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Real-Time PCR Handbook Single-Tube Assays

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Real-Time PCR Handbook Single-Tube Assays Real-time PCR handbook Single-tube assays 96- and 384-well plates 384-well TaqMan® Array cards OpenArray® plates The image on this cover is of an OpenArray® plate which is primarily used for mid-density real-time PCR on the QuantStudio™ 12K Flex system. The figure above shows the commonly used formats for real-time PCR. Contents Basics of real-time PCR 1 Experimental design 2 Plate preparation 3 Data analysis 4 Troubleshooting 5 Digital PCR 6 Basics of real-time PCR 1 Basics of real-time PCR 1.1 Introduction 3 1.2 Overview of real-time PCR 4 1.3 Overview of real-time PCR and 1 real-time PCR components 5 1.4 Real-time PCR analysis terminology 7 1.5 Real-time PCR fluorescence detection systems 11 1.6 Melting curve analysis 15 1.7 Use of passive reference dyes 16 1.8 Contamination prevention 17 1.9 Multiplex real-time PCR 17 1.10 Internal controls and reference genes 19 1.11 Real-time PCR instrument calibration 20 lifetechnologies.com 2 Basics of real-time PCR 1.1 Introduction The polymerase chain reaction (PCR) is one of the most quantitative information on the starting quantity of the powerful technologies in molecular biology. Using PCR, amplification target. Fluorescent reporters used in real- specific sequences within a DNA or cDNA template can be time PCR include double-stranded DNA (dsDNA)- binding copied, or “amplified”, many thousand- to a million-fold dyes, or dye molecules attached to PCR primers or probes using sequence specific oligonucleotides, heat stable DNA that hybridize with PCR product during amplification. polymerase, and thermal cycling. In traditional (endpoint) The change in fluorescence over the course of the reaction PCR, detection and quantification of the amplified is measured by an instrument that combines thermal sequence are performed at the end of the reaction after cycling with fluorescent dye scanning capability. By plotting the last PCR cycle, and involve post-PCR analysis such fluorescence against the cycle number, the real-time PCR 1 as gel electrophoresis and image analysis. In real-time instrument generates an amplification plot that represents quantitative PCR, PCR product is measured at each the accumulation of product over the duration of the entire cycle. By monitoring reactions during the exponential- PCR reaction (Figure 1). amplification phase of the reaction, users can determine the initial quantity of target with great precision. The advantages of real-time PCR include: PCR theoretically amplifies DNA exponentially, doubling • Ability to monitor the progress of the PCR the number of target molecules with each amplification reaction as it occurs in real time cycle. When it was first developed, scientists reasoned that • Ability to precisely measure the amount the number of cycles and the amount of PCR end-product of amplicon at each cycle, which allows could be used to calculate the initial quantity of genetic highly accurate quantification of the amount material by comparison with a known standard. To address of starting material in samples the need for robust quantification, the technique of real- • An increased dynamic range of detection time quantitative PCR was developed and end-point PCR • Amplification and detection occurs in a single is used mostly to amplify specific DNA for sequencing, tube, eliminating post-PCR manipulations cloning, and use in other molecular biology techniques. Over the past several years, real-time PCR has become In real-time PCR, the amount of DNA is measured after the leading tool for the detection and quantification of DNA each cycle via fluorescent dyes that yield increasing or RNA. Using these techniques, you can achieve precise fluorescent signal in direct proportion to the number detection that is accurate within a two-fold range, with a of PCR product molecules (amplicons) generated. Data dynamic range of input material covering 6 to 8 orders of collected in the exponential phase of the reaction yield magnitude. Figure 1. Relative fluorescence vs. cycle number. 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 used to create the plots are a dilution series of the target DNA sequence. 