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

Efficiency of Window Systems

Efficiency of Window Systems Review of Window Shopping: A Study of Desktop Window Switching. ORIGINAL ARTICLE: Warr, A., Chi, Ed H. , Harris, H., Kuscher, A., Chen, J., Flack, R. and Jitkoff. N. (2016) Window Shopping: A Study of Desktop Window Switching. In Proceedings of the 2016 CHI Conference on Human Factors in Computing Systems (CHI 16). ACM, New York, NY, USA. pp 3335-3338. ABSTRACT This is a review of a research article by Warr et al (2016), of Google Inc, Window shopping: A Study of Desktop Window Switching, which is an investigation into the efficiency of window systems. Having multiple windows open is commonplace but users often have problems switching between windows. This report presents an experiment used to evaluate window switching interfaces that are being considered for a new operating system. Its focus is an understanding of the effects of switching on user performances in terms of time to switch and errors in selection. The experiment compares three different styles of window switching interfaces and their effects on user performances. The interfaces chosen were the Cards interface, the Exposà © interface and the Mosaic interface. The results from the experiment suggest that the Mosaic interface allows faster selection than the Cards interface and is less erroneous than the Exposà © interface. SUMMARY OF ARTICLE The report outlines some windows research since the 1980s to the present time. Early research (Bly el al. 1986) showed that even though tiled window systems were faster than overlapping windows, users still preferred overlapping windows. Their conclusion being that it is imperative to create window systems that are both efficient and meet their user preferences. Research by Card and Henderson (1987) argued for designs that reduce the time and cognitive load of switching tasks. Hutchings et als (2004) study showed that users often have 8 or more windows open at any time with a median activation time of 3.8 seconds. Other research looked at arranging and grouping windows. These research works inspired Warr et al to investigate three window switching interfaces. The chosen interfaces were The Cards Interface: These resemble playing cards with small conceptual representation of the window and small amounts of information. They are spread like a stack of cards with the most recent window showing at the front. The Exposà © interface: This arranges the windows in a map, based on their size and original position. The Mosaic Interface: In this interface the windows are paced in order of most recent left to right in a grid form with the windows scaled to size of grid cell. The report details the design and procedures used in the experiment. They used a 3X3 mixed factorial design for the experiment.   This being the three user interface types by three different number of windows open at a time namely 3, 6 and 9 windows. The experiment was carried out to prove two hypothesises about window switching. Hypothesis 1: That the Mosaic interface allows windows to be selected faster than the Cards and Exposà © interfaces Hypothesis 2: The Mosaic interface would have less errors in selection than the Cards and Exposà © interfaces. The experiment was carried out in a lab using 42 participants (27 female and 15 male) ranging in age from 18-60. The participants were volunteers and were selected on the basis that they used laptops as their main computing device, hadnt used any of the selected interfaces previously and on average had more than one window open at any one time. They developed an application which prototyped the interfaces and allowed them to control and gather the data. Trials were carried out using each of the interfaces and selected number of windows. The participant were instructed to switch to predefined windows and the time taken to select the correct window and any errors in selections were recorded. The details of the conditions used are defined. The article displays the results for selection time and errors and shows them in table and graphical format for each interface and number of windows. Using a number of statistical tests the results allowed for partial acceptance of both hypothesis. They suggested that the Mosaic interface allows quicker window selection than the Cards interface that the Mosaic interface has fewer selection errors than the Exposà © interface. In the discussion they imply that the Mosaic interface was faster because the need to scroll in the card selection made it inefficient especially as the number of windows increases. Also in the Exposà © interface as the number of open windows increases the size of the thumbnails in the Exposà © interface decreases making them more difficult to distinguish. There wasnt much difference in number of erroneous selections between the interfaces except the Exposà © interface at the greater number of open windows. They surmise that was due to the fact the thumbnails were small and hard to distinguish. The limitations applied in conducting the experiment makes this a worst case scenario. They conclude that the Mosaic interface enables fast, less error prone window selection. DISCUSSION The article is well laid out. It summarises some similar research previously carried out in