Principles of RNA and DNA estimation M.Pharmacy 1st Semester 2022 (2021-2022) Previous Year's Question Papers/Notes Download - HK Technical PGIMS

❖CONTENT

❑ INTRODUCTION
❑ DNA and RNA quantitation Methods
➢UV absorbance OR Spectroscopy method
➢Fluorescence measurement using nucleic acid-binding dyes
➢Agarose gel electrophoresis
➢Diphenylamine method
❑ CONCULSION
❑ REFFERENCES

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❖INTRODUCTION

• DNA and RNA are quantify to check the concentration and purity of
DNA and RNA present in solution.
• It is important to know the conc. and purity of DNA and RNA for the
use in further applications.
• Accurate determination of nucleic acid concentration and yield is
important for downstream Processing including transfection, cloning,
PCR, and next generation sequencing (NGS).
• Downstream Processing refers to the recovery and purification
of biosynthetic products, particularly pharmaceuticals, from
natural sources such as animal or plant tissue.
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❖INTRODUCTION

• 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 OR Spectroscopy method
• Fluorescence measurement using nucleic acid-binding dyes
• Agarose gel electrophoresis
• Diphenylamine method

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❖Spectroscopy method :-

The most common technique used to determine both nucleic acid concentration
and purity is absorbance.
Absorbance measurements can be used to estimate the concentration of DNA or
RNA in purified samples.
In this method, the nucleic acid sample is placed into a quartz cuvette, which is
then placed inside the UV spectrophotometer.
Small volume spectrophotometers such as the NanoDropTM instrument, DeNovix
DS-11 spectrophotometer, and Blue-Ray Bio EzDrop spectrophotometer enable the
analysis of sample volumes as low as 1 μL using pedestals.
UV light is passed through the sample at a specified path length, and
concentrations of nucleic acids can be directly calculated by measuring absorbance
values at 260 nm against a blank using the Beer-Lambert's equation.

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❖Beer-Lambert's equation:
A = εcl

where;

A = UV absorbance
ε = wavelength-dependent extinction coefficient
c = nucleic acid concentration
l = light path (cm)

• ABSORBANCE MEASUREMENTS, CONTAMINANTS, AND NUCLEIC ACID PURITY
• To evaluate protein contamination, determine the ratio of the absorbance at 260 nm (nucleic
acid absorbance) and 280 nm (absorbance of aromatic rings in protein amino acids, though
phenol will also absorb at 280 nm). High quality DNA will have an A260/A280 ratio of 1.7–2.0.
High quality RNA will have an A260/A280 ratio of ~2.0.

• 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. The A260/A230 ratio should be >1.5 for applications that are
sensitive to chemical interference, including enzyme-based assays such as qPCR and
sequencing. The lower the ratio, the greater the amount of organic compounds or chaotropic
salts in the preparation.

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

Extinction coefficients*:
•dsDNA (pure): 0.020 (μg/mL)-1 cm-1
•ssDNA (pure): 0.027 (μg/mL)-1 cm-1
•ssRNA (pure): 0.025 (μg/mL)-1 cm-1

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❖NanoDropTM instrument

• Can quantify nucleic acid from microvolume of 0.5 μL- 2.0 μL
• Measures DNA and RNA (A260),and protein (A280) conc. And sample purity (260/280 ratio).
• Large conc. range (2 ng/μL-15,000 ng/μL dsDNA) without dilution.

Purity and quantification
➢ Pure DNA sample gives a 260/280 ratio=1.8
➢ For pure RNA 260/280=2
➢ A 260?230 indicates possible contaminates.
➢ A Shift in the wavelength is indicates contamination
absorbing at low wavelengths.
➢ The wavelength of the sample peak should be at 260nm if contaminants
are present the peak may shift.

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❖Fluorescence measurement using nucleic acid-
binding dyes

• These methods use fluorescent dyes that bind DNA or RNA non-specifically, or
selectively bind nucleic material. Differences in spectral characteristics of nucleic
acid-bound fluorophores allow sample concentrations to then be determined.
• 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.
• Fluorescent dyes, such as Pico Green OR SYBR Green are specific for Double
strand DNA.
• These methods are more sensitive than UV absorbance, especially when you
expect low concentrations in your samples and are often used to quantify DNA
for next generation sequencing.

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❖ Agarose gel electrophoresis

• Agarose gel electrophoresis separates DNA and RNA from other
impurities.
• In agarose gel electrophoresis, a sample of the isolated DNA or RNA is
loaded into a well of the agarose gel, which is placed in an electric field.
• The negatively charged nucleic acid migrates toward the anode, separating
DNA and RNA fragments by size and shape.
• Nucleic acid concentration and yield can be determined by comparing the
intensity of sample bands to standards of known amounts.
• Because DNA and RNA absorb light at 260 nm, intensity can be measured
using a UV transilluminator.
• Higher sensitivity can be obtained by labeling nucleic acid samples and
standards with a nucleic acid dye such as ethidium bromide or SYBR®
Green and measuring intensity at the specified wavelength for that dye.

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SCHEMATIC DIAGRAM
OF AGAROSE GEL
ELECTROPHORESIS
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❖Diphenylamine method
Determine the concentration of a given DNA Sample using diphenylamine method.
Principle:
When DNA is treated with diphenylamine under the acidic condition a bluish green colored complex is formed.This
reaction is given by 2 deoxypentose in general. In acidic solution deoxypentose are converted into a highly reactive β
hydroxyl leavulinic aldehyde which reacts with diphenylamine gives bluish green colored complex. The colour intensity
was measured using a red filter at 595nm.
Equipments:
A UV/Visible spectrophotometer, Vortex mixer, Weighing balance, Water bath
Reagents:
Diphenylamine reagent, Calf DNA,Glacial acetic acid, Concentrated sulfuric acid
Standard DNA Solution:thymus
Weigh 1g of diphenylamine and transfer it into a 250 ml amber coloured glass bottle. Add 100 ml glacial acetic acid and
shake well to achieve complete dissolution. Add 2.5 ml of concentrated sulfuric acid. Store the reagent in dark at 2 – 8°C.
Diphenylamine Solution:
Dissolve 1g of diphenylamine in 100 ml of glacial acetic acid and 2.5 ml of concentrated H2SO4. This solution must be
prepared fresh
Buffered Saline:
0.5 mol/litre NaCl; 0.015 mol/litre sodium citrate, pH 7.

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❖PROCEDURE

1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of labeled test
tubes.
2. Pipette out 1 ml of the given sample in another test tube.
3. Make up the volume H2O to 1 ml in all the test tubes. A tube with 1 ml of distilled water
serves as the blank.
4. Now add 2 ml of DPA reagent to all the test tubes including the test tubes labeled
'blank' and 'unknown’.
5. Mix the contents of the tubes by vortexing / shaking the tubes and incubate on a
boiling water bath for 10min.
6. Then cool the contents and record the absorbance at 595 nm against blank.
7. Then plot the standard curve by taking concentration of DNA along X-axis and
absorbance at 595 nm along Y-axis.
8. Then from this standard curve calculate the concentration of DNA in the given sample.

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❖CONCLUSION

• This test is very specific to determine the concentration of DNA and is widely used.

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❖REFFERENCE

1. Bio-Resource http://technologyinscience.blogspot.in.nucleic-
acid-quantification- dnarna.html

www.technologyinscience.blogspot.com
2. Pharmaceutical Biotechnology by U. Satyanarayna.
3. Pharmaceutical Biotechnology by Chandrakant kokare.