Philosophy and Art Essay Example | Topics and Well Written Essays - 4500 words

Philosophy and Art - Essay Example He is a philosopher obsessed with clarity and light.Thus, if a discussion in which light, vision, and its abstract are constitutive of its very logic may be called ocularcentric, then it would be difficult to deny that Descartes' philosophy exemplifies ocularcentrism (Levin 1993). This essay discusses the ocular metaphoric and the part in plays in the lives of the contemporary American society today. It will demonstrate the extent to which vision constitutes the essential analogical figure in the readings of Jay, Bryson and Lacan. The paper will examines the role visual imagery plays in these writers main arguments, analyzes why it is so important to their theoretical framework, and considers the rhetorical work it is used to carry out. This essay will attempt to discuss how ocularcentrism shapes the understanding of what is central to the American society and what is peripheral; what is "visible" and "invisible" within the theoretical framework, and what the nature and limits of it are. Thus, the inherited advantages and disadvantages of this belief in ocularcentrism will be discussed, along with its effect on visual art. It is impossible to separ... modernity to be changed in its concepts, Jay asks for it to be seen as a diverse and complex body rather than a harmonious body of theories and practices. This essay claims that maps and plans are necessary to explain the components of modernity. He goes on to divide the body of the essay into three parts: Cartesian perspectivalism, seventeenth century Dutch art and baroque art. In his essay Jay refutes the concept of Cartesian perspectivalism to be the reigning form of any kind of visual model for modernity. While this claim is amongst the first to be made by critics, it does have its flaws. For starters, Jay refuses to create an argument that can fit into a specialized field of visual presentation. It aims at acknowledging local and international ideas which touch a large cultural stratum. The scale implied by Jay is coarse in its ability to touch such a large and dynamic field which is known not only for its various models but because of the centuries of art it covers. Thus, while Jay's argument can be considered inspiring in its attempt to break the routine form of ideological thinking, it is flawed in its ability to cover such a diverse cultural body. Instead, Jay could have used a concept which is smoothed down to a finer level thus allowing the subtle distinctions that arise in different pieces to be observed. Philosophy is a subject which changes over time. As the world develops, old problems fade away and new ones take their place. Art is a perfect display of the different developments in human sciences. It is representative of the cultural forms in any given society. However, another shift has also occurred bringing down the possibility of the art form from a means of human science or public culture. In American history this can be traced back to the 1980's

The Paralegal Profession Essay Example | Topics and Well Written Essays - 1000 words

The Paralegal Profession - Essay Example Lawyers who handle high profile, media-friendly cases gain the distinct advantage of demanding higher pay and accolades, particularly those who win more cases than their peers. Becoming a lawyer involves a lot of hard work and preparation for students who wish to pursue this career. To begin with, prospective law students must take up a pre-law course, majoring in any field of their choice, before they can proceed to their law degree. Law schools do not require any prerequisite courses for admission. However, most students choose among accounting, economics, philosophy, history, composition and literature, psychology, sociology, political science, religion and logic as their pre-law courses (Abernethy. 1996). Potential law students have many options at their disposal that will enable them to make an informed decision on whether or not to proceed with their desire to pursue a law degree or not. For example, they can sit in on a class or two to get a feel for what is expected from students in a typical law class. They may also join a tour of any law school of their choice or meet with current law students, if they are interested. Abernethy, A. J.D. Ph.D. mentions another option, which is, to â€Å"shadow† a lawyer, following him around for a day or two, just to have an idea of what a typical day for a legal professional is like. It is vital to remember that a lawyer’s daily schedule differs from one day to the next, ranging from a day in court to a long day at the library researching for a case. It is also important to note that the legal profession offers a wide range of fields—from commercial law to tax law to human rights law—so it would help the student considerably if he or she can shadow more than one lawyer (1996). Some students also get the chance to work as â€Å"runners† in a law office before they begin law school. â€Å"Runners offer general clerical assistance but their function usually entails filing papers at the

Real Time Pcr Essay Example for Free

Real Time Pcr Essay PROBE-BASED DETECTION SYSTEMS14 Hybridization probes (also called FRET probes)16 MELTING CURVE ANALYSIS16 Multiplex real-time PCR18 APPLICATIONS OF REAL TIME PCR18 GENE EXPRESSION ANALYSIS18 SNP GENOTYPING19 HIV DETECTION19 CYSTIC FIBROSIS (CF) DETECTION:20 THE ADVANTAGES OF REAL-TIME PCR20 THE DISADVANTAGES21 REFRENCES21 REAL TIME PCR TRADITIONAL PCR The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or cDNA template can be copied, or â€Å"amplified†, many thousand- to a millionfold. In traditional (endpoint) PCR, detection and quantitation of the amplified sequence are performed at the end of the reaction after the last PCR cycle, and involve post-PCR analysis such as gel electrophoresis and image analysis. REAL-TIME QUANTITATIVE PCR (qPCR) In real-time quantitative PCR (qPCR), the amount of PCR product is measured at each cycle. This ability to monitor the reaction during its exponential phase enables users to determine the initial amount of target with great precision. WHAT’S WRONG WITH AGAROSE GELS? * Poor precision. * Low sensitivity. Short dynamic range lt; 2 logs. * Low resolution. * Non-automated. * Size-based discrimination only * Ethidium bromide staining is not very quantitative REAL TIME PCR VS PCR . BASIC PRINCIPLE Quantitative PCR  is carried out in a  thermal cycler  with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited  fluorochrome. The thermal cycler is also able to rapidly heat and chill samples thereby taking advantage of the physicochemical properties of the  nucleic acids  and  DNA polymerase. The PCR process generally consists of a series of temperature changes that are repeated 25 – 40 times, these cycles normally consist of three stages: the first, at around 95  Ã‚ °C, allows the separation of the nucleic acid’s double chain; the second, at a temperature of around 50-60  Ã‚ °C, allows the alignment of the primers with the DNA template;  the third at between 68 72  Ã‚ °C, facilitates the  polymerization  carried out by the DNA polymerase In real-time PCR, * the amount of DNA is measured after each cycle by the use of fluorescent markers that are incorporated into the PCR product. The increase in fluorescent signal is directly proportional to the number of PCR product molecules (amplicons) generated in the exponential phase of the reaction. * Fluorescent reporters used include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that are incorporated into the product during amplification. * The change in fluorescence over the course of the reaction is measured by an instrument that