Wednesday, January 29, 2020
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 Ã¢â¬Å"amplifiedÃ¢â¬ , 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. WHATÃ¢â¬â¢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 PCRÃ is carried out in aÃ thermal cyclerÃ with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excitedÃ fluorochrome. The thermal cycler is also able to rapidly heat and chill samples thereby taking advantage of the physicochemical properties of theÃ nucleic acidsÃ andÃ DNA polymerase. The PCR process generally consists of a series of temperature changes that are repeated 25 Ã¢â¬â 40 times, these cycles normally consist of three stages: the first, at around 95Ã Ã °C, allows the separation of the nucleic acidÃ¢â¬â¢s double chain; the second, at a temperature of around 50-60Ã Ã °C, allows the alignment of the primers with the DNA template;Ã the third at between 68 72Ã Ã °C, facilitates theÃ polymerizationÃ 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 1Ã¢â¬âAmplification 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. DenaturationÃ¢â¬âThe temperature should be appropriate to the polymerase chosen (usually 95Ã °C). The denaturation time can be increased if template GC content is high. 2. AnnealingÃ¢â¬âUse appropriate temperatures based on the calculated melting temperature (Tm) of the primers (5Ã °C below the Tm of the primer). 3. ExtensionÃ¢â¬âAt 70Ã¢â¬â72Ã °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 60Ã °C as the temperature. BASICS OF REAL TIME PCR Baseline Ã¢â¬â The baseline phase contains all the amplification that is below the level of detection of the real time instrument. Threshold Ã¢â¬â where the threshold and the amplification plot intersect defines CT. Can be set manually/automatically CT Ã¢â¬â (cycle threshold) the cycle number where the fluorescence passes the threshold Rn Ã¢â¬â (Rn-baseline) NTC Ã¢â¬â 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 Ã¢â¬Å"hot-startÃ¢â¬ 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 Ã¢â¬Å"virtual backgroundÃ¢â¬ . 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: Ã¢â¬ ¢ Four interchangeable block formats Ã¢â¬ ¢ Optional Automation Accessory amp; Barcode Scanner Ã¢â¬ ¢ Argon ion laser/CCD camera Ã¢â¬ ¢ Easy to Use Software, Multiple Applications Ã¢â¬ ¢ Set up Wizards Ã¢â¬ ¢ QC Filtering/Flag System Ã¢â¬ ¢ Flexible data reports amp; exporting SOFTWARES FOR DATA ANALYSIS AND PRIMER DESIGNING 1 ) Light CyclerÃ ® Relative Quantification Software The first commercially available software was the Light CyclerÃ ® 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 OligoPerfectÃ¢â ¢, 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 5Ã¢â¬â¢ primer 0. 5 ? M 3Ã¢â¬â¢ 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: * ThermostabilityÃ¢â¬â thermostable RTs function at the higher end of (or above) this range and allow for successful reverse transcription of GC-rich regions. * RNase H activityÃ¢â¬â 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 90Ã¢â¬â110% for assay validation. This efficiency range corresponds to standard curve slopes of Ã¢â¬â3. 6 to Ã¢â¬â3. 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 Ã¢â¬ ¢ Low cost assay Ã¢â¬ ¢ Easy design and set up SYBR Green I disadvantages Ã¢â¬ ¢ Non specific system Ã¢â¬ ¢ Not adapted to multiplex Ã¢â¬ ¢ 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 5Ã¢â¬â¢ end of the probe and a quencher to the 3Ã¢â¬â¢ 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 (25Ã¢â¬â40 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 15Ã¢â¬â30 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 3Ã¢â¬â¢ label of FAM, whilst the other has a 5Ã¢â¬â¢ 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 94Ã °C to denature the DNA. 2. Cooling the sample to 60Ã °C. 3. Slowly heating (by increasing the temperature 0. 2Ã °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 94Ã °C to denature the DNA, followed by cooling to 60Ã °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 Ã¢â¬Å"housekeepingÃ¢â¬ 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, weÃ¢â¬â¢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 ,Ã¢â¬â¢ polÃ¢â¬â¢Ã¢â¬â¢ 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 Ã¢â¬Å"in houseÃ¢â¬ 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
Tuesday, January 21, 2020
Adam Sandler is in control -- really The clout of a child-man Monday, April 14, 2003 Posted: 1:07 PM EDT (1707 GMT) Adam Sandler accepts a Kids' Choice Award for best movie actor. Many adult movie critics would disagree. -------------------------------------------------------------------------------- Story Tools -------------------------------------------------------------------------------- RELATED Ã¢â¬ ¢ Review: Jack's back in 'Anger Management' Ã¢â¬ ¢ 'Anger' tops box office with $44.5M LOS ANGELES, California (AP) -- Adam Sandler, who's become one of Hollywood's top box-office draws by playing infantile men prone to fisticuffs and tantrums, displays in real life a shambling politeness and faux-naive modesty. With a shy smile, he often looks down as he speaks, while deflecting questions with self-deprecating jokes. The 36-year-old comic's almost childlike behavior conceals his status as a Hollywood