3 For Research Use Only. Not for use in diagnostic procedures. Basics of real-time PCR 1.2 Overview of real-time PCR This section provides an overview of the steps involved in Two-step qRT-PCR performing real-time PCR. Real-time PCR is a variation Two-step quantitative reverse transcriptase PCR (qRT-PCR) of the standard PCR technique that is commonly used to starts with the reverse transcription of either total RNA or quantify DNA or RNA in a sample. Using sequence-specific poly(A)+ RNA into cDNA using a reverse transcriptase (RT). primers, the number of copies of a particular DNA or RNA This first-strand cDNA synthesis reaction can be primed sequence can be determined. By measuring the amount using random primers, oligo(dT), or gene-specific primers of amplified product at each stage during the PCR cycle, (GSPs). To give an equal representation of all targets in quantification is possible. If a particular sequence (DNA or real-time PCR applications and to avoid the 3� bias of RNA) is abundant in the sample, amplification is observed oligo(dT) primers, many researchers use random primers in earlier cycles; if the sequence is scarce, amplification or a mixture of oligo(dT) and random primers. 1 is observed in later cycles. Quantification of amplified product is obtained using fluorescent probes or fluorescent The temperature used for cDNA synthesis depends on the DNA-binding dyes and real-time PCR instruments that RT enzyme chosen. Next, approximately 10% of the cDNA measure fluorescence while performing the thermal is transferred to a separate tube for the real-time PCR cycling needed for the PCR reaction. reaction. Real-time PCR steps One-step qRT-PCR There are three major steps that make up each cycle in a One-step qRT-PCR combines the first-strand cDNA real-time PCR reaction. Reactions are generally run for 40 synthesis reaction and real-time PCR reaction in the cycles. same tube, simplifying reaction setup and reducing the possibility of contamination. Gene-specific primers (GSP) 1. Denaturation: High temperature incubation is used to are required. This is because using oligo(dT) or random “melt” double-stranded DNA into single strands and primers will generate nonspecific products in the one-step loosen secondary structure in single-stranded DNA. procedure and reduce the amount of product of interest. The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be increased if template GC content is high. 2. Annealing: During annealing, complementary sequences have an opportunity to hybridize, so an appropriate temperature is used that is 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 real-time PCR is small, this step is often combined with the annealing step using 60°C as the temperature. lifetechnologies.com 4 Basics of real-time PCR 1.3 Overview of real-time PCR and real-time PCR components This section provides an overview of the major reaction Magnesium concentration components and parameters involved in real-time PCR In real-time PCR, magnesium chloride or magnesium experiments. A more detailed discussion of specific sulfate is typically used at a final concentration of 3 mM. components like reporter dyes, passive reference dyes, and This concentration works well for most targets; however, uracil DNA glycosylase (UDG) is provided in subsequent the optimal magnesium concentration may vary between sections of this handbook. 3 and 6 mM. 1 DNA polymerase Good experimental technique PCR performance is often related to the thermostable DNA Do not underestimate the importance of good laboratory polymerase, so enzyme selection is critical to success. technique. It is best to use dedicated equipment and One of the main factors affecting PCR specificity is the solutions for each stage of the reactions, from preparation fact that Taq DNA polymerase has residual activity at low of the template to post-PCR analysis. The use of aerosol- temperatures. Primers can anneal nonspecifically to barrier tips and screwcap tubes can help decrease DNA, allowing the polymerase to synthesize nonspecific cross-contamination problems. To obtain tight data from product. The problem of nonspecific products resulting replicates (ideally, triplicates), prepare a master mix that from mis-priming can be minimized by using a “hot-start” contains all the reaction components except sample. The enzyme. Using a hot-start enzyme ensures that no active use of a master mix reduces the number of pipetting steps DNA polymerase is active during reaction setup and the and, consequently, reduces the chances of cross-well initial DNA denaturation step. contamination and other pipetting errors. Reverse transcriptase Template The reverse transcriptase (RT) is as critical to the success Use 10 to 1,000 copies of template nucleic acid for each of qRT-PCR as the DNA polymerase. It is important to real-time PCR reaction. This is equivalent to approximately choose an RT that not only provides high yields of full-length 100 pg to 1 μg of genomic DNA, or cDNA generated from 1 cDNA, but also has good activity at high temperatures. pg to 100 ng of total RNA. Excess template may bring with High-temperature performance is also very important it higher contaminant levels that can greatly reduce PCR for denaturation of RNA with secondary structure.
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