combines thermal cycling with scanning capability. By plotting fluorescence against the cycle number, the real-time PCR instrument generates an amplification plot that represents the accumulation of product over the duration of the entire PCR reaction (Figure 1). Figure 1—Amplification plots are created when the fluorescent signal from each sample is plotted against cycle number; therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. The samples being amplified in this example are a dilution series of the template. TYPES OF PCR Quantitative PCR| Qualitative qPCR| A specific or non-specific detection chemistry allows the quantification ofthe amplified product. | In qualitative qPCR, the goal is to detect the presence or absence of a certain sequence. | The amount detected at a certain point of the run is directly related to theinitial amount of target in the sample| For virus sub-typing and bacterial species identification. Can also be used for allelic discrimination between wild type and mutant, between different SNPs or between different splicing forms. | common pplications of quantitative PCR are gene expression analysis, pathogen detection/quantification and microRNA quantification| Different fluorophores can be used for the two alleles, and the ratio of the fluorophores signals correlates to the related amount of one form compared to the other one. | Quantitative PCR software uses the exponential phase of PCR for quantification. | Specific detection methods such as Double-Dye probe systems are more ofte n used for theseApplications| Overview of real-time PCR Real-time PCR is a variation of the standard PCR technique used to quantify DNA or RNA in a sample. Using sequence-specific primers, the relative number of copies of a particular DNA or RNA sequence can be determined.. Quantification of amplified product is obtained using fluorescent probes or fluorescent DNA binding dyes and real time PCR instruments that measure fluorescence while performing temperature changes needed for the PCR cycles. qPCR STEPS There are three major steps that make up a qPCR reaction. Reactions are generally run for 40 cycles. 1. Denaturation—The temperature should be appropriate to the polymerase chosen (usually 95 °C). The denaturation time can be increased if template GC content is high. 2. Annealing—Use appropriate temperatures based on the calculated melting temperature (Tm) of the primers (5 °C below the Tm of the primer). 3. Extension—At 70–72 °C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in qPCR is small, this step is often combined with the annealing step using 60 °C as the temperature. BASICS OF REAL TIME PCR Baseline – The baseline phase contains all the amplification that is below the level of detection of the real time instrument. Threshold – where the threshold and the amplification plot intersect defines CT. Can be set manually/automatically CT – (cycle threshold) the cycle number where the fluorescence passes the threshold Rn – (Rn-baseline) NTC – no template control Rn is plotted against cycle numbers to produce the amplification curves and gives the CT value. ONE-STEP OR TWO-STEP REACTION qRT-PCR can be one step or two step. 1. Two-step qRT-PCR Two-step qRT-PCR starts with the reverse transcription of either total RNA or poly(A)+ RNA into cDNA using a reverse transcriptase (RT). This first-strand cDNA synthesis reaction can be primed using random hexamers, oligo(dT), or gene-specific primers (GSPs). To give an equal representation of all targets in real-time PCR applications and to avoid the 3? bias of oligo(dT), it is usually recommended that random hexamers or a mixture of oligo(dT) and random hexamers are used. The temperature used for cDNA synthesis depends on the RT enzyme chosen. Following the first-strand synthesis reaction, the cDNA is transferred to a separate tube for the qPCR reaction. In general, only 10% of the first strand reaction is used for each qPCR. . One-step qRT-PCR One-step qRT-PCR combines the first-strand cDNA synthesis reaction and qPCR reaction in the same tube, simplifying reaction setup and reducing the possibility of contamination. Gene-specifi c primers (GSP) are required. This is because using oligo(dT) or random primers will generate nonspecific products in the one-step procedure and reduce the amount of product of interest. O verview of qPCR and qRT-PCR components This section provides an overview of the major reaction components and parameters involved in real-time PCR experiments. * DNA polymerase One of the main factors affecting PCR specificity is the fact that Taq DNA polymerase has residual activity at low temperatures. Primers can anneal nonspecifically to DNA, allowing the polymerase to synthesize nonspecific product. The problem of nonspecific products resulting from mispriming can be minimized by using a â€Å"hot-start† enzyme. Using a hot-start enzyme ensures that no active Taq is present during reaction setup and the initial DNA denaturation step. * Template Anywhere from 10 to 1,000 copies of template nucleic acid should be used for each real-time PCR reaction. This is equivalent to approximately 100 pg to 1 ? of genomic DNA, or cDNA, generated from 1 pg to 100 ng of total RNA. Excess template may increase the amount of contaminants and reduce efficiency. If the template is RNA, care should be taken to reduce the chance of genomic DNA contamination. One option is to treat the template with DNaseI. Ultrapure, intact RNA is essential for full-length, high-quality cDNA synthesis and accurate mRNA quantification. RNA should be devoid of any RNase contamination, and aseptic conditions should be maintained. * Reverse transcriptase The reverse transcriptase (RT) is as critical to the success of qRT-PCR as the DNA polymerase. It is important to choose an RT that not only provides high yields of full-length cDNA but also has good activity at high temperatures. High-temperature performance is also very important for tackling RNA with secondary structure or when working with gene-specific primers (GSPs). * dNTPs It is recommended that both the dNTPs and the Taq DNA polymerase be purchased from the same vendor, as it is not uncommon to see shifts of one full threshold cycle (Ct) in experiments that employ these items from separate vendors. * Magnesium concentration In qPCR, magnesium chloride or magnesium sulfate is typically used at a fi nal concentration of 3 mM. This concentration works well for most targets; however, the optimal magnesium concentration may vary between 3 and 6 mM. * UNG The Uracil-N-Glycosylase is an enzyme that hydrolyses all single-stranded and double-stranded DNA containing dUTPs. Consequently, if all PCR amplifications are performed in the presence of a dNTPs/dUTPs blend, by carrying a UNG step before every run it is possible to get rid of any previous PCR product. * ROX Some thermocyclers require MasterMix containing ROX dye for normalization. This is the case for the ABI and Eppendorf machines, and optional on the Stratagene machines. If you work with such machines, it is easier to work with the ROX dye already incorporated in the MasterMix rather than adding it manually. It guarantees a higher level of reproducibility and homogeneity of your assays. * Fluorescein For iCycler iQ, My iQ and iQ5 machines (BioRad thermocyclers), the normalization method for SYBR Green assay uses Fluorescein to create a â€Å"virtual background†. As in the case for the ROX, it is better and easier to use a MasterMix that contains pre-diluted Fluorescein, guaranteeing higher reproducibility and homogeneity of your assays. REAL TIME PCR SYSTEM: System Features: †¢ Four interchangeable block formats †¢ Optional Automation Accessory amp; Barcode Scanner †¢ Argon ion laser/CCD camera †¢ Easy to Use Software, Multiple Applications †¢ Set up Wizards †¢ QC Filtering/Flag System †¢ Flexible data reports amp; exporting SOFTWARES FOR DATA ANALYSIS AND PRIMER DESIGNING 1 ) Light Cycler ® Relative Quantification Software The first commercially available software was the Light Cycler ® Relative Quantification Software (2001). 