window switching although they say that this is the first comparative study of these specific window overview schemes (p 3336). Their reference material is fairly old. The most recent being a study of their own presented at the CHI conference in 2011. The aim of the experiment, investigating the efficiency of three window interfaces, and the reason for it are well defined. It presents the experiment in distinct sections outlining the design of the experiment, how the participants were chosen, the materials used, the procedures, their interpretation of the results and conclusion. They conclude that the Mosaic interface is the faster and the least error prone of the three chosen interfaces. This is internally valid as it was in a controlled environment however how transferable this is in the real world where prior knowledge and familiarity may affect the results is not measured. It also ignores user preferences that Bly et al considered important. There is no measure of the user experience in this experiment. Purposive sampling of participants appears to have been used. They did not include a group with prior knowledge of the interfaces and were careful throughout the experiment to alleviate any learning effects. This may have been for quick and economic reasons even though participants received a gift card. The sample size may be small because of the criteria used to select the participants and there is no explanation as to why there are almost twice as many women. The data collected from this group cant really be used to generalise about the interfaces. The results may be biased towards non experienced users. They could however be used as a control reference for any future research to externally validate these interfaces with other users. The authors of the article are affiliated to Google inc and Warr, Chi and Harris have published many articles in areas relating to computer interface design. Their previous works has been cited many times. This paper was presented to the CHI 16 Conference on Human Factors in Computing Systems. This is a top conference for HCI where the future of how people interact with technology is discussed. It is a conference for top researchers, designers and corporations. This was an ideal place to present this research paper and may prompt further research in this area. A good knowledge of statistics is required to understand the results as presented in the article. The researchers appear to be confident in their methods and choice of statistical tests but without a knowledge of statistics it is difficult to know whether their choice of statistical tests are biased. In their discussion they explore the reasons why the Mosaic interface may have been faster than the Cards interface. The put forward three credible explanations for this. These factors could be explored in any future studies. They acknowledge the fact that the experiment may not reflect real life scenarios. CONCLUSION Warr, Chi, Harris, Kuscher, Chen, Flack, and Jitkoff presented a detailed experiment in window switching interfaces that were being considered for a new operating system. They suggest that the Mosaic interface allows faster selection than the Cards interface and less erroneous than the Exposà © interface from the results they obtained. The results may not transfer to the general population and does not study the ergonomics of the interfaces and ignores user preferences. However they may inspire further research in the area of window switching. BIBLIOGRAPHY Bly, S. A. and Rosenberg, J. K. 1986. A comparison of tiled and overlapping windows. Proc. CHI86, ACM Press, 101-106. Card S. K. and Henderson, A. 1987. A Multiple, Virtual-Workspace Interface to Support User Task Switching. Proc CHI87, ACM Press, 53-59. Hutchings, D. R., Smith, G., Meyers, B., Czerwinski, M., Robertson, G. 2004. Display space usage and window management operation comparisons between single monitor and multiple monitor users. Proc. AVI04, ACM Press, 32-39.

Athens And Sparta: Similarities And Differences :: essays research papers

Athens and Sparta: Similarities and Differences   Ã‚  Ã‚  Ã‚  Ã‚  One of the greatest city states of all time was the city state of Athens from which we take many of our modern ways. Their government was a full democracy in which they had an assembly was all male citizens over 25 years old, they had the council of five hundred which was chosen by lot of people over 30 and they proposed laws to the assembly. Then there was the court where there were no judges and the juries were very large, the archons which were the nine people with the most power before the assembly took it away from them.   Ã‚  Ã‚  Ã‚  Ã‚  The Athenians descended from the Ionians in the attica region. Their city was mainly located on acropolis'. They had all around education which meant everyone was entitled to education not just the rich. They were also concerned with money and they also had unproductive soil which meant they would have to find other ways to make money, so they became sea traders.   Ã‚  Ã‚  Ã‚  Ã‚  Unlike Sparta their slaves had legal rights which meant they could be freed if the owners of them thought to do so. They also had Draconian laws which were pretty tough laws and sometimes unfair. Since they had slaves they had a lot of time to pursue cultural interests and led their society forward intellectually, so they got into making dramatic plays for their enjoyment.   