Fluorescence filters for PCR applications

PCR or polymerase chain reaction has been revolutionary in the field of biomolecular sciences, allowing for the study of genesis, morphology, and structure of organisms at the smallest possible scales. Here, LASER COMPONENTS discuss the important role that optical filters play in this process

PCR is a commonly used DNA amplification method used to replicate low levels of genetic material from various sample types, ultimately replicating the target sequence over and over again. This is a valuable process in a number of applications, for example amplifying small amounts of biological matter from a crime scene.

How it works

The process begins with the isolation of single stranded DNA (ssDNA); purified double stranded DNA is heated to around 90°C to generate ssDNA by breaking down the hydrogen bonds linking the strands. Next, heat-stable DNA-polymerase (TAQ-Polymerase) is used to replicate the ssDNA chain, beginning the PCR process. Specific primers help the DNA-polymerase bind and restrict which DNA sequences are replicated. The double stranded DNA is then heated, and ssDNA is regenerated. The final step involves cooling and primer/probe binding. The ssDNA and primers or probes (described below) bind, and the cycle continues. Each ssDNA is replicated, so the number of copies increases by a factor of 2 during each round of PCR. The amount of DNA is determined with fluorescent probes.

Fluorescent probes

The fluorescence probes are used to determine reaction efficiency and provide a quantitative analysis of gene expression. This is carried out using either an intercalating dye or hydrolysis probe, which generates fluorescence during PCR under certain conditions. Intercalating dye is introduced to the reaction after the stable polymerase has primed the ssDNA, but before the double strands are replicated. An example dye would be SYBR Green. As the dye binds to the double helix of a DNA strand it fluoresces more intensely, allowing researchers to correlate brighter fluorescence signals to greater yields of DNA in real time.

Optical filters

Optical filters are a critical component in identifying and monitoring this fluorescence. LASER COMPONENTS supplies optical filters specifically designed for use in PCR, from partners Omega Optical. Excitation and emission PCR filters are engineered for extremely high transmission over a desired wavelength band, defined by the fluorophore used to probe the reaction. As there is always some spectral overlap between fluorescence probes, it is ideal to use PCR filters with a high optical density out of band, specifically at the region of crossover to achieve the best signal to noise ratio.  There are countless different analytical setups for molecular cloning today.

From conventional PCR to cutting-edge reverse transcriptase-polymerase chain reactions (RT-PCR), it is now possible to amplify viral RNA as well as DNA. This has proven incredibly useful to virologists studying the sequencing of Sars-CoV-2, otherwise known as COVID-19, in a bid to characterise the virus and to develop novel testing methods.

LASER COMPONENTS’ stock PCR filter set is suitable for any fluorescence application using common dyes. For more bespoke application we have extensive custom capabilities.

Alongside our PCR specific filters, we offer a comprehensive range of other fluorescence filter sets.  Standard sets are available for over 180 dyes and fluorophores. Long pass emission sets and narrowband sets are available for samples with multiple tags.

Fluorescence sets typically consist of an excitation filter selecting the range of excitation wavelengths, a dichroic mirror and an emission filter blocking the excitation light. The excitation filter is a band-pass which transmits the desired wavelengths and reflects or absorbs the unwanted wavelengths. The transmitted light is reflected through 45° by the dichroic mirror, directing the excitation wavelengths to the sample and allowing the emission to transmit back though. Finally, the emission filter blocks the excitation light and transmits the desired fluorescence to the detector.  These filters produced by Omega Optical are characterised by good blocking out of band and high transmission of desired wavelengths to produce a good quality image with a high signal-to-noise ratio.

Filter sets, components, and holders are available for all major microscope manufacturers and models, including Leica, Nikon, Olympus, and Zeiss.

 

 

 

 

 

 

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