2 ) REST In 2002, the relative expression software tool (REST ) was established as a new tool. 3 ) Q-Gene Recently a second software tool, Q-Gene, was developed, which is able to perform a statistical test of the real-time data. Q-Gene manages and expedites the planning, performance and evaluation of quantitative real-time PCR experiments. 4) OligoPerfect A primer design software program such as OligoPerfectâ„ ¢, available on the Web at www. invitrogen. com/oligoperfect, can automatically evaluate a target sequence and design primers for it based on the criteria STEPS OF REAL TIME PCR Real-time reaction mix (final concentrations): 1x 2 x AmpliTaq Gold 0. 5 ? M 5’ primer 0. 5 ? M 3’ primer 0. 2 ? M probe 0. 4 ? Rox reference dye 20 ? l Final Volume (including sample and dH20) STANDARD REAL-TIME PCR PROTOCOL ASSAY DESIGN: This section describes the stages of real-time PCR assay design and implementation. We will identify sources of variability, the role they play in data accuracy, and guidelines for optimization in the following areas: 1Target amplicon and primer design 2. Nucleic acid purification 3. Reverse transcription 4. Controls and normalization 5. Standard curve evaluation of efficiency, sensitivity, and reproducibility Good primer (pair) properties One way to minimize efficiency bias is to amplify relatively short targets. Amplifying a 100 bp region is more likely to result in complete synthesis in a given cycle than, say, amplifying a 1,200 bp target. For this reason, real-time PCR target lengths are generally in the range of 60 bp to 200 bp. In addition, shorter amplicons act as a buff er against variations in template integrity. Primers designed to amplify larger regions are less likely to anneal with the same fragment in a slightly degraded nucleic acid sample. PURIFICATION Phenol-based organic extraction is a very effective method for purifying RNA from a wide variety of cell and tissue types. During sample lysis, phenol and guanidine isothiocyanate disrupt cells and dissolve cell components. while maintaining the integrity of the nucleic acids by protecting them from RNases. Chloroform is added and the mixture is separated by centrifugation, which separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase in the presence of guanidine isothiocyanate, while DNA and protein are driven into the organic phase and interphase. The RNA is then recovered from the aqueous phase by precipitation with isopropyl alcohol. REVERSE TRANSCRIPTION CONSIDERATIONS Most reverse transcriptases employed in qRT-PCR are derived from avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (M-MLV). An ideal reverse transcriptase will exhibit the following attributes: * Thermostability— thermostable RTs function at the higher end of (or above) this range and allow for successful reverse transcription of GC-rich regions. * RNase H activity— RNase H activity can drastically reduce the yield and ratio of full-length cDNA, which translates to poor sensitivity. Several RTs, most notably SuperScript II and III, have been engineered for reduced RNase H activity. NORMALIZATION AND QUANTIFICATION: When analyzing and comparing results of Real-Time qPCR assays many researchers are confronted with several uncontrolled variables, which can lead to misinterpretation of the results. Those uncontrolled variables can be the amount of starting material, enzymatic efficiencies, and differences between tissues, individuals or experimental conditions. In order to make a good comparison, normalization can be used as a correction method, for these variables. The most commonly known and used ways of normalization are : * normalization to the original number of cells, * normalization to the total RNA mass, normalization to one or more housekeeping genes, * normalization to an internal or external calibrator. Normalization to number of cells can actually only be done for cell culture and blood samples. The two majors methods of normalization are the absolute quantification and the relative quantification . Absolute quantification Absolute quantification requires a standard curve of known copy numbers. The amplicon being studied can be cloned, or a synthetic oligonucleotide (RNA or DNA) can be used. The standard must be amplified using the same primers as the gene of interest and must amplify with the same efficiency. The standards must also be quantified accurately. This can be carried out by reading the absorbance at A260, although this does not distinguish between DNA and RNA, or by using a fluorescent ribonucleic acid stain such as RiboGreen. Relative quantification Relative quantification is the most widely used technique. Gene expression levels are calculated by the ratio between the amount of target gene and an endogenous reference gene, which is present in all samples. The reference gene has to be chosen so that its expression does not change under the experimental conditions or between different tissue. There are simple and more complex methods for relative quantification, depending on the PCR efficiency, and the number of reference genes used. STANDARD CURVE TO ASSESS EFFICIENCY, SENSITIVITY, AND REPRODUCIBILITY The final stage before assay employment is validating that all the experimental design parameters result in a highly efficient, sensitive, and reproducible experiment. * Reaction efficiency One hundred percent efficiency corresponds to a perfect doubling of template at every cycle, but the acceptable range is 90–110% for assay validation. This efficiency range corresponds to standard curve slopes of –3. 6 to –3. 1. The graph in Figure shows the measurement bias resulting solely from differences in reaction efficiency.. A standard curve is generated by plotting a dilution series of template against the Ct for each dilution. To some, sensitivity is measured by how early a target Ct appears in the amplification plot. However, the true gauge of sensitivity of an assay is whether a given low amount of template fits to the standard curve while maintaining a desirable efficiency. The most dilute sample that fits determines reaction sensitivity. The standard curve also includes an R2 value, which is a measure of replicate reproducibility. Standard curves may be repeated over time to assess whether the consistency, and therefore the data accuracy for the samples. Real-Time PCR Fluorescence Detection Systems Several different fluorescence detection technologies can be used for realtime PCR, and each has specific assay design requirements. All are based on the generation of a fluorescent signal that is proportional to the amount of PCR product formed. The three main fluorescence detection systems are: * DNA-binding agents (e. g. SYBR Green and SYBR GreenER technologies * Fluorescent primers (e. g. , LUX Fluorogenic Primers and Amplifluor qPCR primers) * Fluorescent probes (e. g. , TaqMan probes, Scorpions, Molecular Beacons) The detection method plays a critical role in the success of real-time PCR. DNA-Binding Dyes The most common system for detection of amplified DNA is the use of intercalating dyes that fluoresce when bound to dsDNA. SYBR Green I and SYBR GreenER technologies use this type of detection method. The fluorescence of DNA-binding dyes significantly increases when bound to double-stranded DNA (dsDNA). The intensity of the fluorescent signal depends on the amount of dsDNA that is present. As dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and