workaholic who's unwaveringly loyal to his friends. "I was in his office 12 hours a day, and he was either working with me, or working on editing something else, or working on one of his other projects," said David Dorfman, screenwriter of "Anger Management," which co-stars Sandler and Jack Nicholson. "He worked nonstop." Sandler has used his clout not only to gain greater control over his own films, but also to boost the careers of former "Saturday Night Live" friends such as Rob Schneider, David Spade and Dana Carvey by guiding their pet projects through the studio system. Dorfman described Sandler as "a benevolent mogul," and Carvey said his 2002 film, "The Master of Disguise," would have gone nowhere without Sandler's help. "I mean, I owe him. I don't really know why he did it, you'd have to ask him. But it was great to have him push it through. I guess I was nice to him on 'Saturday Night Live,' " Carvey said, adding with a laugh: "Thank God." Critical roasting But some Sandler favors backfire. He gave directing duties of "The Master of Disguise" to his longtime production designer Perry Andelin Blake, although Blake had no previous experience. Critics complained that the film was a mess, and Sony was still reworking the movie shortly before its release last summer. Carvey had already starred on the late-night comedy show for five years when Sandler began appearing in 1991, the same year as Spade, whose 2001 film, "Joe Dirt," and upcoming "Dickie Roberts: Former Child Star" were produced by Sandler. Sandler co-stars with Jack Nicholson in "Anger Management." During his five-year run on the show, Sandler was known for impish characters such as Canteen Boy and musical bits such as Operaman.
Sunday, January 12, 2020
A global society is marked by new technologically integrated local and international relations which encourage the study of the effects of globalisation and as well as internationalisation of various disciplines in a society which are involved in social interactions. This interactions may be locally based or may involve international relations some of which have a close relationship to political philosophy, international economies, conflict analysis, international laws and policies as well as sociology. Globalisation makes the world more productive and stable. In the past few decades, the global economy has become increasingly interconnected and integrated. This integration is quite evident in the international markets and it has been on a steady increase since the seventeenth century although in the last few years, globalisation has undergone revolution which has given rise to many controversial issues regarding its effects and impacts on the larger global society (Lindert, Williamson 2001). Some researches have indicated that in the last two decades or so, the increased integration in the global markets has resulted in greater inequality in the way income is distributed among nations around the world. In connection to this, Bourguignon and Morrisson (2000) have also observed that combining inequality within and across countries has brought a significant rise in the global inequality since 1960 thus making the already poor countries poorer while the rich countries get richer through globalisation. Globalisation refers to the way in which nations increasingly become interconnected with one another in terms of economic, political, technological, social and cultural aspects. More often than not, the word globalisation is used to refer to the economic aspect of a nation. Economic globalisation describes the integration of national economies into the international economies via trade, migrations, international market capital flows, foreign investments and increased use of technology. The increasing rate of globalisation as has been experienced in the past two decades or so has led to rapid growth of global trade, financial capital flows, direct foreign investments and cross border trade transactions (Johnston, Taylor, Watts 2002). The major facilitators of this growth include improved transport and communication infrastructure, improved technological know-how, quicker methods of telecommunication and internet services. In addition to this, globalisation has been favoured by removal of trade barriers and policies across nations which previously inhibited international trade among nations. This has now liberalised trade and encouraged more export and import transactions which promote globalisation. The establishment of international trade organisations such as world bank, IMF and WTO have also played a significant role in facilitating globalisation as they are devoted to promoting trade and investment across nations worldwide. The result of this kind of globalised trade is presence of multinational companies (MNCs) whose budgets exceed those of the economies of many nations in which they are situated (Atkison 1999). Globalisation and sustainability. In the recent years, many controversies have emanated from the issue of globalisation. It is now seen an unnecessary evil which is threatening the social and environmental sustainability of the global societies. Globalisation is viewed by some people as an opportunity for national