced as a function of time. Such oligonucleotide labeling probes include, but are not limited to, the 5′-exonuclease assay TaqMan labeling probes (see U.S. Pat. No. 5,538,848), various stem-loop molecular beacons (see e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517, stemless or linear beacons (WO 99/21881), PNA Molecular Beacons (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons, non-FRET labeling probes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise/Amplifluor labeling probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion labeling probes (U.S. Pat. No. 6,589,743), bulge loop labeling probes (U.S. Pat. No. 6,590,091), pseudo knot labeling probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), hairpin labeling probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up labeling probes, self-assembled nanoparticle labeling probes, and ferrocene-modified labeling probes described, for example, in U.S. Pat. No. 6,485,901. Labeling probes can also comprise black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Labeling probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch). Labeling probes can also comprise two labeling probes, wherein for example a fluorophore is on one probe, and a quencher on the other, wherein hybridization of the two labeling probes together on a target quenches the signal, or wherein hybridization on target alters the signal signature via a change in fluorescence. Labeling probes can also comprise sulfonate derivatives of fluorescein dyes with a sulfonic acid group instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (available from Amersham).

(70) The term “sample” refers to biological samples from any source which include nucleic acid or DNA.

(71) The term “real time quantitative PCR” and “real time qPCR”, is used interchangeably with the term “quantitative PCR” (abbreviated “qPCR”), and refers to a method for simultaneous amplification, detection, and quantification of a target polynucleotide using labeled probes during PCR and further includes the protocols in the examples herein and such methods as TaqMan, SYBR Green assays, and the like; whether in a system performing quantitative real-time PCR or semi-quantitative real-time PCR.

(72) The term “target,” refers to a polynucleotide sequence that is sought to be amplified and can exist in the presence of other nucleic acid molecules or within a larger nucleic acid molecule or in a sample. The target polynucleotide can be obtained from any source, and can DNA or cDNA from RNA, or can be methylated, non-methylated, or both.

(73) The term “threshold cycle” or “CT” is defined as a fractional cycle number at which a reporter signal rises above a threshold value, including where DNA quantification by real-time PCR relies on plotting fluorescence against the number of cycles on a logarithmic scale. A threshold for detection of DNA-based fluorescence is, preferably, set 3-5 times of the standard deviation of the signal noise above background. The number of cycles at which the fluorescence exceeds the threshold is called the threshold cycle (Ct) or quantification cycle (Cq).

(74) The term “threshold” or “threshold value” is defined as the reporter signal value that is used for calculation of threshold cycle (CT).

(75) The term “reporter signal” is defined as the signal generated by a PCR product reporter (typically a dye or a labeled probe) which is correlated with the concentration of PCR product, in an assay for measuring biological data, including but not limited to data for cycling reactions. Examples of suitable data include but are not limited to, fluorescent signal data, optical signal data, magnetic signal data, and electronic signal data. Suitable assays include DNA quantification by qPCR. The reporter signal can be generated by a DNA-binding or intercalating dye (e.g. SYBR Green or EvaGreen) that binds to all double-stranded (ds) DNA in PCR, increasing the fluorescence quantum yield of the dye, and leading to an increase in fluorescence intensity measured at each cycle. The assay should be adjusted so that the increasing signal intensity does not interfere with, or prevent, accurate quantification of the target.

(76) The mutant Taq polymerase of the invention is suited to improved efficiency qPCR where the cycling includes using a rapid extension time of one second per cycle. It can be used with the commonly employed method of varying primers and templates for increasing the efficiency of target amplification, to find the most efficient primer-template combination together with the most efficient mutant Taq polymerase of the invention, as assessed in a titration experiment with serial dilutions of DNA template to create a standard curve of the change in (CT), as determined from reporter signal, with each dilution. The slope of the linear regression is then used to determine the efficiency of amplification, which is 100% if a dilution of 1:2 results in a (CT) difference of 1.

(77) The efficiency of qPCR can also be determined by mechanism-based qPCR quantification methods, which do not require a standard curve for quantification. Methods such as MAK2 (see Boggy G, Woolf P J (2010); Ravasi T (ed.). “A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data” PLOS ONE 5 (8): e12355) have been shown to have equal or better quantitative performance to standard curve methods. These mechanism-based methods use knowledge about the polymerase amplification process to generate estimates of the original sample concentration and amplification.

(78) Real-time or qPCR with the mutant Taq DNA polymerase of the invention can also be used to quantify nucleic acids, and monitor gene expression, by relative quantification and absolute quantification. Absolute quantification gives the exact number of target DNA molecules by comparison with DNA standards using a calibration curve; which necessitates that the PCR of the sample and the standard have the same amplification efficiency. The mutant Taq DNA polymerase of the invention provides a faster cycle time with a consistent and increased efficiency, making them suitable for absolute quantification. The same properties of the mutant Taq DNA polymerase of the invention also makes them well-suited relative quantification.

(79) Diagnostic qualitative PCR is applied to rapidly detect nucleic acids that are diagnostic of, for example, infectious diseases, cancer and genetic abnormalities. The properties of the mutant Taq DNA polymerase of the invention in qPCR assays allows significantly improved diagnosis of infectious diseases, newly emerging diseases, such as new strains of flu and coronavirus, and in diagnostic tests.