can be detected using real-time PCR instruments. SYBR Green I advantages †¢ Low cost assay †¢ Easy design and set up SYBR Green I disadvantages †¢ Non specific system †¢ Not adapted to multiplex †¢ Non suitable for qualitative qPCR Primer-Based Detection Systems Primer-based fluorescence detection technologies can provide highly sensitive and specific detection of DNA and RNA. In these systems, the fluorophores is attached to a target-specific PCR primer that increases in fluorescence when incorporated into the PCR product during amplification. * Amplifluor Real-Time PCR Primers Amplifluor real-time PCR primers are designed with both a fluorophore and quencher on the same primer. The primer adopts a hairpin configuration that brings the fluorophore in close proximity to the quencher. The fluorescent signal increases when the primer is unfolded and the fluorophore and quencher are de-coupled during incorporation into an amplification product. Figure: Ampliflour primer PROBE-BASED DETECTION SYSTEMS Probe-based systems provide highly sensitive and specifi c detection of DNA and RNA. However, dual-labeling and complex design specifi cations make them expensive and more diffi cult to use than primer-based systems or DNAbinding dyes. TaqMan probes = Double-Dye probes TaqMan probes, also called Double-Dye Oligonucleotides, Double-Dye Probes, or Dual Labelled probes, are the most widely used type of probes. A fluorophore is attached to the 5’ end of the probe and a quencher to the 3’ end. The fluorophores is excited by the machine and passes its energy, via FRET (Fluorescence Resonance Energy Transfer) to the quencher. TaqMan probes can be used for both quantification and mutation detection, and most designs appear to work well. TaqMan ASSAY DENATURATION ANNEALING OF PRIMERS AND PROBE POLYMERIZATION AND PROBE CLEAVAGE Molecular Beacons In addition to two sequence-specific primers, molecular beacon assays employ a sequence-specific, fluorescently labeled oligonucleotide probe called a molecular beacon, which is a dye-labeled oligonucleotide (25–40 nt) that forms a hairpin structure with a stem and a loop . A fluorescent reporter is attached to the 5 end of the molecular beacon and a quencher is attached to the 3 end. The loop is designed to hybridize specifically to a 15–30 nucleotide section of the target sequence Figure: Moleculer Beacon They are highly specific, can be used for multiplexing, and if the target sequence does not match the beacon sequence exactly, hybridization and fluorescence will not occur a desirable quality for allelic discrimination experiments. Hybridization probes (also called FRET probes) Roche has developed hybridization probes for use with their LightCycler. Two probes are designed to bind adjacent to one another on the amplicon. One has a 3’ label of FAM, whilst the other has a 5’ LC dye, LC red 640 or 705. When the probes are not bound to the target sequence, the fluorescent signal from the reporter dye is not detected. However, when the probes hybridize to the target sequence during the PCR annealing step, the close proximity of the two fluorophores allows energy transfer from the donor to the acceptor dye, resulting in a fluorescent signal that is detected. FRET probe principle and light cycler MELTING CURVE ANALYSIS Melting curve analysis can only be performed with real-time PCR detection technologies in which the fluorophore remains associated with the amplicon. Amplifications that have used SYBR Green I or SYBR GreenER dye primers can be subjected to melting curve analysis. Dual-labeled probe detection systems such as TaqMan probes are not compatible because they produce an irreversible change in signal by cleaving and releasing the fluorophore into solution during the PCR; however, the increased specificity of this method makes this less of a concern. The level of fluorescence of both SYBR Green I and SYBR GreenER dyes significantly increases upon binding to dsDNA. By monitoring the dsDNA as it melts, a decrease in fluorescence will be seen as soon as the DNA becomes single-stranded and the dye dissociates from the DNA. Figure: Melting curve analysis can detect the presence of nonspecifc products, as shown by the additional peaks to the left of the peak for the amplified product in the melt curve. How to perform melting curve analysis To perform melting curve analysis, the real-time PCR instrument can be programmed to include a melting profile immediately following the thermocycling protocol. After amplification is complete, the instrument will reheat your amplified products to give complete melting curve data. Most real-time PCR instrument platforms now incorporate this feature into their analysis packages. In general, the program steps will be: 1. Rapid heating of the amplified sample to 94 °C to denature the DNA. 2. Cooling the sample to 60 °C. 3. Slowly heating (by increasing the temperature 0. 2 °C/second) the sample while plotting fluorescence signal vs. temperature. (As the temperature increases and the dsDNA strands melt, the fluorescence signal will decrease. ) Figure: Example of a melting curve thermal profile setup on an Applied Biosystems instrument (rapid heating to 94 °C to denature the DNA, followed by cooling to 60 °C. ) Multiplex real-time PCR In multiplex real-time PCR, more than one set of gene-specific primers is used to amplify separate genes from the template DNA or RNA in a single tube. Typically, multiplex reactions are used to amplify a gene of interest and a â€Å"housekeeping† gene (e. g. , #-actin or GAPDH), which is used as a normalize for the reaction. Because more than one PCR product will be quantified in the same tube, different fluorescent reporter dyes are used to label the separate primers or probes for each gene. More Samples Analyzed per Plate. Target and normalizer in same reaction and Less sample consumed. APPLICATIONS OF REAL TIME PCR GENE EXPRESSION ANALYSIS A sample gene expression analysis using a multiplex TaqMan assay is presented in the following sections. In this example, we’re interested in the relative expression of three genes in the polyamine biosynthesis pathway, ornithine decarboxylase (ODC), ODC antizyme (OAZ), and antizyme inhibitor (AZI), in two different samples, sample A and sample B. 1. RNA was isolated from sample A and sample B. 2. RNA was reverse transcribed into cDNA. 3. The amount of the target genes (ODC, OAZ, and AZI) and the reference gene (b-actin) was determined in each of the cDNA samples using a multiplex qPCR assay. 4. Data were analyzed and the relative expression of each of the target genes in the two samples was calculated. EXAMPLE BRCA1 is a gene involved in tumor suppression. BRCA1 controls the expression of other genes. In order to monitor level of expression of BRCA1, real-time PCR is used. SNP GENOTYPING In order to perform SNP genotyping, two specific probes labeled with different dyes are used, the first for the wild type allele and the second for the mutant allele. If the assay results in the generation of only the first fluorescent color, then the individual is homozygous wild type at that locus. If the assay results in the generation of only the second fluorescent color, then the individual is homozygous mutant. And finally, if both fluorescent colors are produced, then the individual is heterozygous. At the end of the reaction, hydrolysis probes are digested. The quality of a hydrolysis probe is given by the hybridization efficiency, the quenching of the intact probe and the cleavage activity of Taq polymerase. HIV DETECTION Nowadays HIV is strikingly spreading out whole the world. so in order to diminish its distribution , it is necessary to detect it as soon as possible amp; for this purpose, Real time PCR is recommended by scientist. In this method ,’ pol’’ gen of the virus, is amplified in thermocycler. 