PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR
PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR
Susan J.
Ahn
,
José
Costa
and
Janet Rettig
Emanuel*
Department of Pathology, Yale University School of Medicine,
PO Box 208023, Lauder Hall 219C, New Haven
, CT 06520-8023,
USA
Received January 2, 1996;
Revised and Accepted May 2, 1996
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 PicoGreentm (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 [mu]g/[mu]l) by diluting it 1:20 in water or buffer; a practical lower limit of
detection is 50-100 ng DNA in a 50-100 [mu]l microcuvette. The
A
260
/
A
280
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 Itm (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 laser-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 [lambda] 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.
Eco
RI (New England Biolabs, Beverly, MA) digests were performed with 5 U per sample
in a 10 [mu]l reaction mix at 37oC 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 [mu]M each primer, 50 [mu]M each dNTP, 0.02 U/[mu]l 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
A
260
/
A
280
of each sample was read against a TE blank in a Lambda-2 Spectrophotometer (Perkin Elmer Corp., Norwalk, CT), fitted with a 100 [mu]l quartz microcuvette. DNA samples were diluted 1 [mu]l into 100 [mu]l of TE. A reading of 0.020, the lower confidence level of the
instrument, represented 100 ng of DNA in the cuvette and a concentration of 100
ng/[mu]l DNA in the original sample.
Ethidium bromide was diluted to a final concentration of 0.2 [mu]g/ml and SYBR-Green I was diluted 1/10 000 for staining of agarose gels.
Electrophoresis was at 150 V for 1 h in an MPH apparatus (IBI, New Haven, CT).
An IS-1000 Digital Imager (Alpha-Innotec, San Leandro, CA) with an EB-1 or SG-3 band pass filter collected images generated by 254 nm
UV transillumination. Quantitation standards were commercially purchased
genomic DNA or a 100 bp ladder marker (Research Genetics, Huntington, AL)
calibrated for DNA content (10 ng/[mu]l per band) as well as fragment size.
REFERENCES
1 Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Ehrlich, H.A. (1988) Science239,487-491.
