Quantitative assay of total dsDNA with PicoGreen reagent and real-time fluorescent detection.
We describe a quantitative assay of dsDNA based on real-time PCR measurement of fluorescence due to the interaction of PicoGreen dye with dsDNA. An aliquot of 1 to 5 ml of the sample is mixed with 45 ml of diluted PicoGreen reagent within an optical PCR tube. This is placed into the real-time apparatus set to read SYBR Green I dye at the end of three cycles of 94 degrees C for 30 s and 65 degrees C for 30 s. The averaged fluorescence value is converted into DNA amount using a calibration curve prepared with lambda-DNA standard. The calibration curve has a dynamic linear range from 0.20 to 50 ng and a standard deviation variability below 5.0%. In conclusion, this method allows reliable determinations on minimal amounts of DNA from biological samples and PCR products in clinical applications of molecular biology.
Blotta
Ida
I
Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy. cantafor@iss.it
Prestinaci
Francesca
F
Mirante
Sabrina
S
Cantafora
Alfredo
A
D010582:Bacteriophage lambda / Q000235:genetics; D002138:Calibration; D003199:Computer Systems; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004279:DNA, Viral / Q000032:analysis; D005091:Exons / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis*; D005470:Fluorometry / Q000379:methods* / Q000592:standards; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D011973:Receptors, LDL / Q000235:genetics; D012015:Reference Standards; D013696:Temperature
D004279:DNA, Viral; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D011933:Reagent Kits, Diagnostic; D011973:Receptors, LDL; D004247:DNA