6 patient have been studied. infection in these patients was confirmed by ELISA amp; western blot. * Sampling amp; RNA extracting from patients. * Cloning of target segment by using Xba I amp; Hind III. And 180 bp primers. * Standard virus mRNA was extracted. * Quantitative analysis of HIV virus by SYBR-green Real Time RT-PCR. CYSTIC FIBROSIS (CF) DETECTION: Cystic f ibrosis (CF) is the most common inherited disease among Caucasian populations with an incidence of ~1 in 2500 births. A3 base pair (bp) deletion, designated DF508, accounts for nearly 70% of CF cases and causes severe manifestations of the disease. It results in the absence of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and this error prevents normal processing and translocation of the polypeptide chain to apical membranes of epithelial cells. This deletion can be detected by molecular beacons in real time PCR. Figure:Examples of specific molecular beacon fluorescence increase during real-time PCR in samples containing single lymphoblasts homozygous normal for CF (green), heterozygous DF508 (blue), or homozygous DF508 (red). A) Fluorescent signal from the molecular beacon detecting the normal allele. (B) Fluorescent signal from the molecular beacon detecting the DF508 allele. Dashed lines indicate the threshold of 200 units (~10 SD above baseline readings) used for determining CT values. THE ADVANTAGES OF REAL-TIME PCR * The ability to monitor the progress of the PCR reaction as it occurs in real time * The ability to precisely measure the amount of amplicon at each cy cle * An increased dynamic range of detection * The combination of amplification and detection in a single tube, which eliminates post-PCR manipulations. Rapid cycling times (1 hour) * High sample throughput (~200 samples/day) * Low contamination risk (sealed reactions) * Very sensitive (3pg or 1 genome eq of DNA) * Broad dynamic range (10 1010 copies) * Reproducible (CV lt; 2. 0 %) * Allows for quantitation of results * Software driven operation * No more expensive than â€Å"in house† PCR ($15/test) THE DISADVANTAGES * Current technology has limited capacity for multiplexing. Simultaneous detection of 2 targets is the limit. * Development of protocols needs high level of technical skill and/or support. Requires Ramp;D capacity and capital) * High capital equipment costs ($ 50,000 -160,000). REFRENCES * http://www. icmb. utexas. edu/core/DNA/qPCR/QiagenRT-PCR. pdf www. icmb. utexas. edu * http://books. google. com. pk/books? id=-v-U-mXWg-gCamp;printsec=frontcoveramp;dq=real+time+pcramp;hl=enamp;sa=Xamp;ei=Bph1UezKIceDhQeUh4CwCAamp;ved=0CDAQ6AEwAQ#v=onepageamp;q=real%20time%20pcramp;f=false books. google. com. pk * PCR/Real-Ti me PCR Protocols www. protocol-online. org Real-Time Pcr: An Essential Guide Google Books books. google. com * * http://www. gene-quantification. e/bio-rad-CFX96-bulletin-5589. pdf www. gene-quantification. de * https://www. google. com. pk/#output=searchamp;sclient=psy-abamp;q=fret+rt-qpcramp;oq=fret+in+rtamp;gs_l=serp. 1. 1. 0i22i30l2. 1583. 4622. 1. 10196. 6. 6. 0. 0. 0. 0. 551. 2584. 3-3j1j2. 6. 0 0. 0 1c. 1. 9. serp. 97Wjtm9UCU4amp;psj=1amp;bav=on. 2,or. r_cp. r_qf. amp;fp=f6d28cf5fd703914amp;biw=1366amp;bih=600 www. google. com. pk * BioTechniques Real-time PCR for mRNA quantitation www. biotechniques. com * http://env1. gist. ac. kr/joint_unugist/file/g_class11_real_time_pcr_vt. pdf env1. gist. ac. kr

History Of Intruder Knowledge Versus Attack Sophistication Information Technology Essay

History Of Intruder Knowledge Versus Attack Sophistication Information Technology Essay Intrusion detection is a necessary security infrastructure for any organization. Its a process of noticing or monitoring the events like imminent threats or unexpected new attacks, standard security practices, acceptable policies and existing attacks that occur in a network or computer. Detecting process is mainly based on signs of incidents. The process which attempts to block these detected incidents is known as intrusion prevention. Both the Intrusion Detection System (IDS) and Intrusion Prevention System (IPS) are principally focused on log information, identifying incidents, blocking incidents, reporting incidents to administrator. The regular problems when handling IDS is analysis of system generated events, because in a busy network there will be so many events to analyse with help of some monitoring tools and devices but its very hard manage due to unwanted outcomes, undetected threats and unmanageable threats. These threats can cause a serious damage to the network or organi zation. Research Question and Objectives: Every organisation recurrently face problem because of threats. As an Information Systems Security student I would like to do some research in Intrusion detection system. My main aim is to do an experiment on the Network Intrusion Detection System (NIDS) with help of Snort to detect network based attacks. Presently how the security infrastructure of the organizations is facing problems with imminent threats and malicious attacks? How it can be reduced by intrusion detection system? In what way the tools and techniques can be used to experiment the network based attacks? The research objectives are planning and implementing IDS, Monitoring for critical security threats and detecting them network wide, detecting malicious users on the network, proactive administration, regular network maintenance, 24/7 security event management, Signature and protocol tuning, alerting and preventing the detected threats. Hopefully all these objectives can be achieved by implement a network security with Snort. Snort is a flexible, small, light-weight and cross platform tool which is very suitable for NIDS. While working on this research network may also need some other computer running with tools like Suricata and Bro which are also familiar for NIDS and Experiment will also examine the integration of OSSEC with the analyst console Sguil. Literature Review: The Intrusion Detection Systems (IDS) are vital modules of defensive methods to protect a network or computer system from abuse. Network intrusion detection system examines all inbound and outbound network activities and notices the attack in network or computer. IDS are a passive monitoring system it alerts when distrustful activity takes place. It inspects the network traffic and data. It identifies the probes, exploits, attacks and vulnerabilities. It responds to the malicious events in several ways like displaying alerts, events log or paging an administrator. It can reconfigure the network and reduce the effect of the malicious activities like worms and virus. It precisely looks at intrusion signatures or hacker signatures so that it can distinguish worms or viruses from general system activities. Intrusion detections are categorized as misuse detection, anomaly detection, passive and reactive system, network based system and host based system. This picture shows history of Intruder Knowledge versus Attack sophistication Source: http://www.cert.org/archive/pdf/IEEE_IDS.pdf Misuse detection: In misuse detection IDS investigates the gathered information and compares it to huge databases of attack signature. Primarily IDS look for particular attack which was already documented. It is very similar to anti-virus because the detection software has good collection of intrusion signature database and it compares packets against the database. Anomaly detection: In anomaly the administrator provides the baseline, network traffic load state, typical packet size, breakdown and protocol. Anomaly detector compares the inspected network segment to normal baseline