UV/ Vis spectroscopy Determines the purity of the isolated DNA The absorbance at 260nm is due to the DNA species, while the absorbance at 280nm is due to the protein species

Absorbance ratio (A260/A280)Gives the relative measurement of DNA and protein content in the isolated DNAIf the value of A260/A280:= 1.8 1.9Pure of DNA free of protein< 1.8 1.9Indicates protein contamination> 1.8 1.9Indicates the presence of RNAAcid HydrolysisReagents1.0 M HCl-causes the dissociation of DNA into its component-causes depurination1.0 M NaOH- neutralizes the solution; needed so that it wouldnt affect the testsWhy not use alkaline hydrolysis? Pentoses of DNA does not have an OH group at the 2nd carbon No formation on monophosphate intermediate DNA is stable in alkaline hydrolysisWhy is the DNA-HCl mixture heated to 100 degree Celsius? to be able to destroy the hydrogen, phosphodiester and glycosidic bonds results dissociation of DNA into its components: phosphate group purine and pyrimidine deoxyribose

Results of hydrolysis: breaking of bonds associated with the DNA molecule1. hydrogen bonds-heating to temperature above 80 degrees Celsius2. phosphodiester bonds-heating the temperature above 90 degrees Celsius-acids with pH less than 23. Glycosidic bonds-acids with pH less than 2

Test for deoxyribose (dische test)Standard : deoxyribose standard solutionReagents : diphenylamine reagent (contains conc. H2SO4)Positive result: blue colored solutionPrinciple: 1. Dehydration of deoxyribose by H2SO4 (formation of 5 hydroxy-levulinaldehyde)2. Complexation with diphenylamine (blue color)Test for phosphateStandard : phosphate solutionReagents : Conc. H2SO4, conc. HNO3, 2.5% ammonium molybdate solutionPositive result: yellow precipitate (ammonium phosphomolybdate)Principle: Precipitation of phosphateTest for purines (murexide test)Standard : solid guanineReagents : conc. HNO3, 10% KOHPositive result: red purple residuePrinciple: 1. Oxidation of purine by conc. HNO3 (forming dialuric acid and alloxan)2. Condensation reaction of alloxan to form alloxanthin3. Neutralization, forming red purple murexide (potassium salt of purpurate)

Test for pyrimidines (wheeler Johnson)Standard : cytosine solutionReagents : bromine water, Ba(OH)2Positive result: purple precipitate; negative for thymine because of methyl groupPrinciple: 1. Bromination of pyrimidine to form dialuric acid2. Neutralization of dialuric acid by Ba(OH)2 to form barium salt of Dialoric acid