PCR works by an amplification process- as each round of replication progresses, the amount of DNA increases by a factor of 2. This rapidly increases the amount of DNA in the sample. By using primers that are specific to the sequence of interest (small DNA molecules that tell an enzyme where to start replicating), only one particular DNA sequence is amplified.
- Fluorescence probes are used to determine how much relevant DNA is in the sample.
- Fluorescence intensity is proportional to the amount of the specific DNA sequence in the sample.
- Fluorescent moieties are attached to short, single-stranded DNA probes that bind to specific sequences in the DNA sample.
- Fluorescence can be turned on or off depending on whether the fluorescence probe is bound to the DNA.
Polymerase Chain Reaction (PCR) and Reverse-Transcriptase PCR (RT-PCR) are DNA-amplification methods used in a large number of markets, including
- Healthcare – disease detection, cancer genotyping
- Defense – biological warfare detection
- Forensics – crime scene analysis
- Food systems – bacterial contamination
Optical filters are crucial in PCR systems, particularly in the fluorescence detection phase. These filters ensure that only the specific wavelengths of light required for excitation and emission are transmitted, significantly enhancing the accuracy of DNA quantification.
- Custom Excitation Filters: Different light sources, such as lasers, LEDs, and broadband sources, require specific filters for optimal performance. Omega Optical provides custom excitation filters that offer precise wavelength control and minimal overlap, improving signal-to-noise ratios by over 30% compared to standard filters. This is essential for achieving accurate DNA amplification results.
- Custom Dichroic Filters: These filters reflect certain wavelengths while transmitting others, playing a crucial role in directing light within PCR systems. Omega Optical’s custom dichroics are tailored to work seamlessly with excitation and emission filters, ensuring efficient light steerage and enhanced performance.
- Custom Emission Filters: These filters are designed to block unwanted wavelengths while allowing the specific emission wavelengths to pass through. Omega Optical offers emission filters with extremely steep edges and extended blocking, tailored to the number of fluorophores in the system. This customization ensures maximal signal-to-noise ratios, which is critical for high-precision DNA analysis.
Fluorescent probes are used in PCR to detect the amount of specific DNA sequences in a sample. The fluorescence intensity is proportional to the amount of DNA, providing a clear indication of DNA amplification. Common probes include SYBR Green, which binds non-specifically to double-stranded DNA, and more specific probes like TAQman and molecular beacons, which provide fluorescence only when bound to target sequences. These probes rely on high-quality optical filters to achieve accurate detection.
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Custom solutions for PCR
Custom Excitation Filters
Custom Dichroics
Depending on the geometry of your PCR optical system, you may or may not need a dichroic filter. This special type of mirror reflects some wavelengths and transmits other wavelengths. Below are three examples of dichroics for FAM and ROX. These are designed to work with the excitation and emission filters for light steerage. They are typically only specified to steer (either reflect or transmit) in the ranges of the excitation and emission bandpass filters. Although they do contribute to the blocking of out-of-band wavelengths, this is not their primary purpose.
Custom Emission Filters
Similar to the excitation filters, the emission filter blocking range depends on whether a broadband (i.e. arc-lamp) or discrete (i.e. LED) is being used. Another factor for emission filters is how many fluorophores (channels) are in the system. The greater the number of channels, the narrower the bandpass and the steeper the edges of the bandpass need to be. The number of channels also dictates how far the blocking needs to be. If there is no emission at a particular wavelength, blocking isn't required at that wavelength for good performance.
The customer in green had 5 channels so the emission filters are narrower and steeper than in the other examples. They also block farther into the NIR for maximal signal to noise. The customer in blue, with only 2 channels, did not need extended blocking and could use slightly wider bandpasses.
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