and examines the anomalies. Passive and Reactive systems: In passive systems IDS perceive a potential security breach, signal alerts and information of logs. Coming to reactive system IDS reacts to the distrustful and malicious activities either by shutting down the user or by reprogramming the firewall to stop or block network traffic from a malicious source. Network based IDS: IDS are network or host based solutions. Network based intrusion detection systems (NIDS) is an independent platform which categorizes network traffic and examines multiple hosts. They are hardware appliances hence they consists of network intrusion detection capabilities. It does consist of hardware sensors which are located along the network or demilitarized zone. NIDS gains access over network traffic by connecting to network hubs and switches and they are configured got network tap or port mapping. The sensor software will examine all the data packets which are going in and out of the network. NIDS are comparatively cheaper solutions that HIDS. It also need less training and administration but it is not as flexible as HIDS. NIDS system must have a good bandwidth Internet access and regular updates of latest worms and virus signatures. Best example is Snort Host based IDS: Host based intrusion detection systems (HIDS) are not suitable for real time detection. It has to be configured properly to use in real time. It has software agents which are installed on individual host computers within the system. It analyse the packets going in and out from that specific computer where the intrusion detection software is installed. It also examines the application logs, system calls and file system changes. HIDS can provide some addition features which not there in NIDS. For instance HIDS are capable to inspect activities which are only able to implement by administrator. It detects the modifications in the key system files and can also examine the attempts to overwrite key files. Trojans and backdoors installation can be detected and stopped; these particular intrusions are not generally seen in NIDS. HIDS systems must have internet access and also frequent updates of worms and virus signatures. Certain application based IDS are also a portion of HIDS. Best examp le is OSSEC. IDS Protection Source: http://www.cert.org/archive/pdf/IEEE_IDS.pdf Intrusion detection system (IDS) vs. Intrusion prevention system (IPS): Most of them believe like IDS IPS works similar and IPS is future way of IDS. But it is like comparing an apple and banana. These two solutions are very different from each other. IDS is passive it monitors and detects but IPS is active prevention system. The IDS drawbacks can be overcome by implementation, management and proper training. IDS is a cheaper implementation that IPS. However, by looking at IPS benefits most of them believe that IPS is following generation of IDS. The main point to remember is that no single security device can prevent all attacks at all the time. IDS and IPS works satisfactory when they are integrated with some addition and current security solutions. The combination of firewall and IDS gives protection to system so IPS is usually considered as next generation IDS. Presently IPS also has both types of HIPS and NIPS as like IDS. IPS can some more actions like dropping the malicious data packets, sending an alarm, reorganizing the connection and/or stoppi ng the traffic from the malicious IP address, correcting CRC errors and few more like cleaning up unwanted network and transport layer options. Snort: Snort is free and open source software which is used for network intrusion detection (NIDS) and network intrusion prevention system (NIPS). Martin Roesch was the creator of snort in 1998 but now it is maintained by a network security software and hardware company known as Sourcefire. Roesch is the founder and Chief technical officer of Sourcefire. The latest version is 2.9.0.5 and it was released on 6th April 2011. It is written in C language and cross-platform so that can run on any operating system. It is also a licensed by GNU general public license. Over a decade Snort has been recognized as the best prominent software in the security Industry. Snort is a great piece of software used for NIDS. It has ability to perform real time traffic analysis, protocol analysis, content matching, Internet Protocol networks packet log and content search. It can even examine probes or attacks, buffer overflows, OS fingerprinting, common gateway interface, stealth port scans and server message block probes. Snort mainly configured in three modes network intrusion detection, sniffer and packet logger. In NIDS mode it can examine network traffic and inspect it against ruleset provided by the user. As a sniffer it read all network data packets and displays them on the user console. As a packet logger it writes all log packets to the harddisk. Some 3rd party tools like Snorby, RazorBack and Base interface with snort for administration, log analysis and reporting. Snort provides dramatic power, speed and performance. It is light weight and protects against latest dynamic threats by rules based detection engine. Its source code and ruleset are regularly revised and tested by worldwide security professionals. It is most popular for IDS and IPS solutions with more than 205,000 registered users. There are minimum 25 companies that are incorporate with Snort for network security assistance. Snort vs. Suricata vs. Bro Source:http://blog.securitymonks.com/2010/08/26/three-little-idsips-engines-build-their-open-source-solutions/ Suricata and Bro: Suricata is also an open sources which is used for IDS and/or IPS. Open Information Security Foundation (OISF) has developed it. First standard release was in July 2010. It was written in C language and can run in Linux, Mac and Windows operating systems. It was licensed by GNU general public license. Suricata is a new tool when compared with other Opensource IDS and very best in all as shown in the above figure. As its new software there are no much research papers and journals. Bro is open source and UNIX based, it is used for NIDS. It was written by Vern Paxson and licensed by BSD. It runs on any Linux based operating system. These two tools are very good very there is no much research and literature on them. But these two are quite good when compared to Snort. OSSEC and SGUIL: OSSEC is an open source HIDS. It does log analysis, rootkit detection, windows registry monitoring, active response and integrity checking. It offers IDS for all Linux, Mac and Windows Operating systems because it has centralized cross platform. It was written by Daniel B in 2004. SGUIL is a pool of free software modules for Network Security Monitoring and IDS alerts. It was written in Tcl/Tk and run on any OS which supports Tcl/Tk. It integrates with Snort and generates alert data and session data from SANCP. Full content can be retrieved my running Snort in packet logger mode. Sguil is an application of Network Security Monitoring (NSM) Critical evaluation: The gathered information from different sources gives a brief idea of research. Literature covers all the aims and objectives of the research which was drawn and supported from the pool of journals, research papers, white papers, blogs and wikis. Introduction gives the over idea of the research going to takes place. Research question focuses on the field of interest and research area. Objectives mentions the clear tasks what are going to be achieved and its designed as a step by step procedure like starting with planning and implementation of IDS and later the steps that have to be achieved in the research area and ends with the some necessary applications like Snort, OSSEC and SGUIL