(80) Real time or qPCR with the mutant Taq DNA polymerase of the invention can also be used to assay gene expression and provide meaningful information relating to food safety, food spoilage and fermentation and microbial risk assessment of water quality (drinking and recreational waters) and in public health protection.

(81) Real time or qPCR with the mutant Taq DNA polymerase of the invention may also be used to amplify taxonomic or functional markers of genes in environmentally relevant samples to help determine, e.g., the amount of microorganisms in a sample, and/or, can identify different families, genera, or species based on the marker. Real time or qPCR with the mutant Taq DNA polymerase of the invention may also be used for functional markers (protein-coding genes) to show gene expression within a community, which may reveal information about the environment.

(82) Real time or qPCR with the mutant Taq DNA polymerase of the invention may also be used to detect agricultural pathogens, including those attacking plant propagules or seedlings. Real time or qPCR with the mutant Taq DNA polymerase of the invention may also be used in systems that allow detection and discrimination of small amounts of pathogens like the Phytophthora ramorum, an oomycete that kills Oaks and other species, even when mixed in with the DNA of the host plant.

(83) Real time or qPCR with the mutant Taq DNA polymerase of the invention may also be used to detect GMOs given the sensitivity and dynamic range offered in detecting associated sequences, using specific primers that amplify not the transgene but the promoter, terminator or even intermediate sequences used during the process of engineering the vector. As the process of creating a transgenic plant normally leads to the insertion of more than one copy of the transgene its quantity can also be assessed with the mutant Taq DNA polymerase of the invention.

(84) The use of qPCR with the mutant Taq DNA polymerase of the invention allows both the quantification and genotyping (characterization of the strain, carried out using melting curves) of a virus. The degree of infection, quantified with the mutant Taq DNA polymerase of the invention as the copies of the viral genome per unit of the patient's tissue, is relevant in many diagnoses.

EXAMPLES

(85) In gene expression analysis with the Taq DNA polymerase mutants of the invention, one typically performs RNA extraction form the sample followed by reverse transcription, to generate cDNA as the target. The cDNA target can be efficiently quantified with the Taq DNA polymerase mutants of the invention, and one of the methods describe above, where a reporter signal is monitored for threshold and the CT for the sample(s) is determined.

(86) Production of Mutants

(87) Taq DNA polymerase mutants were generated by conventional inverse PCR mutagenesis. All mutants are sequenced verified, expressed in E. Coli, and purified. A C-terminal His tag was added to all Taq DNA polymerase mutants and the wild type for ease of purification.

(88) Exemplary Probe qPCR

(89) Probe qPCR was performed under the following conditions:

(90) The target for the qPCR was the SARS-Co N gene, 2019-nCoV_N2, published by the United States Center for Disease Control for the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel.

(91) TABLE-US-00001 Forward Primer: 2019-nCoV_N2 Forward Primer: (SEQ ID NO: 63) TTACAAACATTGGCCGCAAA Reverse Primer: 2019-nCoV_N2 Reverse Primer: (SEQ ID NO: 64) GCGCGACATTCCGAAGAA Probe: 2019-nCoV_N2 Probe: (SEQ ID NO: 65) FAM-ACA ATT TGC CCC CAG CGC TTC AG-BHQ1

(92) Initial target concentration was 10 copies of synthesized Covid 19 N gene per reaction (Twist Bioscience, CA).

(93) Each 20 μl reaction contains 4 μl of 50 ng/μl Taq DNA polymerase, 1 μl of 10 μM Forward primer, 1 μl of 10 μM reverse primer, 1 μl of 5 μM Labelling Probe, 1 μl of 10 copies/μl of Covid 19 N gene, and 2 μl of 10× reaction buffer (which makes final of composition of 20 mM Tris-HCl, 80 mM Tris-Acetate, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 3 mM Mg-Acetate, 0.1% Triton®-X-100, pH 8.8 @ 25° C.) with water comprising the remaining 10 μl.

(94) The qPCR machine used was the Prime Pro 48 Real-time qPCR machine (Cole-Parmer, UK). The reaction protocol was: 95° C. 30 sec denature, followed by 40 cycles of [95° C. 4 sec annealing, 60° C. 1 sec extension]. Fluorescent signal was collected during each cycle the 60° C. extension step.

(95) Results are shown in FIGS. 1A to 1II. The wild-type result (WT) is included with each panel, showing that it does not display significant signal. The mutants identified that generated significant signal are the following: V62S, V64S, A70F, F73A, A77F, P253G, E255K, D257R, A259F, A271F, L288S, E289K, S357I, L361S, L376S, P382G, T385I, G418P, R419D, E421K, L461S, A472F, E497K, L498S, E524K, D551R, R556D, S679I, L789S, E189K/E507K/E742K. The DNA and protein sequence of each of these mutants are in the sequence listing (see sequence listing guide above).

(96) All patents, applications and references above are hereby incorporated by reference. The specific processes, methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, any of the terms “comprising”, “including”, containing”, etc. are to be read expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference, and the plural include singular forms, unless the context clearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

(97) The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.