See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/318744511

DNA QUANTIFICATION

Chapter · July 2016

CITATIONS READS

0 3,441

3 authors, including:

Karen Pachchigar Akshay Khunt

College of basic science & humanities, Sardarkrushinagar Dantiwada Agricultural … Sardarkrushinagar Dantiwada Agricultural University
20 PUBLICATIONS   40 CITATIONS    5 PUBLICATIONS   5 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Green synthesis of Nano particle View project

Tagging of Wilt Resistant Gene(s) in Castor (Ricinus communis L.) View project

All content following this page was uploaded by Karen Pachchigar on 28 July 2017.

The user has requested enhancement of the downloaded file.

DNA QUANTIFICATION Pachchigar, K. P. et al., 2016

DNA QUANTIFICATION
Karen P. Pachchigar1, Akhshay Khunt 2 and Bhilocha Hetal3
1
Assistant Professor, and 2,3 P.G. Scholar,
Department of Genetics and Plant Breeding, C. P. College of Agriculture,
S.D. Agricultural University, Sardarkrushinagar - 385 506

Background:
 Quantification and assessment of DNA/RNA and Protein purity and concentration, is
first entry step in most of molecular biology protocol routinely employed in many lab.
 Molecular biologists routinely work with DNA. RNA, and have devised some simple,
fast spectrophotometric assays for these molecules.
 With this concerned use the UV absorbance of biological samples to obtain qualitative
and quantitative information especially for nucleic acid was first proposed by
Warburg, O. and Christian W. (1942).
 The results of numerous molecular screening and assay methods often rely on the
overall quality of the genomic DNA (gDNA) input material. Downstream application
of DNA is purely dependant on how better we able to qualitatively as well
quantitatively DNA with robust and affirmative analysis. For this purview for
quantifying the amount of nucleic acid in a preparation are (A) spectrophotometric
estimation (B) flourometric determination and (C) DNA quantification using
NanoDrop.
 Now a days UV analysis of DNA/Protein has been benchmark method for rapid
quantification and assaying purity of sample for various purpose.
Principle:
 Most biological molecules do not intrinsically absorb light in the visible range, but
they do absorb ultraviolet light
 The heterocyclic bases of DNA are
aromatic and absorb in the ultraviolet
region of the electromagnetic
spectrum. It is noteworthy that λmax for
all Watson-Crick bases is between 250
nm and 280 nm.
 So, following Lambert-Beer’s law, it is
possible to estimate the concentration
and quantity of the oligonucleotide in
solution.
 Quantitative estimation of nucleic acid
can be carried out with simple
relationship that molar extinction
coefficient (ε) for dsDNA is 0.020
(μg/ml)−1 cm−1 , single-stranded DNA
it is 0.027 (μg/ml)−1 cm−1, for single-stranded RNA it is 0.025 (μg/ml)−1 cm−1
 So, it is to be note that molar extinction coefficient (ε), will decrease as ring system of
adjacent purines and pyrimidines become stacked in polynucleotide chain. Therefore
free nucleotide bases have maximum molar extinction coefficient (ε) and further

4 ICAR Sponsored summer school on Allele mining in crops: Methods and Utility
18th July-7th August, 2016
DNA QUANTIFICATION Pachchigar, K. P. et al., 2016

decrease as ssDNA to most lower for dsDNA to triple stranded DNA like H-DNA to
G-quartat qudruplex DNA.
 Checking quality of sample for detection of Protein, or other contaminant with
DNA/RNA sample is most common practice. Usually, this is carried out by checking
ratio of absorbance at different wavelength.
 For standard practice for DNA sample usually A260/A280 are used, as if ratio is 1.8
indicate good pure quality of DNA, while ratio of 2.0 indicate contamination of RNA
in sample and same ratio less than or 0.6 is indicating protein in sample.
 Following table giving idea about characteristic nature of DNA and Protein, at
different wavelength:
Wavelength Characteristic nature Remarks
(Nm)
215-230 Minimum absorbance Measurements are generally not performed at this
for nucleic acids. wavelength because commonly used buffers and
Peptide bonds in solvents, such as Tris, also absorb at these
proteins absorb light. wavelengths. Contaminant phenolate ion, thiocynate
and other organic compound absorb at this range.
260 Nucleic acid have Purines absorbance maximum is slightly below 260;
maximum absorbance pyrimidines maximum, is slightly above 260. Purines
at this wavelength have a higher molar absorptivity than pyrimidines.
Therefore, the absorbance maximum and absorptivity
of a segment of DNA depends on its base
composition.
270 Phenol absorbs Phenol may be contamination
strongly
280 Protein absorbance Aromatic amino acid strongly absorb at 280 Nm.
peak
330 or Non protein and Presence of particulate matter causing light scattering
higher Nucleic acid
absorbance range