which are very important to achieve the most out of Intrusion detection. Literature review covers almost each and every necessary step that is required in the research area. It is also very relevant to the research area and completely confined to it without any deviations. Intrusion detection and different types of IDS are clearly explained. Host based intrusion detection systems and Network based intrusion detection systems are clearly explained with help of graphical images. The differences between IDS and IPS are mentioned and it also explains why IPS is more powerful. Lastly main application like Snort, Suricata, Bro, OSSEC and SGUIL are completely covered with features. But the interesting finding during literature search is Suricata and Bro. Both are very good for IDS and they are having more advanced features than the Snort. However there is very less research done it that area. So there is a need of qualitative data by taking interviews of some security professionals and lectures. At last, in brief literature covers all the parameters of research question, objectives, methods and outcomes of different IDS and applications which are suitable for IDS are well organized and documented. Research Methods and Methodology: I would like do the research according to Inductive process because I am sure about the topic and I want to know the outcomes of the experiment. As inductive research moves from specific point to general I selected it and start working. In this research I am planning to implement an experiment in small network with some applications. I am using these methodology and methods for the sake of researching, investigating and evaluating the research area. I have got some set of research problems and classifications. According to explanatory research action I have set some aims to achieve. As a next step collected a pool of information required, organized the required out of it, analysed information and evaluated the literature, planning the experiment in all possible ways to detect more threats even in a busy traffic network. Now it is an important time to start my experiment before that I have to do some qualitative research by conducting interviews about Suricata and Bro because I need some assistance on suricata and bro to take a advantage of it. I am not interested on survey because as they are new applications people might know less about it and I thing its waste of doing. Case study and field study are also better to do because they can have depth look at issue or problem. But problem with field study is they may consume more time and they are very expensive. Quantitation method will be used analysing some numerical values, graphs and proportions. Experiment design can be categorized by certain criteria Controlled experiment, Cross-sectional designs, Quasi experimental designs and Pre experimental designs Methodologies discussed in the literature review are from user view so I might vulnerable to attack and have plan well for the implementation of experiment. These vulnerabilities can be fixed face to face interviews with security professionals and can also do by narrowing hypothesis. After the experiment the observations and analysis must be tested with hypothesis of proposed theory. Finally I will use both quantitative and qualitative methods for data collection process. I have planned to continue my experiment with the same Inductive research approach. Objectives Methods Planning and implementation of IDS Literature review, research papers and interviews Detection process Literature review, case study and research papers Network maintenance, proactive administration and security Management Literature review, white papers, blogs, case studies Signature and Protocol tuning Interviews, updates from, on-going researchs and literature reviews Implementing of security management tools Interviews, case studies and some more qualitative approaches Budget: Issues of access and ethics: Potential outcomes: Expected Impact: The experiment impact would be more informative and extremely useful in the field of intrusion detection. Research will clearly show the intrusions events and blocks them even at the busy network traffic time. It may also show some new advantages because of the suricata and bro. In my opinion this research is going to detect and block all the intrusions up to date. Depending upon the qualitative approach some more methods of suricata and bros can be implement to network to get the best out of it. Conclusion: The research at first started with a study of intrusion detection and then after I have drawn some boundaries with that following objectives. During literature collection I found some other interesting tools like Suricata and Bro which are predominately better that Snort. Though they are good but I couldnt find much literature and research area with them. So finally I decided to do an experiment on IDS with a small network consisting of Snort IDS and secondarily I am planning to keep one computer with Suricate IDS and other with Bros IDS and see the difference of these three tools from another angle. If I am successful dissertation can end up like Snort vs Suricata vs Bro or else minimum I can be successful with Snort. Using the research methodology of data collection and critical evaluation the literature work is investigated and evaluated. Lastly the outcomes of the theory are assumed from the research. I have already spoken to Neil regarding my dissertation idea and selected him as my supervisor. Finally I thank Neil Richardson and Louise Webb for providing ne this opportunity.

Free Essays - Evil and Good in Othello :: Othello essays

Evil and Good in Othello Life in general is often used as a system of ways to define what kind of person you are by its end. Shakespeare takes that theory into test upon his characters in his work of the famous play Othello. Through the verbal twists and turns along with the addition of color symbolisms, the personalities of Othello, Iago, Desdemona are revealed to their fullest extents, along with their own balance of good and evil within. When this is realized by this famous Shakespearian work, the judgment of good and evil is carried out, and as a result of mass purging of emotions, neither prevails in the resolution. Othello, due to his Moorish nature but at the same time morally white and untainted, can be considered grey with the opening of the play, but possesses the potential to become either the most brilliant white or the darkest black. From the way that he is described by Iago and sometimes Brabantio, he is a dark beast lurking in the shadows, but he is as white as he can be by the Duke. Grey is a color not quite white nor black, hesitation and confusion wavering behind his eyes. This confusion is caused by his naiveté at trusting people too easily, and Iago eagerly takes this weakness to his advantage. So that when Iago manipulates Othello, Othello unknowingly gives in to the temptation, even going as far as telling Iago "I am bound to thee for ever" (III. iii. 242). Othello at this point is completely taken in with Iago's mind poisoning and willingly submits to him, yielding to his trickeries. Inevitably with a little push from Iago, Othello slowly goes down the path o f dark and pure blackness, with murder evident in mind. With Iago's tampering of his inner moralities, Othello turns black like a speeding snowball, once Iago set him on the right path. Everything else Othello had done the damage himself; Iago only suggested the notion in the most subtle of ways. Thus he sometimes "breaks out to savage madness" as Iago put it, when being put under such pressure (IV. i. 65). He is so far gone that he even has epileptic fits hearing of Desdemona's infidelity.

