9. Application Note for DNA Quantification
9. Application Note for DNA
Quantitation
9.1 Introduction
Comparison of two different detection techniques for DNA quantitation:
Absorbance vs. Fluorescence
The quantitation of small
amounts of dsDNA is important
for a variety of biological
applications. These include
standard molecular biology
techniques, such as
synthesizing cDNA for library
production and purifying DNA
fragments for subcloning, as
well as diagnostic techniques,
such as quantitating DNA
amplification products and
detecting DNA molecules in
drug preparations.
9.1.1
Common techniques for the quantitation of DNA
The most commonly used technique for measuring nucleic acid concentration is
the determination of
absorbance at 260 nm (A
260
).
The major disadvantages of
the absorbance method are the relative contribution of nucleotides and single-
stranded nucleic acids to the signal, the interference caused by contaminants
commonly found in nucleic acid preparations, the inability to distinguish between
DNA and RNA and the relative insensitivity of the assay (an A
260
of 0.1
corresponds to a 5 µg/ml dsDNA solution, referring to 1 cm pathlength for
measurements in a cuvette).
Hoechst (bisbenzimide) dyes
are sensitive
fluorescent nucleic acid stains. H33258 is selective for dsDNA, but does not show
significant fluorescence enhancement in the presence of protein and allows the
detection and quantitation of DNA concentrations as low as 10 ng/ml DNA.
Cyanine dyes such as YO-PRO
TM
-1 and YOYO
-1
(cyanine monomers and
dimers) from Molecular Probes are also known for the quantitation of nucleic
acids - 0.5 ng/ml DNA in solution is the detectable concentration.
June 2002
Operating Manual for GENios, GENios FL, and GENios
Plus
No: I 112 904 Rev No: 1.1
9-1