Ã‚  Ã‚  Ã‚  Ã‚  There were three classes of people in Athens the first class were Citizens which were above age 19, then there were Metics which were considered the middle class. Then there were slaves which was about as low as you could go.   Ã‚  Ã‚  Ã‚  Ã‚  So all in all you will see Athens is a lot different then Sparta which I will tell you about now.   Ã‚  Ã‚  Ã‚  Ã‚  Sparta government was a lot different then Athen's they had the five ephors who were overseers and were the real rulers of Sparta. Then came the council of Elders which was made up of 28 men over 60 and they proposed laws to the assembly. Then there were kings who were elected by the assembly and served as high priests, judges, and army commanders. Then came the Assembly which were all citizens over 30 who elected officials and voted on major policies. Their government was Totalitarian.   Ã‚  Ã‚  Ã‚  Ã‚  The Spartans descended from the Dorians and were located in the Laconia region. Their city was built in a valley and had no walls because they said they could defend from an invaders. Unlike the Athenians their soil supported farming but they were culturally backward, which means we never got anything out

Althea Gibson :: Biography Biographies

Althea Gibson As we look at our past, we are better able to see the positive advances that we have made. One of the greatest obsticles that we have overcome is the barrier of race. Over the last several decades African Americans have had a huge impact on many areas of society such as politics, educatoin and most definately athletics. On August 27, 1927, Althea Gibson was born. She did not enter the world to a life of glamor. From the beginning, her life was a tough one. Her family resided in Harlem during the 1930’s and 40;s. Times were very difficult for the young girl. Her family was on welfare and she, herself was a client of the society for the Prevention of Cruelty to Children. Because of home life traumas, Althea frequently skipped school. The times that she did go, she struggled though the day. While she was growing up, she also ran away from home numerous times. As any young child would do, Althea searched for an outlet from the everyday hardships of school and home life. This is where she discovered tennis. It all began at local recreation programs where she took up table tennis, or ping pong. Althea had talent right from the beginning. She realized that she, a girl who’s life had been nothing but rough roads and abrupt turns, could be good at something. As she continued to gain interest as well as perfect her table tennis skills, Althea began to compete, and win several local tournaments. Because of these victories,Althea began to get noticed. One man, a musician by the name of Buddy Walker, saw Althea play one day and had a revolation. He figured that if Althea was so good at table tennis, then she would excel in regular tennis as well. At the time, no one realized just how beneficial this idea would be for the young Harlem girl, Althea Gibson. Through donations raised to pay for her membership and lessons, Althea became a member of an African American club called the Harlem Cosmopolitian Tennis Club. It was here that she aquired the skills needed to compete in tennis matches at a higher, more competative level. At the time that Althea was emerging as a recognized tennis player, African American’s opportunities were somewhat limitied. One organization called the American Tennis Association provided tounament opportunities for African American tennis players. In 1942, Althea Gibson had her first landmark Althea Gibson :: Biography Biographies Althea Gibson As we look at our past, we are better able to see the positive advances that we have made. One of the greatest obsticles that we have overcome is the barrier of race. Over the last several decades African Americans have had a huge impact on many areas of society such as politics, educatoin and most definately athletics. On August 27, 1927, Althea Gibson was born. She did not enter the world to a life of glamor. From the beginning, her life was a tough one. Her family resided in Harlem during the 1930’s and 40;s. Times were very difficult for the young girl. Her family was on welfare and she, herself was a client of the society for the Prevention of Cruelty to Children. Because of home life traumas, Althea frequently skipped school. The times that she did go, she struggled though the day. While she was growing up, she also ran away from home numerous times. As any young child would do, Althea searched for an outlet from the everyday hardships of school and home life. This is where she discovered tennis. It all began at local recreation programs where she took up table tennis, or ping pong. Althea had talent right from the beginning. She realized that she, a girl who’s life had been nothing but rough roads and abrupt turns, could be good at something. As she continued to gain interest as well as perfect her table tennis skills, Althea began to compete, and win several local tournaments. Because of these victories,Althea began to get noticed. One man, a musician by the name of Buddy Walker, saw Althea play one day and had a revolation. He figured that if Althea was so good at table tennis, then she would excel in regular tennis as well. At the time, no one realized just how beneficial this idea would be for the young Harlem girl, Althea Gibson. Through donations raised to pay for her membership and lessons, Althea became a member of an African American club called the Harlem Cosmopolitian Tennis Club. It was here that she aquired the skills needed to compete in tennis matches at a higher, more competative level. At the time that Althea was emerging as a recognized tennis player, African American’s opportunities were somewhat limitied. One organization called the American Tennis Association provided tounament opportunities for African American tennis players. In 1942, Althea Gibson had her first landmark

