ESTIMATION OF DNA AND RNA

BY MEASUREMENT OF SUGAR
Introduction
General History of Nucleic Acid

1868 -- Miescher---Nuclein

1880—Fischer—Purine and Pyrimidine

1889—Altmann-- Nucleic acid

1910– Levene—Ribose and deoxyribose

Nucleic Acid Components and Family
 Types of
Nucleic Acid

DNA RNA
(Deoxyribose Sugar) (Ribose Sugar)
Double Stranded Single Stranded
General Structure of DNA and RNA
Deoxyribonucleotide and Ribonucleotide
Structure
DNA
History of DNA Structure Invention

Watson and Crick in 1953

 Components, Structure, Alignment,
Types of DNA

Components—Heterocylic Bases (A,T,G,C),

Deoxyribose Sugar

Structure —Double Helix

Alignment —Bases are Stacked Inside

Types —A DNA, B DNA and Z DNA

Bases of DNA
Base Pairing Between DNA Bases
Deoxyribose Sugar
Structure of DNA
Types of DNA
Sources and extraction of DNA
 DNA
Estimation

Qualitative Quantitative
Estimation Estimation
Qualitative DNA Estimation
 Quantitative
DNA
Estimation

By By
UV Spectrometric Colorimetric
Method Method
Significance of quantitative DNA estimation
Quantitative DNA Estimation
by UV Spectrometry
Quantitative DNA Estimation
by
Colorimetric Method
 History of DNA Estimation

Dische’s Test,1930

Burton’s Test,1955
Principle
Reaction Scheme
Chemical Structure of Reaction
Components
Requirements
Diphenylamine (DPA)
Standard DNA
Method
 Process

• Process 1

• Process 2
 Process 1

By taking one standard DNA

Standard DNA and unknown DNA will be

treated with DPA following same working table

Process 2
Working table
Result
Calculation and Comment
 Sensitivity and Specificity
• Extreme sensitivity coupled with a low
reagent blank.
• Most satisfactory.
• A260/ A595 =4:1
 Advantage and Disadvantage
• Easy to perform

• The only potential hazard for which

precaution is necessary is in handling the acid.
• Sometimes purine base containing nucleotide
responds more.
 Precaution
Glacial acetic and sulphuric acids are used—care
must be taken.
DPA reagents and the reaction mixtures are kept
at dark as they are light sensitive.
RNA
 History of RNA Structure
• 1959--Severo Ochoa– RNA synthesis

• 1965--Robert W. Holley—Yeast t-RNA sequenced

• 1967--Carl Woese—catalytic RNA

• 1976--Walter Fiers—Phage genome (RNA)
sequenced
Components, Structure, Types of
RNA

• Components —ribose sugar, bases

(unusual base Uracil) and modified bases
like

• Structure—single stranded

• Types—m-RNA, t-RNA, r-RNA.

RNA Structure
RNA structure components
Types of RNA
Source and extraction of RNA
 Estimation of RNA

QUALITATIVE QUANTITATIVE
ESTIMATION ESTIMATION
Qualitative RNA estimation
 Quantitative
RNA
Estimation

UV spectrometry Colorimetric
Significance of RNA quantification
Quantitative RNA estimation by UV
spectrometry
Hyperchromic shift of DNA
 COLOURIMETRIC METHOD OF
RNA ESTIMATION
(BY ESTIMATING RIBOSE SUGAR CONTENT)
 History of RNA estimation

• Bial’s test, 1902

• Kamali and Manhouri, 1968

Principle
Reaction Scheme
Chemical Components of Reaction
 Requirements

• RNA stock solution.

• Orcinol

• FeCl3 or CuCl2

• Distilled water

• Spectrophotometer
Orcinol
Standard RNA
Method
 Process

• Process 1

• Process 2
 Process 1
Only one standard is taken compared with O.D
of unknown.
 Process 2
Series of standard concentrations are made
according to the working table.

The same treatments are given to standard DNA

solutions as well as unknown DNA solution.
Working Table
Result
Calculation and Comment
 Specificity and sensitivity
• The yield and purity of RNA preparation can be
assessed by measuring the absorbance of
ultraviolet light by a solution of nucleic acid.
• A pure RNA solution should give a 254nm:
280nm of 2; one unit of A254 measured in 1cm
light path length is equivalent to 5µg/ml.
• If the concentration of the sample is high the
sample is diluted by using n-butanol.
• Xylose can be taken as standard
 Precaution

• Acid must be taken care of.

 Advantage and Disadvantage
• Very specific and sensitive process.

• Other broken sugars may interfere.

• Sometimes only purine base containing
nucleotides respond more