PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR

PCR has become a powerful tool for genetic analysis and many applications for gene sequence quantitation are based on this technology ( 1–3). Standardized reaction conditions require accurate quantitation of input DNA as well as optimization of chemical and cycling parameters. In this study we discuss PicoGreen (Molecular Probes, Eugene, OR) fluorescence enhancement as a useful assay for template DNA quantitation and PCR product formation. Spectrophotometry is the principal method for evaluating quantity and quality of nucleic acids. In aqueous solution, DNA has maximal absorbance near 260 nm with an extinction coefficient of 50; protein absorbs light strongly near 280 nm. The concentration of a sample can be read directly (in μg/μl) by diluting it 1:20 in water or buffer; a practical lower limit of detection is 50–100 ng DNA in a 50–100 μl microcuvette. The A260/A280 ratio provides an estimate of DNA purity; values of 1.7–2.0 predict ‘clean DNA’. However, single-stranded DNA, RNA, PCR primers and dNTPs, or aromatic organic compounds such as phenol interfere by absorbing light in this range. Fixed tissue samples with substantial protein crosslinking and DNA preparations containing added enzymes or protein stabilizers are difficult to evaluate spectrophotometrically ( 4). Intercalating fluorochromes, such as ethidium bromide or Hoechst 33258, selectively bind to dsDNA. The sensitivity of Hoechst 33258 is ∼25 ng of DNA per assay, but preferential association with domains of high A–T content or reduced binding to DNA fragments <500 bp may result in skewed analysis ( 5). Accurate evaluation may require sophisticated or dedicated equipment since both dyes photobleach easily and fluorescence enhancement of DNA binding is low, leading to high background readings. These compounds are carcinogenic and pose handling and disposal problems. Electrophoretic array is the most common means of evaluating molecular distribution of both simple and complex DNA samples. When stained with ethidium bromide, transillumination with 254 nm UV light permits CCD camera visualization of a single agarose gel band containing ∼5 ng or a polydisperse sample containing 25–50 ng of dsDNA. SYBR-Green I  (Molecular Probes, Eugene, OR) is a proprietary fluorescent dsDNA-specific stain that has an emission peak at 520 nm following excitation at 254 or 497 nm. Image collection and analysis with 254 nm transillumination requires the use of an optical quality band-pass filter to eliminate infrared interference. SYBR-Green I is more sensitive than ethidium bromide with a limit detection of ∼50 pg per band or ∼250 pg per lane polydisperse dsDNA. Argon la er-activated gel scanning or capillary electrophoresis is more sensitive ( 6), but far more costly. Gel analysis allows evaluation of genomic DNA integrity, completeness of restriction endonuclease digestion and quantity of late cycle PCR products. However, this method is impractical for routine or high throughput DNA quantitation ( 7). PicoGreen is a fluorochrome that selectively binds dsDNA and has characteristics similar to that of SYBR-Green I. It has an excitation maximum at 480 nm (lesser peaks in the short-wave UV range) and an emission peak at 520 nm. When bound to dsDNA, fluorescence enhancement of PicoGreen is exceptionally high; little background occurs since the unbound dye has virtually no fluorescence. PicoGreen is very stable to photobleaching, allowing longer exposure times and assay flexibility. However, the molecular structure of the dye is proprietary and the mode of binding is not fully characterized, so potential handling hazard is unknown. We evaluated PicoGreen for quantitation of multiple DNA sample types. We examined the linearity of binding and the effective detection range for different species of ‘high molecular weight’ DNA standards (human placental, calf thymus and λ phage; with or without restriction digestion) and DNA isolated from a variety of tissue types preserved under different protocols. We also assayed ‘low molecular weight’ dsDNA (∼150 bp PCR products) in the presence or absence of reaction primers, dNTPs and Taq polymerase. Oligonucleotide primers were evaluated for interference with quantitation in some samples. Control DNAs were from commercial sources. EcoRI (New England Biolabs, Beverly, MA) digests were performed with 5 U per sample in a 10 μl reaction mix at 37 C for 2 h. We obtained sample DNA by organic extraction from flash-frozen or paraformaldehyde-fixed paraffin-embedded surgical remainder tissues. PCR mixtures contained 0.2 μM each primer, 50 μM each dNTP, 0.02 U/ μl AmpliTaq polymerase (Perkin-Elmer Corp., Wilton, CT) and TaqStart MAb (Clontech, Palo Alto, CA). Primers were removed from PCR reactions with Microcon 30 (Amicon, Beverly, MA). The A260/A280 of each sample was read against a TE blank in a Lambda-2 Spectrophotometer (Perkin Elmer Corp., Norwalk, CT), fitted with a 100 μl quartz microcuvette. DNA samples were diluted 1 μl into 100 μl of TE. A reading of 0.020, the lower confidence level of the instrument, represented 100 ng of DNA