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Acid Hydrolysis and Chemical
Characterization of DNA
Group 5:Hung, Wei Yi
Jimeno, Krizia RoseKintanar, Armando Victor
Lopez, Angela PatriciaLoyola,Juan Paolo
Acid Hydrolysis
DNA Hydrolysis
The breaking of the DNA double-helix bonds by the addition of water
Complete hydrolysis of chromosomal nucleic acids gives the inorganic phosphate, 2-deoxyribose and four different heterocyclic bases: Adenine, Guanine, Thymine, Cytosine
Chemical Hydrolysis
Uses strong acid at high temperature to break down the stable structure of DNA into its components
Conditions:› Acid: 6M HCl› Temperature: 100° C for 1 hour
These conditions break both of the phosphate ester bonds and also the N-glycosidic bond between the deoxyribose and the purines and pyrimidine bases.
The products of this mixture are the 4 bases, phosphoric acid, and deoxyribose which then polymerises to produce a brown sticky tar.
Dische TestTest for Deoxyribose
Deoxyribose
Monosaccharide Present in the DNA structure Discovered in 1929 by Phoebus Levine
Dische Test
It can identify DNA chemically. Positive result: blue solution Reagents used: diphenylamine reagent Principle: conversion of pentose to a
molecule that reacts with the diphenylamine reagent
Dische Test
Test for Phosphate
Test for Phosphate
A phosphate group consists of a central phosphorous surrounded by four oxygens.
Test for Phosphate
Nucleotides are building blocks of Nucleic acids.
Three compounds are needed in the formation of a Nucleotide: deoxyribose; a base; and a phosphate group.
They combine through 2 condensation reactions.
Test for Phosphate
Basic structure of a nucleotide:
Test for Phosphate
Nucleic Acids are formed by the linking of 2 or more nucleotides by the formation of Phosphodiester bonds.
The Phosphodiester bond is formed by the condensation reaction between the –OH group of the phosphate group in a nucleotide and a –OH group on the carbohydrate ring of another nucleotide.
Test for Phosphate
DNA is formed from 2 polynucleotides. It has a double helical structure with
bases on the inside and phosphate outside of the helix.
They are held together by the pairing of nucleotides bases through H-bonding.
Test for Phosphate
Test for a phosphodiester bond linked at C3 or C5 in the ribose.
Positive result: Yellow precipitate (Phospho-ammonium molybdate) (NH4)3PO4·12MoO4.
Principle: The Phosphate ions react with the ammonium molybdate to form a phosphomolybdate complex.
Murexide TestTest for purine
Murexide Test
In the test for presence of purines, DNA is reacted with Nitric acid since Purines are known to be readily soluble in dilute acid. Nitric acid oxidized it leaving a yellow precipitate upon evaporation; however it turned red when moistened with a base, a positive result for presence of purine bases
Wheeler-Johnson Test
Pyrimidines
Pyrimidines that occur in DNA are cytosine and thymine. Cytosine and Uracil are the pyrimidines in RNA.
HN
NH
O
O
Uracil
HN
NH
O
O
CH3
Thymine
NH2
NH
HN
O
Cytosine
Test for Pyrimidines (Wheeler-Johnson Test)
A qualitative test for the presence of either uracil or cytosine.
Positive result: Purple colored solution Reagents used: Excess Bromine water,
Excess Ba(OH)2
Principle involved in Wheeler-Johnson Test (Standard)
Uracil/cytosine form dibromoxyhydrouracil when treated with bromine water. When treated at ordinary temperature with an excess of barium hydroxide the two
atoms of bromine are replaced by hydroxyl groups and isodialuric acid is formed. Isodialuric acid then undergoes a rearrangement into dialuric acid. Both isodialuric and dialuric acids give a violet-blue precipitate with barium
hydroxide.
Wheeler-Johnson Test (DNA hydrolyzate)
This compound gives a negative result with Wheeler-Johnson Test .
Principle:
Dische Test› Dehydration of deoxyribose forming
hydroxyl levulinaldehyde› Complexation reaction with diphenylamine
Principle
Murexide Test› Oxidation of purine by concentrated HNO3
forming dialuric acid and alloxan› Condensation reaction leading to formation
of alloxanthan› Neutralization leading to formation of
murexide or ammonium purpurate
Principle
Wheeler Johnson Test› Formation of dialuric acid by neutralization
Phosphate Test› Addition, Precipitation and Complexation