and international economic growth while others feel that it is a threat to economic prosperity, political sovereignty and cultural integrity (Jussilla, Cullen 2002). People in developed countries are particularly concerned with the fact that globalisation poses a threat to unskilled workers who are bound to be left jobless with the increased technological methods of production in the contracting industries. On the other hand, the developing nations are mostly concerned about the loss of political powers and sovereignty as well as loss of economic prosperity which is bound to be brought about by globalisation. According to Bhalla (2002) the whole concept of globalisation is surrounded by ideological issues both positive and negative which in the long run affect the people or societies in the globalised world. , the controversial aspect of globalisation can not be easily resolved. Globalisation and environmental sustainability. Environmental sustainability refers to the aspect of addressing the needs of the current global societies fully while being cautious not to compromise the needs of the societies which are to come,that is, future generations. According Heinberg (2005), globalisation has directly or indirectly led to several environmental issues such as global warming, increased deforestation, depletion of the ozone layer, destruction of water catchment areas, biodiversity, most natural resources have reached or are nearing depletion levels, pollution of water, air and the entire environment among others. For instance, globalisation has enabled MNCs to invest in countries which have few or no environmental conservation by-laws and this results in high environmental degradation as well as depletion of natural resources. However, the WTO in response to this effect has argued that the large amounts of income or capital flows earned from globalisation are capable of catering for the environmental degradation and this is bound to improve the quality of the environment to even higher standards that it were in before. In addition, globalisation has led to increased industrialisation all over the globe and this has resulted in the much dreaded effects of depletion of the ozone layer and global warming. Global warming comes as a result of release of industrial pollutants from the manufacturing industries into the environment. Such pollutants include carbon monoxide, carbon dioxide, oxides of sulphur and so forth. The effects of global warming are quite evident all over the globe as they have led to diverse changes in weather and climate resulting in drought and famines in some parts of the world among other effects. The depletion of the ozone layer occurs as a result of release of chemicals such as freons and fluorocarbons in to the air which react with ozone layer hence thinning it (Bellow 2001). However,there are several positive effects of globalisation in relation to environmental sustainability. Precisely, globalisation has led to improved methods of energy utilisation, substitution of resources especially those occurring naturally, innovation of metal recovery and material recycling methods, dematerialisation of resources among other positive practices (Eiezen, Maxine 2006). Globalisation has particularly been putting great emphases on dematerialisation and this has helped industries to improve their efficiency in production, minimise and manage their waste production and reduced the production costs through use of cheap systematic procedures. In addition, the use of improved technological methods has made companies to adopt newer and more efficient methods of energy utilisation and as a result, the energy requirements in industrialized countries have substantially reduced in the recent years. Moreover, many non-renewable energy resources have been substituted with renewable sources of energy and this has greatly reduced the risk of depletion of such resources in the near future. Although the improved technology has great positive effects on the environmental sustainability, globalisation is negatively associated with the exportation and use of technologies and activities that can have detrimental effects on the environment. All these environmental effects of globalisation have either a direct or an indirect impact on the global societies. Lack of clean water for instance is a negative effect which is likely to cause diseases and suffering to the larger population. On the other hand, improved energy efficiency, advanced technology and industrialisation translates a greater advantage to the people living in the globalised world in terms of better quality consumer products, free exchange of goods, capital and services between nations among others. Globalisation and social sustainability. On the aspect of social sustainability, globalisation plays a very important role in terms of improving or lowering the social status of the people living in globalised world. Experts have argued that globalisation tends to impose a greater disadvantage to the poor countries while at the same time it favours the already developed nations (Bellow 2001). They add that although globalisation is believed to favour free trade among nations, it lead to much