Measuring Nucleic Acid Concentrations
The concentration of nucleic acids in solution can be determined by UV spectroscopy, either qualitatively or quantitatively, since DNA absorbs light strongly at 260 nm. Qualitatively, double and single-stranded DNA and RNA have been assigned average absorbance values. At 260 nm, 1 OD unit has been estimated to contain the following:
double-stranded DNA 50 µg/ml,
single-stranded DNA 33 µg/ml,
single-stranded RNA 40 µg/ml.
Pure solutions of any one of the above nucleic acids could easily be assayed for concentration. The difficulty arises when the preparation has more than one type of nucleic acid. For instance,
plasmids isolated from E. coli typically include RNA unless an RNase is added during the protocol to degrade the contaminant. More problematic is the presence of contaminating protein. Since protein also absorbs in the 260 nm range, nucleic acid solutions tainted with protein yield false concentration values. Prior to determining the concentration, nucleic acids should be phenol/chloroform extracted to remove contaminating proteins.
Quantitatively, the concentration of nucleic acids can be measured using Beer’s Law, which states:
A = εbc,
where “A” is absorbance, “b” is the light path (cm), “c” is the concentration of the solute (M), and ε is the extinction coefficient. Each molecular species of DNA has its own extinction coefficient which is dependent upon its length and base composition. The extinction coefficient is determined by summing the individual ε for each base. The extinction coefficients for the individual bases are:
adenine 15200
cytosine 7050
guanine 12010
thymine 8400
Beer’s Law is only practical if the DNA molecule has been highly purified and has a known sequence, such as with a synthesized oligonucleotide. Beer’s Law can be applied to larger DNA
molecules, but the calculation of ε is usually cumbersome. For larger DNA molecules, the qualitative measurement is more practical.
Estimation of DNA Concentrations
The determination of the concentration of DNA or RNA in solution is a fundamental task in molecular biology. DNA is usually the limiting reagent in most experiments; therefore, knowledge of its concentration is critical. Restriction endonuclease reactions, ligations, transformations, etc., are all extremely dependent upon the concentration of the DNA. Determination of the concentration of DNA can be estimated either by qualitatively comparing the fluorescence of DNA bands in an agarose gel to a standard or by spectrophotometric means.
Qualitative Estimation
DNA fluoresces in the presence of ethidium bromide, and the intensity of the fluorescence is proportional to its concentration. As such, comparison of relative fluorescence of unknown DNA to known standards can be used as a rough estimation of DNA concentration. This comparison is usually made following the electrophoresis of the standard (e.g., 1 µg of Lambda DNA cleaved with the restriction endonuclease HindIII) and the unknown. The mass of 1 µg of a Lambda-HindIII standard provides convenient references for comparison (Table 8.2).
Table 8.2 A Commonly Used Electrophoretic Standard and the Masses of Individual Fragments
Kb Fragment µg of DNA/1 µg total
23.31 480 ng
9.42 194
6.56 135
4.36 90
2.32 48
2.03 42
0.56 12
0.13 3
An alternative method for estimating DNA concentration relies on the lower limit for visually detecting DNA in a gel. It has been estimated that a band of DNA below 5 ng is not detectable by the human eye. Serially diluting DNA to extinction, followed by electrophoresis, can also be used to measure DNA concentration. This technique is particularly useful if there is more than one species or fragment of DNA in a sample. In this manner, the concentration of individual bands can be estimated.
Quantitative Estimation
DNA, RNA, and protein strongly absorb ultraviolet light in the 260 to 280 nm range. UV spectroscopy can be used as a quantitative technique to measure nucleic acid concentrations and protein contamination. Nucleic acids absorb strongly at 260 nm and less strongly at 280 nm while proteins do the opposite. The general rules for determining the concentrations of nucleic acids at 260 nm are:
(1) 1 OD unit of double-stranded DNA is 50 µg/ml;
(2) 1 OD unit of single-stranded DNA is 33 µg/ml; and
(3) 1 OD unit of single-stranded RNA is 40 µg/ml.
Proteins absorb strongly at 280 nm where 1 OD unit is 1 mg/ml. When using UV spectroscopy for estimating DNA concentrations, it is very important to remove all protein and RNA from the DNA solution. Good estimations can only be made on clean preparations.
An estimate of the purity of a DNA preparation can be made by measuring the absorbance at both 260 and 280 nm. Pure solutions of nucleic acids will absorb approximately twice as much at 260 nm as at 280 nm. Experimentally, the ratio of 260 nm/280 nm of a pure DNA solution is between 1.8 and 2.0. As protein contamination increases, the ratio decreases. Additionally, the presence of contaminating organic solvents, such as phenol, can affect estimations of concentration and purity.