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