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Deoxyribonucleic acid, also referred to as DNA, is a bunch of nucleotides, perfectly organized to form a double helix. At a molecular level, DNA is fundamental in understanding evolution. DNA is like a footprint to the body, making proteins. Forensics, modern biology, genetics, epidemiology, genealogy, and genetic engineering are dependent on DNA and its fingerprinting.
RNA is a biomolecule, which is similar to DNA. At the same time, it is single-stranded. The main function of RNA is to transform the information stored in DNA into proteins. It regulates the activity of genes in development, cellular differentiation, and changing environments. RNA has applications in disease control, reverse genetics, tumors, animal production of commercial interest, parasites, and tumors.
Quantification of DNA and RNA is a significant step as it aids to know the amount of DNA present, which is essential in performing restriction digests, PCR and RAPDs. Before proceeding to any application, quantification after DNA isolation is important. There are some methods used for the quantification after DNA extraction. This article brings the quantification methods into focus.
Mostly, the methods are common for DNA and RNA. There are six methods principally used for the quantification of DNA. They are as follows:
This method is one of the commonly used ways in the quantification of DNA. This involves measurement of absorbance/transmittance of light through a liquid for determination of the concentration of substances in the liquid.
To different degrees, molecules absorb different wavelengths of light and many molecules possess particular wavelengths in which they absorb maximally. Through UV-transparent cuvettes and spectrophotometer, absorbances are measured. Initially, the absorbance of the buffer that the DNA is dissolved in, is measured. This is ‘blank’ and it provides the background absorbance. Then, the absorbance of the sample is measured.
The absorbance measures provide the concentration of DNA and the presence of contaminants in the DNA. DNA and RNA absorb maximally at 260 nm. Proteins absorb best at 280 nm. The chaotropic salts and organic compounds possess the absorbance maximum at 230 nm.
The A260/A280 ratio is utilized as an indicator of the purity of DNA. For an ideal DNA, this number should be in the range of 1.8 - 2. The A260/A230 ratio is best if it is greater than 1.5.
Usually, A260 of 1 is equivalent to 50 microgram/milliliter pure double-strand DNA. The formula used for estimation of DNA is as follows:
Concentration (microgram/milliliter) = A260 reading * dilution factor * 50 microgram/milliliter
This method does not need any reagents. Besides, it is simple and quick. But, there is lesser sensitivity at lower concentrations of DNA. In addition, it is not able to distinguish between RNA and DNA.
Fluorescence dyes, which bind to DNA are used for the quantification of DNA. Dyes like SYBRGreen and PicoGreen are specific to double-strand DNA. This is in contrast to the UV absorbance method as it measures all kinds of nucleic acids. Compared to the UV absorbance method, this method is more sensitive as it is used when there is a lower sample concentration. It is often used in quantifying DNA for next-generation sequencing.
In contrast to the UV absorbance method, the fluorescence method needs a standard curve, a set of samples with known fluorescence and their respective fluorescence. The fluorescence of the sample is compared to that of known dye and plotted in the curve, yielding the quantity of DNA. Although this method consumes more time, there is no need for manual calculation as several fluorometers are equipped in performing the estimation for sample concentration.
When the quantity of DNA is too small, the gel electrophoresis technique is employed for quantification. In addition to the quantification, this method is also useful to view whether the DNA is intact or in the correct size. This information is not provided by an absorbance-based method and there is no need for a fluorometer or spectrophotometer.
A gel containing intercalating dye (ethidium bromide) is poured and a DNA ladder is chosen with known concentrations. Commercial DNA ladders inform the concentration of each band in the ladder. Then, the samples are run at various dilutions and thereafter, they are quantified based on the intensities of the band relative to the intensity of the ladder. It is preferred to compare the band intensity of the fragment in the ladder that is closest in size to the sample DNA. This method is suited for the quantification of DNA, which is to be used in PCR amplification. Based on the presence of other bands or streaking, this method provides an indicator of RNA or DNA contamination. To the contrary of absorbance-based methods, this method does not inform about chaotropic salts and contaminating proteins.
Quantification of DNA through capillary electrophoresis is the same as agarose gel electrophoresis, with the exception that this system is smaller and automated. The run time is only a few minutes per sample. Only 1-2 microliters of sample are needed for the technique. The DNA fragments move through nanofluidic or microfluidic channels during the run and the sample is split by electrophoresis. Compared to larger fragments, smaller fragments move quickly. Using a fluorescent dye that intercalates into the DNA, the fragments are quantified over time. Before the larger fragments, smaller ones are quantities first. This method precedes next-generation DNA sequencing and microarray studies. At the same time, it is not used much for the standard plasmid preparation.
Diphenylamine is used as a quantification agent for DNA. Diphenylamine reacts with deoxyribose sugars under acidic situations and forms a blue complex, which can be quantified at 595 nm.
The method is less sensitive, so it is not used much. Besides, it is a time-consuming method. But, the measurements are made in the visible range and can be read by a standard ELISA reader, if none of the instruments are available.
Fluorometric probes are utilized by Quantitative PCR (qPCR) and the probes bind target DNA in the annealing phase of PCR. For RNA quantification, the PCR template is cDNA (complementary DNA), which is synthesized by reverse transcription of RNA.
Specialized thermal cyclers are utilized to monitor the fluorescence signals during amplification. The estimated fluorescence is proportional to the total quantity of RNA. A standard curve is used as a tool to quantify the amount of RNA and DNA in test samples. In digital PCR methods, a standard curve is not required.
Real-time PCR can quantify specific target sequences based on the amount of amplifiable nucleic acid. This is advantageous in the quantification of genomic DNA, where small and sheared RNA and DNA fragments are ignored. It detects the inhibitors within the reaction mixture and it is inherently specific to the fluorometric probes.
There are some things to consider, before choosing the particular quantification method. The desired method needs to be chosen based on time, downstream application, and availability of the instrument. Thus, time, cost, expected DNA/RNA concentration, and equipment are the significant factors. The method’s suitability and pros and cons of each method should be carefully analyzed before the start of the quantification process.