Simple - easy monitoring and validation of DNA extraction protocols
Specific - minimal interference with sample detection
Optimized - ideal for blood, urine and sputum samples
Sensitive - specially designed for real-time PCR assays
DNA Extraction Control enables users of diagnostic assays to validate their extraction step. DNA Extraction Control contains a known concentration of cells that contain the control DNA sequence. Cells containing internal control DNA are "spiked" into the lysis buffer with the sample prior to DNA extraction
Following DNA extraction, the reaction mix is added to the extracted DNA prior to amplification. All components required for amplification of sample DNA should also be added. Presence of internal control DNA confirms the success of the extraction step, and reduces the chance of obtaining a false negative result in the sample DNA.
DNA Extraction Control 560 is suitable for use with commercially available silica-membrane DNA extraction kits and CHELEX matrices and has been tested on a wide range of real-time PCR platforms including ABI-7500, LightCycler 480®, RotorGene-Q™ and MX3005P®.
DNA Extraction Control 560 uses Cal Fluor® Orange 560 and is also available with Quasar®670 or Cal Fluor® Red 610, to fit in with existing protocols. CAL Fluor and Quasar dyes are performance-optimized fluorophores for multiplex real-time PCR.
A common practice in real-time PCR is to add a known amount of “spiked” control DNA after DNA extraction. This monitors PCR inhibition but has no value as an extraction control. The ideal situation is to have the test sample and internal control undergo the same processing prior to real-time PCR (fig. 1). Bioline have specially developed a DNA Extraction Control (DEC), which more closely mimics the test sample, as compared to spike controls. Genetic material from the test sample and the DEC is simultaneously extracted by common extraction methods, with the extraction control being as sensitive to inhibition and extraction failure as the test sample.
The DEC cells are of a known concentration, containing the Internal Control DNA sequence. This sequence contains no known homology to any organism and, importantly, has minimal interference with detection of sample DNA. The DEC cells are spiked into the lysis buffer with the target sample, prior to DNA extraction. Control Mix (primers and probe) is then added to the reaction mix before amplification. Signal derived from the Internal Control DNA confirms the success of the extraction step (fig. 2). DEC also monitors co-purification of PCR inhibitors (fig. 3) that may cause biased or false amplification patterns.
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