How To Get To School Without Saying The F Word :: essays research papers

Ingredients: Yourself and the city of New York Please! Don’t scream too loud at the sunlight piercing your protesting eyes, torn from the throes of a trip in Rome. Monotonously say, â€Å"Yes Mom,† to the screaming she-devil dragging you out of bed. Place your feet on the cold floor and yelp. Scramble for your floppies; make sure they’re under the bed. No other place will do. You will now be forced to grope underneath the bed for this necessary accessory. Make sure your hand encounters something cold and slimy that you can’t identify. But please! DON’T SAY THE F WORD. Remember, Mom is watching. Remove your hand, wipe on tissue and slowly make your way to the bathroom. Make sure it is occupied; hopefully by your brother but if he is not available, your sister will do. If it is your father, go back to your room and wait for either sibling. Once your brother or sister is in the bathroom, bang on the door at least twice. Repeat if desired. Have them stay inside for another five minutes, pace the hallway in impatience and wish to be an only child. You may also wish to die but this is quite drastic and not necessary. Once the occupant leaves the bathroom, glare at them angrily and then stalk inside. Jump into the shower, turn the wrong knob so that cold water pours over your face. But please! DON’T SAY THE F WORD. Someone might be standing right outside. Make sure your shower is just long enough so that you have to skip breakfast. Emerge from your shower, don a towel and stare at yourself in the mirror. Complain about your looks, wallow in self-pity and begin to cry hysterically. Stop crying. Stare at yourself in the mirror, analyze your face. Say, â€Å"It could be worse.† Blame the media for your insecurities, and give yourself a pep talk. Try to brush your teeth without incident. Whistle a little, be happy. Don’t trip on the way out the bathroom. Tip-toe on the cold, hard floor to your room where your little sister lies in ambush in the corner . She will dart out screaming and scare you to death, them run shrieking down the hallway. Please! DON’T SAY THE F WORD! Her young, impressionable mind does not need the profanity. Quickly grab the first T-shirt and jeans you find in the Everest of piles that is your clothing.

Rap/Hip-Hop: The Effects on Today’s Youth Essay

Abstract Rap/Hip-Hop first became popular among African American and Latinos among youth in the streets of New York during the 1970’s. It was their way of expressing themselves in a friendly manner in the form of speech, fashion, and personal style. Rap/Hip-Hop music was a way for them to get their message out, by telling stories of their lifestyles. However, over the years Rap/Hip-Hop music has become much more popular for its explicit lyrics, reference of drugs, alcohol and sexual explicit videos. Many may ask â€Å"Could Rap/Hip-Hop music have a negative influence on today’s youth, because of its unhealthy messages†? This research paper will examine the effects of rap/hip-hop music on today’s youth and whether or not it has a negative influence. Brenda L. Williams-Capers Professor Dana Taylor English Composition II December 11, 2011 Over the year’s many researchers have conducted studies on the effects of rap/hip-hop on today’s youth. The results have been mixed; however there are indications that some forms of Rap/Hip-Hop music could have negative influence on today’s youth. This is due to the fact that youth start to adapt the negative aspects of the gangster rap, by displaying aggressive behaviors and committing crimes of violence. Rap gets a bad rap because some songs contain explicit lyrics, drugs, alcohol and sex. A study conducted by Ralph J. DiClemente (2003) indicated that teen girls who spend more than 14 hours per week watching the sex and violence depicted in the â€Å"reel† life of â€Å"gangster† rap music videos are more likely to practice these behaviors in real life. Also, this study suggested that those teens were also three times more likely to hit a teacher, over 2.5 times more likely to get arrested, twice as likely to have multiple sexual partners, and 1.5 times more likely to get a sexually transmitted disease, use drugs, or drink alcohol. This is an indication that today’s youth that listen to these songs over and over again for long period of times could have behavior and aggressive issues. Tanner (2009) indicated that there is a sense of injustice that rap is often said to involve young people with the police and courts. The study also indicates