adolescent psychology Essays -- essays research papers

The actual definition of an adolescent psychiatrist â€Å"is a Doctor of Medicine or Doctor of Osteopathy who specializes in the diagnosis and, if indicated, the treatment of disorders of thinking, feeling, and/or behavior affecting children, adolescents, and their families.†   Ã‚  Ã‚  Ã‚  Ã‚  For someone to become an adolescent psychiatrist it takes on average nine to ten years of special training and schooling. It requires graduating from high school, then going to college and getting a bachelors degree in either art or science, then four years of medical school, with a year of interning, followed by three years of training at a resident level in medicine and neurology, and then two years of college dealing with training and internships dealing with children, adolescents, and their families. Even though it is not required one can take the American Board of Psychiatry and Neurology (ABPN) examination which will give one more certification and put then higher up on the professional ranking also with being certified with the ABPN examination one is expected to be able to diagnose and treat all problems within the patient.   Ã‚  Ã‚  Ã‚  Ã‚  The degrees one must have to become an adolescent psychiatrist are the following either: and Bachelors in art or science and a Medical degree. An adolescent psychiatrist also has the degree to council and diagnose adults and children as well, not just adolescents. An adolescent psychiatrist can also do family, group, and couple psychotherapy.   Ã‚  Ã‚  Ã‚  Ã‚  In America the job opportunities for an adolescent psychiatrist are excellent. In 1980 the government requested the statistics for the need of adolescents psychiatrist in America by the Graduate Medical Education National Advisory Committee and they â€Å"projected a need in 1990 for 9,000 child and adolescent psychiatrists, with a supply of only 4,100 (a ratio of 45.6%). The 1. report was even more urgent: it projected a need in the year 2010 for 32,075 child and adolescent psychiatrists, but a supply of only 3,942 (a ratio of only 12.3%).† There are many opportunities in high schools, they are needed to try and to help the students go through all the drama and stress that they may go through during their high school term. Another place is at colleges for many different reasons one reason is ... ...ids are able to get help and some guidance with whatever their problem may be.   Ã‚  Ã‚  Ã‚  Ã‚  An adolescent yearly income varies on many things such as where you work such as in a clinic where one can charge very little or nothing at all, but in a private practice one can charge up to three-hundred dollars an hour.   Ã‚  Ã‚  Ã‚  Ã‚  The average treatment time is twelve session, and one hour a session, but in some cases (that is getting more common every year) if the disorder or disorders has been going on for a long 3. period of time or the if it is confusing and complicated or if the disorder or disorders continue the sessions may be extended for a period of time.   Ã‚  Ã‚  Ã‚  Ã‚  As an adolescent psychiatrist one will learn the following: how to have a positive self esteem. Learn how to talk one on one with all ages. How to observe and examine changing roles in both males and females, and also how to â€Å"identify stressful and crisis situations for adolescents and how to manage them.† These are just some of the ways to put the abilities that an adolescent psychiatrist to a helpful use.

Macbeth and Lady Macbeth Essay

Macbeth is a story consisting of very complex, intricate and unique characters. Two of which are Macbeth and Lady Macbeth. It has been constantly debated as to who among the two of them has a stronger character. In my opinion Macbeth has a stronger character. This I will justify as I go along with my argument. Macbeth and Lady Macbeth as mentioned above have several similarities and differences between them. The most common similarity they share is that they both a fickle minded and they can both be easily manipulated into doing/committing evil acts such as murdering a king just because 3 witched predicted that they would be king queen. Next, both Macbeth and Lady Macbeth have a very commanding and authoritative personality which helps solidify the statements that either of them makes. In other words they are able to use their commanding personality to create trust and make people believe them. Lastly, they are both very loyal and charming hosts. They both impress their guests very well and are able to gather lots of praises and applaud for them no matter where they go. Lady Macbeth on the other hand has a few strong characteristics as well. She is able to manipulate Macbeths mind into doing her will easily. Next, she is very ambitions which makes her very determined to get whatever she wants. But all these so called strong characteristics in Lady Macbeth are more towards the negative side. So I think that it is not appropriate to call her as having a stronger character than Macbeth. Next I will explain why Macbeth is in my opinion the stronger character. There are many qualities that can be associated to people with strong characters but in my argument I have decided to highlight only a few major characteristics which are follows. Being courageous and honorable, thinking before acting, feeling remorseful for the bad acts he commits and lastly having courage to face the consequences of all his actions. Courage and bravery are 2 qualities which are synonymous with people of strong personalities. I believe that this is a very important quality to be reckoned with when identifying people with strong qualities because it in a way glorifies the character and gives more meaning to the word â€Å"strong†. So a good example to show this would be in the following extract from the story. For brave Macbeth–well he deserves that name– Disdaining fortune, with his brandish’d steel†¦ (Act 1 scene 1 conversation between sergeant and Duncan) We can clearly interpret from this quote that the sergeant refers to Macbeth as being a very valiant and strong warrior, he also says that Macbeth moves through the battle field fearlessly slaying all in his way. Another quality of Macbeth which makes his character stronger is his nature to think of the consequences of his actions. In the story Macbeth thinks of what would be the consequences he would have to face if he killed Duncan to become king. He finds out that firstly it is wrong to commit murder and also he finds out that if he kills Duncan he will have to live with a guilty conscience for the rest of his days. I believe that this quality is just as important as the above 2 because it shows that a â€Å"strong† character is able to think one step ahead before committing his actions, meaning that he knows whether what he is doing is right or wrong. We can see Macbeth showing this quality through the following lines. Cannot be ill, cannot be good: if ill. Why hath it given me earnest of success, Commencing in a truth? We see in thorough this quote that Macbeth is truly questioning as to whether or not the witches are right and whether or not he should believe them. Which helps solidify the point that he thinks before he acts. Another strong characteristic that Macbeth possesses is that he feels guilty for the bad actions that he commits which in our context is the killing of King Duncan and several other people. But I believe that this can also be seen as an act to courage in a way, because it takes extraordinary courage to commit murder and that too killing the King himself. Another way to view this is a positive act is that fact that Lady Macbeth forced Macbeth into committing this acting by challenging his manly hood, and it was because he wanted to keep his honour that he was forced to commit such a horrid act. On the other hand Lady Macbeth’s sole aim was to reap the benefits of this act. So I believe that Macbeth’s ability to show that he feels guilty for his actions and is willing to take an action to rectify those actions makes him a stronger character. So in conclusion I would like to state that Macbeth has a much stronger character than that of Lady Macbeth and that all the qualities that I have stated above make Macbeth a much stronger character than Lady Macbeth in all rights.