DNase Hydrolysis Test Study Guide

DNA Composition

  • Components
    • DNA is composed of:
    • Nitrogenous bases
    • Deoxyribose sugar
    • Phosphates
  • Structure
    • Ribose sugars and phosphates are linked by covalent bonds to form the sugar-phosphate backbone of the molecule.

Deoxyribonuclease (DNase)

  • Definition
    • DNase is an enzyme secreted by certain bacterial species.
  • Role
    • DNase is considered a bacterial virulence factor that contributes to infection.
    • It breaks the covalent bonds between phosphate and the deoxyribose sugar molecules.
    • The breaking of these covalent bonds in the DNA backbone leads to:
    • Depolymerization of DNA into short nucleotide chains.
  • Types of DNase Enzymes
    • There are two types of DNase enzymes which cleave at different locations:
    • One type breaks the bond between the 5' carbon atom of deoxyribose sugar and the phosphate.
    • The other type cleaves the bond between the phosphate and the 3' carbon atom of the deoxyribose sugar.

Detection of DNA Hydrolysis

DNase Agar

  • Components Required for Detection
    • DNA:
    • Serves as the substrate for the enzyme.
    • Methyl Green Dye:
    • Binds to polymerized, uncleaved DNA.
    • When intact, the media appears blue/green.
    • The dye will not bind to small DNA fragments or short nucleotide chains.

Results Interpretation of DNase Test

DNase Negative Result

  • Test Setup
    • Bacteria are streaked onto DNase agar and incubated overnight.
  • Observed Result
    • If the agar surrounding the bacterial growth remains green/blue, methyl green dye has remained bound to DNA, indicating:
    • Methyl green dye only binds to polymerized DNA.
    • Therefore, DNA has not been cleaved.
    • Conclusion:
      • There is no zone of clearing, indicating that the result is DNase negative.

DNase Positive Result

  • Test Setup
    • Bacteria are streaked onto DNase agar and incubated overnight.
  • Observed Result
    • If the agar surrounding the bacterial growth becomes clear, this indicates that:
    • The methyl green dye has been removed, meaning DNA must have been cleaved.
    • DNase has been secreted by the bacteria, which cleaved the DNA around it.
    • Conclusion:
      • A clear zone around the bacteria indicates that the result is DNase positive.

Summary of Findings

  • DNase Production
    • The DNase is produced and secreted by the bacteria.
    • The DNA around the bacteria is cleaved, confirming the presence of DNase.
  • Dye Disassociation
    • The methyl green dye cannot bind to the small DNA fragments produced, resulting in a zone of clearing around the bacteria.
  • Control
    • An uninoculated control can be used for comparison in the experiment.

Visual Representation of the Test

  • The DNase test can be visualized through color changes in the agar:
    • Blue/green indicates no cleavage and a negative result.
    • Clear zones indicate cleavage by DNase and a positive result.