that youth who listen to rap music have a stronger connection to property crimes than the crimes of violence, and the likelihood of committing those crimes are probably increased by poverty rather than listening to rap/hip-hop music. In a radio broadcast, Ed. Gordon (2006) mentioned two studies which addressed the effect that rap music has on today’s youth. The first study conducted by The Prevention Research Center of the Pacific instituted for Research and Evaluation in Berkeley, Calif., indicated that rap songs were linked to alcohol addictions, drug use, and violence among youth. Also, a study conducted by The Center on Alcohol Marketing and Youth, Georgetown University, also suggested that youths are exposure to alcohol thought targeted marketing advertising for beer and ale by 66 percent for youth between the age of 12-20 and 81 percent market exposure to distilled spirits. Nearly two-thirds of rap songs mentioned illicit drugs and nearly half of rap songs mentioned alcohol compared to other types of music. The overexposure to alcohol was connected to cognacs and brandies, both which are linked lyrics in hip-hop and rap music, which rappers glorify and advertise in their songs. Rap music is also used to advertise clothing, sneakers, and cars. Rappers are walking billboards and today’s youth want that same swagger. Cited in The Brown University Child and Adolescent Behavior Letter (2003), a study was conducted by a group of researchers from Iowa State University and The Texas Department of Human Services who examined over 500 students who had listened to seven violent songs and eight non-violent songs. This study measured student’s aggressive thoughts and their feelings. The results were that violent songs led to more aggressive interpretation of words that also increased the speed in which the students read aggressive vs. non-aggressive works and increased the proportion of word fragments that filled in to make aggressive words. Their study also indicated that the violent songs increased violent thoughts causing youth to act out. The popularity of Rap/Hip-Hop music and Hip/Hop styles has now reached the Middle East. Iraqis youth in are now starting to imitate hip-hop lifestyles of American soldiers. Youth have now started to adapt hip-hop styles such as, dancing, wearing baggy pants, slangy English, and cutting $ signs into their close cropped hair. Tattoos are now also becoming popular among Iraqis youth. This goes against Islam’s strictures on baring their skin. Not all Iraqis welcome the culture of the American Hip/Hop style, because they feel that Iraqis youth are now beginning to reject authority and other forbidden behaviors, like engaging in forbidden love affairs. Iraqis youth feel that their eyes are now open to the outside world since living under Saddam Hussein’s dictatorship. Iraqis youth are now also starting to use the internet and are watching satellite TV, which allows them better understanding of American cultures, both positive and negative aspects. In a book written by Michael Dyson, â€Å"Reflecting Black: African-American Cultural Criticism. He addressed the question† Is Rap music really as dangerous as many critics argue:† Michael Dyson (1993) states that Rap music is a form of profound musical, cultural, and social creativity. He also stated that Rap music expresses the desire of young black people to reclaim their history. He defenses the negative impact on Rap/ Hip-hop music, which the general public claims that Rap/Hip-Hop expresses and causes violence among youth. It is in his opinion that many rappers have spoken against the violence in Rap/Hip-Hop. Rappers KRS-One spoke against black on black crimes in a song titled â€Å"Stop the Violence† and â€Å"Self Destruction†. Rapper KRS-One wanted youths to realize that not all Rap/Hip-Hop music called for violence behavior among youth. Also, rappers such as Will Smith and his partner DJ Jazzy Jeff rapped positive messages to youth about having fun in the summer time, not glorifying drugs, alcohol or sex. Some feel that rap can be negative and positive on today’s youth. Michael Dyson (1993) feels that Rap/Hip-Hop music is getting a bad rap. Could that be true, or is Rap/Hip-Hop really unhealthy for today’s youth? Over the year’s research, books and studied have been conducted to determine if violent and explicit lyrics in Rap/Hip-Hop music really causes or promotes violence in today’s youth. Since it’s beginning in the 1970’s Rap/Hip-Hop has gone from being fun to listen to now being a explicit rated albums. In today’s time Rap/Hip-Hop glorifies drugs, violence, and sex at an alarming rate. Today’s youth are also embracing the lifestyles’ of rappers in their language and attitudes, with their want of money, cars, drugs and alcohol that rappers talk about in their lyrics causing them to become materialistic teenagers. What must we do as concerned parents? We must be aware of what our children are watching and listening to. Parents must be involved in what their children are purchasing and they must ask questions to understand what their children are experiencing. In the 1990’s C. Dolores started a crusade to stop the sale of offensive rap music to minors. Mrs. Tucker outspokenness earned her enemies, however she remained relentless with her crusade against what is