 For a pure RNA sample, the A230:260:280 should be around 1:2:1, and for a pure DNA
sample, the A230:260:280 should be around 1:1.8:1.
 This ratio is used as a secondary measure of nucleic acid purity. The A260/A230 ratio
values for pure samples are often higher than the respective A260/A280 ratio values.
 Strong absorbance around 230nm can indicate that organic compounds or chaotropic
salts are present in the purified DNA. A ratio of 260nm to 230nm can help evaluate
the level of salt carryover in the purified DNA. The lower the ratio, the greater the
amount of salt present.
 As a guideline, the A260/A230 ratio should be greater than 1.5, ideally close to 1.8.
Urea, EDTA, carbohydrates and phenolate ions all have absorbance near 230nm.
 The TRIzol® reagent which can be used for RNA extraction, although more
commonly used for RNA preparation is a phenolic solution which absorbs in the UV
spectrum at 230 nm and around 270nm.
 Guanidine HCL which is used in the extraction of RNA will absorb at 230nm while
guanidine isothiocyanate, used for RNA isolations will absorb at 260nm.

5 ICAR Sponsored summer school on Allele mining in crops: Methods and Utility
18th July-7th August, 2016
DNA QUANTIFICATION Pachchigar, K. P. et al., 2016

Figure 1: UV ABSORBANCE SPECTRUM FOR DNA AND PROTEINS.

A. DNA. B. Protein (BSA).
C. The absorbance spectrum for a mixture of DNA and protein.

DNA QUANTIFICATION USING FLOURIMETER:

 In current era of NGS technologies, most of sequencing manufacturer recommends to
have dsDNA-specific fluorometric quantitation methods.
 It is important to choose fluorometric methods of DNA quantitation that are specific
to double-stranded DNA (dsDNA), since single-stranded DNA (ssDNA) is not a
suitable substrate for most library preparation technologies.
 Bisbenzmide (Hoechst Dye-H33258) exhibits changes in fluorescence characteristics
in the presence of DNA that allow accurate DNA estimation. The dye is non-
intercalating, binds to the minor groove of DNA (with marked preference to AT rich
regions and gives 458 nm.
 The Hoechst 33258 and Hoechst 33342 dyes have complex, pH-dependent spectra
when not bound to nucleic acids, with a much higher fluorescence quantum yield at
pH 5 than at pH 8.
 Since RNA does not generally bind to H33258, the measured fluorescence is a direct
indicator of DNA concentration, provided a clean DNA standard of known
concentration is used.
 Sensitivity of H33258 decreases with nuclease degradation, increasing GC content or
denaturation of DNA.
 In preparations with minimal RNA contamination or sometimes samples with
unusually high GC content EtBr fluorescence is the choice. Here, sensitivity is ~20
times less compared to Hoescht dye. EtBr have 302 or 546 excitation and 590
emission and fluorimeter capable of detecting the same can be used for quantification.
 Techniques can accurately quantitate as little as 25 pg/mL of dsDNA in a fluorometer
or 250 pg/mL (typically 50 pg in a 200 µL volume) in a fluorescence microplate
reader.

QUANTIFICATION OF DNA, RNA and Protein:

 Concentration of dsDNA= 50 µg/ml x Dilution factor x (Absorbance at 260 Nm-
absorbance at 320 Nm)
 Concentration of ssDNA = 33 µg/ml x Dilution factor x (Absorbance at 260 Nm-
absorbance at 320 Nm)
 Concentration of RNA = 40 µg/ml x Dilution factor x (Absorbance at 260 Nm-
absorbance at 320 Nm)
 Concentration of Protein (mg/ml) =[(1.55 x A280) – (0.76 X A260)] X Dilution factor

6 ICAR Sponsored summer school on Allele mining in crops: Methods and Utility
18th July-7th August, 2016
 DNA QUANTIFICATION Pachchigar, K. P. et al., 2016

Material:
 DNA/RNA/Protein sample
 Buffer

Method:
1. Start on spectrophotometer and adjust it to wavelength 260 nM.
2. Auto-zero spectrum with blank of TE buffer for DNA/RNA or PBS buffer Protein
sample.
3. Withdraw buffer and rinse cuvette once with distilled water.
4. Fill cuvette with max volume with DNA/RNA/Protein sample.
5. Run absorbance spectrum in range of 230 to 330 Nm.
6. Run Peak value and find point with maximum absorbance in range of scan and note it
7. Further, take absorbance at 230, 260 and 280 Nm and note it.
8. Check quality of sample by taking ratio of A230/260/280, A260/280 and A260/230.
9. Calculate quantity of sample as per equation and note it.

******

7 ICAR Sponsored summer school on Allele mining in crops: Methods and Utility
18th July-7th August, 2016

View publication stats