Molecular Biology Lab Guide

TIPS FOR NUCLEIC ACID PURIFICATION: DNA

Written by MicroDok

DNA PURIFICATION BASICS

In today’s world of DNA (deoxyribonucleic acid) analysis by multiplex and real-time PCR and the search for rare events, the importance of high-quality, purified DNA cannot be underestimated. Quality analytes are necessary for quality downstream assay results. Understanding how DNA purification systems work and then choosing the best chemistry for your application is essential.

COMMON CHALLENGES IN DNA PURIFICATION

  1. Lysing samples to release DNA.
  2. Separating DNA molecules from other molecules such as lipids, proteins, carbohydrates and RNA.
  3. Maintaining the integrity of DNA molecules.

DNA sources differ in their challenges. Foodstuffs, such as chocolate, have compounds that can inhibit downstream assays if these compounds are carried over with the purified DNA. Isolating DNA from Gram positive bacteria requires efficient lysis of the thick peptidoglycan bacterial cell wall. [Gram negative bacteria has a less or thin peptidoglycan layer than Gram positive bacteria] Make sure that the DNA purification approach that you use addresses the challenges that your sample presents.

IMPORTANT TIPS FOR HANDLING BLOOD AND TISSUE SAMPLES FOR DNA ISOLATION

Freeze blood and tissue samples until use to minimize damage to DNA from nucleases. Blood, when thawed, needs to be completely mixed before removing an aliquot for DNA purification.

DNA purification can be thought of in four basic steps:

LYSE >>> BIND >>> WASH >>> ELUTE

DNA PURIFICATION: LYSE, BIND AND WASH

Lyse: Disrupt Cells or Tissues

Tissues or cells can be lysed through any of the following methods:

  • Enzyme treatment
  • Mechanical disruption
  • Detergent treatment

Lyse: Denature or Inactivate Proteins

This can be done by using:

  • Ionic detergents, heat, reducing agents, urea and guanidine
  • Proteases (e.g., Proteinase K)

Lyse: Inactivate Endogenous Nucleases

This can be done by using:

  • Chelating agents (e.g., EDTA)
  • Proteases (e.g., Proteinase K)

Bind and Wash: Separate the DNA

  • Remove other nucleic acids (e.g., RNA)
  • Remove proteins, using:
    • Organic Extraction
    • Salting Out
    • Bind to Solid Support, using:
      • Silica Matrix
      • Anion Exchange Columns
      • Magnetic Particles

Source: PROMEGA

www.promega.com

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