inequality between the developed and the under developed countries since the developing countries are unable to compete favourably with the developed countries in the international markets (Agyeman, Bullar, Evans 2003). In addition, the increased use of technology is bound to render many people jobless especially the unskilled workers, since most of the work which was previously done manually by the people is now easily done by machines. This reflects negatively on the society especially in the poorer countries since when people do not get jobs to do, they are likely to suffer from starvation, poor health, illiteracy among other social problems (Walker 2005). On the positive side, globalisation allows free movement of people across nations as they trade and this has promoted cultural diversity, intermarriages, tourism and so forth. This has further promoted peace,understanding and unity between individuals as well as nations. It is thus clear that globalisation has some critical impacts on the social lives of individuals an groups living in the industrialised countries. Identities and powers based on globalisation and sustainability. The last one decade or so has presented a turbulent phase for the global societies marked by globalisation and resurgence in the identity politics or religion based politics. Globalisation being a process of ongoing capitalism-industrialisation has had some adverse effects on the poor strata of the society. According to Heiberg (2005), the nature of globalisation is contradictory in that it divides the world as much as it unites it. Movements fundamental to globalisation are based on empathy, ideology and identities. The lives of people living in global societies are largely shaped by conflicting trends of globalisation and identity and current events in the world indicate that community identities are threatened by the forces of globalisation. One effect of globalisation on the societal political structure is the emergence of socialist states which came into being in the mid-20th century ushering in the beginning of an industrial society. The intensified rate of globalisation has led to a rise of America as a lone super-power in the world, decline of the power and authority exercised by the United Nations, decline in the amount of Non-Aligned movement, a rise in Fascist-fundamentalist politics among countries and worsening of the problems experienced by the poor in the society. While globalisation is believed to result in a democratic world economy, the political democracy system in the world is stifled in form of emasculation of the potential world government. As the national economies become more and more globalised, the world political powers become more and more centralised into one or a few nations of the world. This power monopoly is a negative effect of globalisation as it leads to a decline in democratic norms and projection of identity politics. Identity politics involve competition over scarce resources which could in principle be taken to mean struggle for political power or economic wealth resources. This kind of competition seems to favour only the rich and mighty residing in the wealthy and powerful countries leading to a form of discrimination. As a result, globalisation makes the people in rich countries continue becoming richer as those in poor countries become more poor by the day. In this case it is clear that although globalisation is capable of empowering a nation both economically and politically, the power distribution is unequal and this puts some nations at a higher political power advantage than others. Conclusion. Beyond doubt, globalisation has led to significant increase in per capita income of different nations which have fully accepted this process and applied effectively the technological advancements which come with it. It can however be seen that, globalisation has pushed most developing or Third world countries along the path of diminishing sustainability and this has not in any way been improved by the recent regulations made in the globalisation processes. For instance, the deregulation of global trade in the recent past has led to a decrease in the environmental and social-cultural constraints associated with globalisation but this kind of deregulation has failed to address the issues of sustainability. In conclusion, it is clear that the current globalisation process is quite unsustainable but with a few policies to govern it properly, the process is capable of bringing much success in the long run to all nations world wide as well as to people as individuals in the globalised world. Reference. Atkinson, G., Dubourg, R. , Hamilton, K. , Munasinghe, M. , Pearce D. , 1999. Measuring sustainable development. UK: Edward Elgar Publishers. Agyeman, J. , Bullard, R. D. , Evans, B. , (eds. ). 2003. Sustainability: Development in an Unequal World, London: Earthscan. Bello, W. F. 2001. The Future in the Balance: Essays on Globalization and Resistance. Oakland, Calif: Food First Books. Bhalla, A. S. 2002. Globalisation and Sustainable Development: A Southern African PerspectiveÃ¢â¬ , International Journal of Technology Management & Sustainable Development, vol. 1, no. 1, pp. 40-57.