called gangster rap. Her concerns were that the lyrics were misogynistic and threatened the moral foundation of the African American community. Conclusion Rap/Hip-Hop music became popular in the 70’s, since than it has changed the world; youth in every corner of the globe have adopted the culture, the speech and the style. Because Rap/Hip-Hop music has become so popular, many people think that this type of music is making today’s youth more violent, due to the glorification of violent, explicit language, sex, disrespect for authority, and disrespect towards women. Just as parents teach their kids not to do drug, they should also teach their kids that rapper is only for entertainment, it is a business for rappers to sale albums to make money. It is a business for them. It is all about album sales. We should all follow Mrs. Tuckers’ example by taking a stand against anything that could have a negative effective on our youth. Rappers should realize that they are role models to most of our youth and stop thinking about their pockets. References DiClemente, R.J., Wingood, G.M., Bernhardt, J.M., Robillard, A., Harrington, K., Davies, S.L., and Hook, E.W. A Prospective Study of Exposure of Rap Music Videos and African American Female Adolescents’ Health. American Journal of Public Health. (2003). Dyson, M.E., Reflecting Black: African-American Cultural Criticism. Minneapolis, MN, USA: University of Minnesota, 1993 p.38 Gordon, E., N News, News & Notes, 8 May 2006 Tanner, J.; Asbridge, M.; & Wortley, S. (2009). â€Å"Listening to Rap: Cultures of Crime, Cultures of Resistance.† Social Forces, 88(2): 693-722. The Brown University Child and Adolescent Behavior Letter 2003 www.newblackmagizine.com

Terrorism: An Emergency?

The September 11th terrorist attack on the World Trade Center will forever be in the minds of every American. It is a reality which tells us that it can happen again, anytime and anywhere. The impact of such act also tells us that it is something the government and the nation must pay attention to and be prepared for.For many years now, terrorism is still an existent problem which gobbled up a substantial amount from the government resources. Not only that, but it also caused countless fatalities and destroyed properties. It has also put the government in an unsure position as to how it can be eliminated. The worsening situation regarding terrorism has the government taking up drastic steps for the nation’s security and considering it an emergency by which every nation must be prepared to face.The Office of the Press Secretary of the United States (2007) announced about additional grant guidance and application kits for three grant programs (Port Security Grant Program (PSGP), Transit Security Grant Program (TSGP), and Emergency Management Performance Grant (EMPG)), which totaled to $827 million this year. According to Michael Chertoff, Homeland Security Secretary, the additional influx of federal dollars will be for the enhancement of security measures. Additionally, the money will enable the emergency managers to have more tools to â€Å"build on national preparedness goals.†Allotment of these funds shows us how serious the prevention of terrorism is. The United States government will prioritize funding for training and public awareness campaigns, reducing the risks of improvised explosive devices and radiological, biological and chemical weapons, and securing transit systems. Moreover, grant funding will further improve the government’s emergency management capabilities.According to the American National Red Cross (2001), there are many things which could happen after a terrorist attack which calls for emergency action. First is that the re can be casualties and damages to properties such as buildings. Second is the involvement of the local, state and federal units due to the criminal nature of the event. Third is the possibility that the health and mental health resources can be strained or overwhelmed. Next, the prolonged existence of public fear, international implications and consequences and extensive media coverage. Fifth is the possibility of evacuation. And lastly, the clean-up which may take up very long.With regards to weapons of mass destruction (WMD), Taylor (2000) analyzed the use of WMD as protective measure against terrorism. The author cited the Nunn-Lugar-Domenici Act, which the U.S. Congress passed in 1996, and which requires the local and state governments to have access to equipment and training needed to fight against acts of terrorism. It involves access to the use of WMD such as chemical, radiological and biological. A large amount of money is funded for the program in order to train law enfor cement and emergency response agencies in dealing with terrorist attacks.Taylor (2000) added that the production of WMD might increase the number of casualties from terrorist attacks as the US population was vulnerable to such attacks. The Legionnaire’s disease alone, which struck American Legion conventioneers, tells us of the worst possible outcome of bioweapons. As this shows that the public can be attacked with these weapons, the enemies could use these for their terrorist attacks.Weapons of mass destruction include chemical, radiological and biological weapons. Their effects could be enormous. Lives will be lost with the deployment of such dangerous weapons. And as terrorists have access to these weapons and more, they can use it to further their attacks, resulting to more and more casualties. This is clearly an emergency both ways because the 40 percent of terrorist attacks around the world is targeted at the United States despite the fact that the country has no quarre ls with other countries.   