Universal SYBR Green Quantitative PCR Protocol. Technology Overview SYBR based Quantitative PCRWith the development of thermal cyclers incorporating fluorescent detection, PCR has a new, innovative application. In routine PCR, the critical result is the final quantity of amplicon generated after the process. Real time or Quantitative PCR and RT PCR use the linearity of DNA amplification to determine absolute or relative amounts of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation. In quantitative PCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log linear phase of amplification, the amount of fluorescence increases above the background. Volume 11, March 2011. Your 1 source for the helicopter marketplace. R22R44R66 Sales and Service Center, Simplex R44 Spray System. Back in 2013 Adobe moved its most valuable software, including Photoshop, Illustrator, and Premiere, to a subscription only model. If you want to use the industry. The point at which the fluorescence becomes measurable is called the Quantification Cycle Cq or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against Cq. The amount of DNA or c. DNA in an unknown sample can then be calculated from its Cq value. A The different phases of the reaction Baseline The initial concentration of template is low therefore, the fluorescence intensity is too low to be detected and only the background signal is evident. Exponential After the target yield has reached the detection threshold, shown as the red threshold line, the course of the reaction can be followed through the exponential phase. Linear As the concentration of template increases, the available DNA polymerase concentration reduces and the reaction rate decreases. Plateau There is insufficient free enzyme to continue amplification and so after this point, the reaction is at the maximum yield, or the plateau phase. B Individual reactions are characterized by the cycle at which fluorescence first rises above the threshold, which is referred to as the Quantification Cycle Cq. If the starting material is abundant, amplification is observed in earlier cycles, and the Cq is lower. If the starting material is scarce, amplification is observed in later cycles, and the Cq is higher. This correlation between fluorescence, Cq, and amount of amplified product enables quantification of the template over a wide dynamic range. Real time PCR also lends itself to relative studies. A reaction may be performed using primers unique to each region to be amplified and tagged with different fluorescent dyes. Several commercially available quantitative thermal cyclers include multiple detection channels. In this multiplex system, the amount of target DNAc. DNA can be compared to the amount of a housekeeping sequence e. GAPDH or actin. SYBR Green q. PCR Applications Mass Screening. XXMicroarray Validation. XXMultiple target genesfew samples. XSNP detection. NRAllelic discrimination. NRPathogen detection. XMultiplexing. NRViral load quantification. NRGene expression analysis. XGene copy determination. NREnd point genotyping. NRin vitro quantification. NRNR not recommended. X recommended. XX preferred method. For additional information, please see the q. PCR Technical Guide or SYBR Green I based q. PCR technical animation. Assay Considerations. DNA Preparation. The single most important step in assuring success with PCR is high quality DNA preparation. Integrity and purity of DNA template is essential. Quantitative PCR involves multiple rounds of enzymatic reactions and is therefore more sensitive to impurities such as proteins, phenolchloroform, salts, EDTA, and other chemical solvents. Contaminants can also interfere with fluorescence detection. The ratio of absorbance values at 2. DNA purity. Pure DNA has an A2. A2. 80 ratio of 1. Lower ratios indicate the presence of contaminants such as proteins. Template. Very few copies of target nucleic acid equivalent to about 1. DNA or c. DNA are needed to initiate q. PCR. To minimize contamination with reaction inhibitors, the starting template amount should be kept to the minimum required to achieve accurate quantification. When the starting material is RNA, primer design and DNase I treatment will reduce signals that may be generated from g. DNA contamination. Primer Design. Whether using a ds. DNA binding dye or a probe based detection chemistry, designing high quality primers is one of the most crucial pre experimental steps in q. PCR. Specific primers for PCR should be designed with the aid of primer design software to eliminate the complications introduced with primer dimers and secondary structures. Lower primer concentrations decrease the accumulation of primer dimer formation and nonspecific product formation, which is critical in using SYBR Green I dye in quantitative PCR. NTPs. Standard PCRq. PCR mastermixes contain d. ATP, d. CTP, d. GTP, and d. TTP. However, some mixes are available that replace d. TTP with d. UTP. Products from previous reactions run with d. UTP will contain uracil instead of thymine. These are then susceptible to cleavage by Uracil DNA Glycosylase UNG. Therefore, prior incubation of subsequent reactions with UNG prevents carry over contamination between reactions. To be effective, all reactions in the laboratory must use d. UTP. Magnesium Concentration. Magnesium chloride Mg. Cl. 2 is necessary for reverse transcriptase, Taq DNA polymerase, and Taq DNA 5 to 3 exonuclease activity. Optimum Mg. 2 concentrations for reactions containing DLP are usually between 3 6 m. M. Lower magnesium chloride concentrations usually result in the formation of fewer nonspecific products. Some Ready. Mix solutions are provided at a 2. X concentration of 7 m. Guitar Amplifier Software For Pc. M magnesium chloride final concentration 3. M. In some cases, a vial of a 2. M magnesium chloride solution is provided for further optimization of the final magnesium chloride concentration if necessary. A reaction mix that does not contain Mg. Cl. 2 may sometimes be required so that a low concentration can be used, e. Scorpion Probe detection. Reverse Transcriptase. A reverse transcriptase enzyme that provides high yields of c. DNA, while retaining activity at high temperature, is critical to the success of RT q. PCR. Performance at high temperatures helps to ensure that regions of RNA with significant secondary structure are destabilized and accessible for hybridization and subsequent amplification. When performing one step RT q. PCR, high temperature performance allows the use of gene specific primers with high melting temperatures Tm, which increases reaction specificity. When performing two step protocols, it is important to ensure that the enzyme results in a linear and proportional yield of c. DNA from RNA. Minimizing pipetting can decrease variability. Some Ready. Mixes contain primers and other reagents needed to perform RT, for example, Ready. Script c. DNA Synthesis Mix RDRT. Taq DNA Polymerase. As with selecting the most appropriate reverse transcriptase for the RT, selection of the appropriate enzyme is vital. A fundamental problem with natural Taq DNA polymerase is that the enzyme has residual activity at low temperature. Non specific primer binding leads to non specific product formation as a result of this residual polymerase activity. Antibody blocked or chemically blocked Taq DNA polymerases hot start help to rectify this situation by preventing enzyme activity until the high temperature, denaturation step begins. Refer to the PCR Mix Selection Guide to define the best hot start polymerase for your application. Internal Reference Dye by Instrument Type.