Saturday, January 4, 2020
The Emancipation Proclamation was a document signed into law by President Abraham Lincoln on January 1, 1863, freeing the slaves then held in the states in rebellion to the United States. The signing of the Emancipation Proclamation did not free a great many slaves in a practical sense, as it couldnt be enforced in areas beyond the control of Union troops. However, it signaled an important clarification of the federal governments policy toward slaves, which had been evolving since the outbreak of the Civil War. And, of course, by issuing the Emancipation Proclamation, Lincoln clarified a position which had become contentious during the first year of the war. When he had run for president in 1860, the position of the Republican Party was that it was against the spread of slavery to new states and territories. And when the slave states of the South refused to accept the results of the election and triggered the secession crisis and the war, Lincolns position on slavery seemed confused to many Americans. Would the war free the slaves? Horace Greeley, the prominent editor of the New York Tribune, publicly challenged Lincoln on that issue in August 1862, when the war had been going on for more than a year. Background of the Emancipation Proclamation When the war began in the spring of 1861, the declared purpose of President Abraham Lincoln was to hold together the Union, which had been split by the secession crisis. The stated purpose of the war, at that juncture, was not to end slavery. However, events in the summer of 1861 made a policy about slavery necessary. As Union forces moved into territory in the South, slaves would escape and make their way to the Union lines. The Union general Benjamin Butler improvised a policy, terming the fugitive slaves Ã¢â¬Å"contrabandsÃ¢â¬ and often putting them to work within the Union camps as laborers and camp hands. In late 1861 and early 1862 the U.S. Congress passed laws dictating what the status of the fugitive slaves should be, and in June 1862 the Congress abolished slavery in the western territories (which was remarkable considering the controversy in Ã¢â¬Å"Bleeding KansasÃ¢â¬ less than a decade earlier). Slavery was also abolished in the District of Columbia. Abraham Lincoln had always been opposed to slavery, and his political rise had been based on his opposition to the spread of slavery. He hadÃ expressed that position in the Lincoln-Douglas Debates of 1858 and at his speech at Cooper Union in New York City in early 1860. In the summer of 1862, in the White House, Lincoln was contemplating a declaration that would free the slaves. And it seemed that the nation demanded some sort of clarity on the issue. The Timing of the Emancipation Proclamation Lincoln felt that if the Union army secured a victory on the battlefield, he could issue such a proclamation. And the epic Battle of Antietam gave him the opportunity. On September 22, 1862, five days after Antietam, Lincoln announced a preliminary Emancipation Proclamation. The final Emancipation Proclamation was signed and issued on January 1, 1863. The Emancipation Proclamation Did Not Immediately Free Many Slaves As was often the case, Lincoln had been faced with very complicated political considerations. There were border states where slavery was legal, but which were supporting the Union. And Lincoln did not want to drive them into the arms of the Confederacy. So the border states (Delaware, Maryland, Kentucky, and Missouri, and the western part of Virginia, which was soon to become the state of West Virginia) were exempted. And as a practical matter, the slaves in the Confederacy were not free until the Union Army took possession of a region. What would typically happen during the later years of the war was that at the Union troops advanced, slaves would essentially free themselves and make their way toward the Union lines. The Emancipation Proclamation was issued as part of the presidentÃ¢â¬â¢s role as commander-in-chief during wartime, and was not a law in the sense of being passed by the U.S. Congress. The spirit of the Emancipation Proclamation was fully enacted into law by the ratification of the 13th Amendment to the U.S Constitution in December 1865.