The population is even more vulnerable to an attack using WMD.According to John Bolton (2002), Under Secretary of State for Arms Control and International Security, there are risks in using WMD. The worst thing that could happen is when the weapons fall into the hands of terrorists. To prevent this from happening, a strategy called the New Strategic Framework was formed. Under the framework, defensive systems which aim to protect against missile attacks will be created, nonproliferation and counterproliferation measures will be enhanced, nuclear weapons will be reduced and cooperation with Russia to eliminate terrorism will be prioritized.John Wolf, Assistant Secretary of State for Nonproliferation (2002), added that these weapons of mass destruction are a threat to the United States forces around the world. The primary concern, according to Wolf, is protection and security from WMD. This entails some steps. First, there should be reduction and cessation of WMD production. The United States’ objective is to control and dispose excess materials of WMD. The second step is stopping Iran’s acquisition of these materials.It is believed that Iran wants to improve and develop its WMD and missile programs. Third step is stopping the proliferation of nuclear and missile in and from South Asia. Wolf stated that approximately one million troops face off on the India-Pakistan border. Concerns were raised over the possibility that the WMD and missiles there might fall in the wrong hands.Several actions are needed to be taken to prevent further proliferation of WMD and missiles. One is to make sure that the suppliers of WMD materials and missiles end their cooperation. Another is to ensure that security is at its maximum against WMD and missile proliferation. The effectiveness of the export control of the states should also be enhanced. They should also secure their WMD and missiles and help other states that do not have enough res ources.Terrorism is a rare phenomenon that must be put in perspective. Everyone knows that one attack using WMD can cause massive casualties. According to Pete du Pont (cited in Taylor, 2000), 15 terrorist incidents happen each year in the United States. Statistics also showed that approximately 42 Americans die while 115 are injured from international acts of terrorism since 1982. What the United States should do, Taylor noted, is to implement military restraint overseas but respond to terrorist incidents against U.S. targets.Should there be a terrorist attack using WMD, there are three things that the government must address. First, responses of the government must minimize the injury and death and implement actions which prevent the public from harm. This means that the attack site should be isolated, the agent class is identified, exposure levels is evaluated and those who are exposed evacuated to facilities where they can be treated. The government must make sure that the indiv iduals in the attack site cooperate with it (Taylor, 2000).Second, evidence should be gathered which will be used for later prosecutions. This should be the primary concern of law enforcement officials. They should identify the attackers. Third is mitigation. How to prevent such incident from happening should be determined (Taylor, 2000).The magnitude of these terrorist attacks and the use of WMD call for every emergency measures from both the state and local levels. One problem which hinders to the success of emergency management, according to Taylor (2000), is the difficulty of getting public support. Effective emergency management program will be the solution to the program, and this requires public support. The public should be educated about what they can do in case terrorist attacks happen. Additionally, they should be taught about the WMD and what they should do in each case.Considering the effects of terrorism, and the things it can do to a country, it should be seen as an e mergency alongside other types of disasters. This paper does not posit that terrorism should be prioritized over other disasters, but the government must also allot resources and actions to prevent or be ready in case it happens.ReferencesAmerican National Red Cross. (2001). Terrorism: Preparing for the unexpected. Retrieved on December 16, 2007 from http://www.redcross.org/services/disaster/0,1082,0_589_,00.htmlBolton, John. (2002). The new strategic framework: A response to 21st century threats. U.S. Foreign Policy Agenda, 7, 2.Office of the Press Secretary. (2007). DHS announces additional $260 million in supplemental grants funding. Retrieved on December 16, 2007 from the Homeland Security web site, http://www.dhs.gov/xnews/releases/pr_1187294574562.shtmTaylor, Eric R. (2000). Are we prepared for terrorism using weapons of mass destruction? Government’s half measures. Policy Analysis, 387, 1-17.Wolf, John. (2002). U.S. approaches to nonproliferation. U.S. Foreign Policy A genda, 7, 2.