Determining the concentration of Nucleic acid

in molecular biology, quantitation of nucleic acids is commonly performed to determine the

average concentrations of DNA or RNA present in a mixture, as well as their purity.

The process of determining the conc. of DNA or RNA and their purity in a mixture is called
nucleic acid quantification.

Reactions that use nucleic acids often require particular amounts and purity for optimum
performance. Accurate determination of nucleic acid concentration and yield is important for
downstream applications including transfection, cloning, PCR, and next generation sequencing
(NGS). These applications often have a specific target nucleic acid concentration for optimal
performance. Inaccurate quantification can increase variability in downstream assays and affect
quality of results.

DNA and RNA quantitation can be performed using any of the following methods:

 UV absorbance / Spectrophotometry (optical density)

 Fluorescence measurement (using nucleic acid-binding dyes)

 Agarose gel electrophoresis

 PCR

1. Spectrophotometry:

Spectrophotometry measures the proportion of light of a certain wavelength that is absorbed by a

sample. The absorbance values allow both qualitative and quantitative analysis of the sample,
e.g. by determining the purity of a solution or the concentration of a certain analyte.

Spectrophotometric analysis is based on the principles that nucleic acids absorb ultraviolet light
in a specific pattern. In the case of DNA and RNA, a sample is exposed to ultraviolet light at a
wavelength of 260 nanometres (nm) and a photo-detector measures the light that passes through
the sample. Some of the ultraviolet light will pass through and some will be absorbed by the
DNA / RNA. The more light absorbed by the sample, the higher the nucleic acid concentration in
the sample. Typical wavelengths for measurement are 260 nm and 280 nm. In addition,
measurements at 230 nm and 340 nm can provide further information.

Since nitrogenous bases absorb UV light, the more concentrated the DNA solution, the more UV
light it will absorb. A solution containing 50 µg per ml of double strand DNA has an absorbancy
(optical density) of 1.0 at a wave length of 260 nm.
Spectrophotometers and microspectrophotometers calculate the nucleic acid concentration using
the following formula:

Concentration (µg/ml) = A260 reading x conversion factor (constant)

The conversion factor is

50 µg/ml for dsDNA,

40 µg/ml for RNA and

33 µg/ml for ssDNA.

if you diluted your samples before making the measurements you need to multiply the
concentration with your dilution factor.

Nucleic acid concentration (μg/mL) = A260 × dilution factor × constant

For example, for a double-stranded DNA solution diluted 40× for measurement, with an
A260 reading of 0.20, the estimated concentration of the undiluted sample would be 0.20 × 40 ×
50 = 0.4 mg/mL or 400 μg/mL.

Once you know the concentration of your sample, you can calculate its yield as follows, e.g. to
determine if your PCR reaction generated a sufficient amount of nucleic acids for your
downstream application:

Total yield can then be obtained by multiplying the nucleic acid concentration by the final total
purified sample volume.

DNA yield (µg) = DNA concentration × total sample volume (mL)

Purity analysis:

Nucleic acids extracted from cells normally require purification to remove protein impurities.
To evaluate DNA purity, measure absorbance from 230nm to 320nm to detect other possible
contaminants. The most common purity calculation is the ratio of the absorbance at 260nm
divided by the reading at 280nm.

Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. A reading of 1.6 does not render the
DNA unsuitable for any application, but lower ratios indicate more contaminants are present.

DNA purity (protein contaminants) = A260 reading ÷ A280 reading

To evaluate chemical contamination, the ratio of the absorbance at 260 nm and 230 nm can be
used. Residual chaotropic salts and organic solvents, which can inhibit PCR, are known to
absorb light in the 230 nm range.

DNA purity (chemical contaminants) = A260 reading ÷ A230 reading

A ratio of 2- 2.2 is good if ratio is lower then it indicate contamination with phenol or
carbohydrates etc.

2. Fluorometry:
Fluorometry is a technique used to identify and quantify analytes in a sample by adding
fluorophores that bind to the analyte of interest to the sample, exciting them with a beam of UV
light and detecting and measuring the emitted wavelength. By comparing the sample
fluorescence to known standards, the analyte can be quantified.

This method is useful for cases where concentration is too low to accurately assess with
spectrophotometry and in cases where contaminants absorbing at 260 nm make accurate
quantitation by that method impossible.

What are Fluorophores?

Fluorophores are microscopic molecules, which may be proteins, small organic compounds, or
synthetic polymers that absorb light of specific wavelengths and emit light of longer
wavelengths.

DYE FOR:
dsDNA ssDNA RNA
RiboGreen
PicoGreen OliGreen
PicoGreen: is an alternative ultrasensitive fluorescent nucleic acid stain for quantitating
doublestranded DNA in molecular biological procedures such as DNA amplification, cDNA
synthesis for library production, and DNA fragment purification for subcloning. The PicoGreen
reagent exhibits an emission maximum at 530 nm when bound specifically to double-stranded
DNA (unbound PicoGreen reagent exhibits minimal fluorescence in solution). The detection
range of PicoGreen when bound to double-stranded DNA is 1 to 1000 ng/mL.
RiboGreen: is a sensitive fluorescent nucleic acid stain for determining the RNA concentration
in solutions. The RiboGreen reagent exhibits minimal fluorescence when free in solution.

How to determine the concentration?

Each nucleic acid dye has a specific excitation and emission wavelength. The DNA or RNA
sample is measured using a fluorometer, and nucleic acid concentrations are then calculated by
comparing fluorescence emission of the sample to a fluorescence curve generated using
standards of known nucleic acid concentration. Different samples types (genomic DNA, plasmid
DNA, etc.) each require their own standard curves. As with absorbance methods, calculated
concentrations must be corrected for dilution factor as required.

(Fluorometers usually calculate the nucleic acid concentration for you. If not, you can calculate it
by comparing the fluorescence of your sample against the standard curve. As for
spectrophotometry, don’t forget to multiply the concentration you get with the dilution factor and
calculate the yield by multiplying the concentration with the total sample volume.)

The purity of nucleic acids can’t be calculated with fluorometry as it only detects the
fluorophores bound to the nucleic acids and therefore can’t detect contaminants.

Considerations when selecting a fluorescent nucleic acid dye for quantitation:

 Specificity: different dyes are optimized to measure dsDNA, ssDNA, RNA, or small
RNAs (e.g., miRNA) based on their mechanism of binding and spectral properties - make
sure you select a fluorescent dye that binds to your target of interest.
 Sensitivity: highly sensitive fluorescent dyes allow you to use less sample or measure
very low concentrations of nucleic acid.
 Dynamic range: some reagents can be used to detect nucleic acid at low concentrations,
but may lose linearity at higher concentrations; the reagent to be used has to be suitable
for the expected concentration range of your samples.

Spectrophotometry vs. fluorometry

Fluorometers can’t measure the purity of nucleic acid samples, only their concentration. Another
downside compared to spectrophotometry is that it’s more costly because of the expensive assay
kits and more time consuming as you need to mix your samples with the fluorescent dye before
analysis.

Its advantages over spectrophotometry are that it’s way more sensitive, providing better results
with diluted samples, e.g. after nucleic acid extraction. The lower detection limit of the
NanoDropTM spectrophotometer is for example 0.4 ng/µl, whereas the Qubit fluorometer has a
lower detection limit of 0.005 ng/µl.