Molecular Microbiology

Melting curve analysis in Real-time PCR

Melting curve analysis and detection systems Melting curve analysis can only be performed with realtime PCR detection technologies in which the fluorophore remains associated with the amplicon. Amplifications that have used SYBR® Green I or SYBR® GreenER™ dye can be subjected to melting curve analysis. Dual-labeled probe detection systems such as TaqMan® probes are not […]

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Real-time PCR probes

TaqMan® probe signal production Whether an MGB or non-MGB probe is chosen, both follow the same pattern for signal production. In the early PCR cycles, only the low, quenched reporter signal is detected. This early signal, automatically subtracted to zero in the real-time PCR software, is termed “baseline”. If the sample contains a target, eventually

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Real-time PCR fluorescence detection systems

Real-time PCR fluorescence detection systems Real-time fluorescent PCR chemistries Many real-time fluorescent PCR chemistries exist, but the most widely used are 5” nuclease assays such as TaqMan®Assays and SYBR® Green dye–based assays (Figure 1). The 5” nuclease assay is named for the 5” nuclease activityassociated with Taq DNA polymerase (Figure 2). The 5” nuclease domain

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Real-time PCR analysis technology

BaselineThe baseline of the real-time PCR reaction refers to the signal level during the initial cycles of PCR, usually cycles 3 to 15, in which there is little change in fluorescent signal. The low-level signal of the baseline can be equated to the background or the “noise” of the reaction (Figure 1). The baseline in

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Real-time PCR primer design

Good primer design is one of the most important parameters in real-time PCR. This is why many researchers choose to purchase TaqMan® Assay products—primers and probes for real-time PCR designed using a proven algorithm and trusted by scientists around the world. If you choose to design your own real-time PCR primers, keep in mind that

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REAL-TIME PCR COMPONENTS

DNA polymerasePCR performance is often related to the thermostable DNA polymerase, so enzyme selection is critical to success. 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 during reaction setup, allowing the polymerase to synthesize nonspecific product.

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STEPS INVOLVED IN PERFORMING REAL-TIME PCR

Real-time PCR is a variation of the standard PCR technique that is commonly used to quantify DNA or RNA in a sample. Using sequence-specific primers, the number of copies of a particular DNA or RNA sequence can be determined. By measuring the amount of amplified product at each stage during the PCR cycle, quantification is

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INTRODUCTION TO REAL TIME POLYMERASE CHAIN REACTION (RT PCR)

The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or complementary DNA (cDNA) template can be copied, or “amplified”, many thousand- to a million-fold using sequence-specific oligonucleotides (or primers), heat-stable DNA polymerase, and thermal cycling. In traditional (endpoint) PCR, detection and quantification

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HOW TO DESIGN PRIMERS FOR YOUR PCR EXPERIMENT

Primers are short stretches of DNA that target unique sequences of a DNA molecule and help identify a unique part of a genome or a gene to be copied further. They are usually 18 to 25 nucleotides long. Primers can be synthesized and used for your PCR (polymerase chain reaction) and other molecular biology experiment

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STEPS INVOLVED IN TRANSFORMING BACTERIAL CELLS

There are different types of steps involved in the transformation of a bacterial cell in gene cloning techniques. Bacterial cells are transformed when they are infused with exogenous DNA from another organism, and this makes them competent enough to express the gene product (or protein) of the gene they are carrying. Usually, the exogenous DNA

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