DNA Extraction, Detection and Quantification

DNA Extraction, Detection, and Quantification

Overview

  • Importance of DNA extraction, detection, and quantification in genetics.

  • Key personnel involved includes Chris Madden and WRIGHT LAB.

Nucleic Acids

  • Types of Nucleic Acids:

    • DNA (Deoxyribonucleic Acid)

    • RNA (Ribonucleic Acid)

    • Proteins

    • Key processes:

      • Transcription: Process of synthesizing RNA from DNA.

      • Translation: Process of synthesizing proteins from RNA.

Genotype vs Phenotype

  • Genotype:

    • The sequence of nucleotides in genetic code inherited from parents.

  • Phenotype:

    • Observable characteristics of an organism.

  • Influenced by:

    • Genotype

    • Epigenome

    • Environmental effects on both genotype and phenotype.

DNA Extraction

Basic Steps

  1. Collection of the sample.

  2. Lysis of the cell and nuclear membrane to release DNA.

  3. Removal of membrane fragments, protein, and RNA.

  4. Concentration of Purified DNA.

  • Various methods used to isolate nucleic acids.

Blood Sample Preparation

  • Whole Blood Collection:

    • Use anticoagulant EDTA to prevent clotting and inhibit enzyme activity; chelates ions like Mg2+.

    • Heparin is not recommended as it may interfere with results (inhibits Taq polymerase in PCR).

Types of Specimens

  • DNA can be extracted from various specimens:

    • Whole blood, bone marrow, fresh/frozen tissue, tumors, skin, fetal chorionic villi, umbilical cord, FFPE (Formalin Fixed Paraffin Embedded) tissue, saliva, amniotic fluid, cultured cells.

Specimen Handling for DNA Extraction

  • Correct handling is crucial to prevent DNA degradation.

  • Specimen storage guidelines:

    • Whole blood and bone marrow: Room temperature for 4 days.

    • Buffy coat: Room temperature for 4 days or frozen at -80°C for years.

    • Fresh tissue good for 2 days at 4°C; frozen at -80°C for years.

    • FFPE tissue: Indefinitely.

    • Saliva: Stabilizing agents for years.

DNA Extraction Methods

General Methodology

  • All methods require:

    • Lysis of the cell and nuclear membrane.

    • Removal of protein and debris.

    • Isolation of the DNA.

  • Common methods:

    • Phenol/Chloroform method

    • Column-based methods

    • Salt extraction

    • Magnetic bead extraction

Sample Preparation Techniques

Whole Blood Preparation

  • Steps:

    • Remove RBCs by lysing other cells using NH4Cl/Tris-HCl.

    • Centrifuge and handle nuclear pellets carefully.

    • Add SDS and proteinase K for nuclear lysis.

Buffy Coat Preparation

  • Centrifuge whole blood; isolate and store the buffy coat sample.

Tissue Extraction

  • Fresh/Frozen Tissue:

    • Cut tissue into small pieces; resuspend in lysis buffer with Proteinase K.

  • FFPE Preparation:

    • Dissolve paraffin, wash in EtOH, and utilize lysis buffer with Proteinase K.

Saliva and Amniotic Fluid

  • Directions for centrifugation and handling of both specimens to preserve nucleic acids.

Precipitation Techniques

Ethanol Precipitation

  • Nucleic acids precipitated using ethanol due to reduced solubility in less polar solvents.

Safety Protocols

Chemicals Handling

  • Importance of safety while using Phenol and Chloroform:

    • Requirements for personal protective equipment (PPE).

DNA Detection, Quality, and Quantification

Spectrophotometry

  • Measuring nucleic acid purity using absorbance at:

    • 260 nm for nucleic acids

    • 280 nm for proteins

  • 260/280 ratio:

    • Ideal >1.8 for DNA; >2.0 for RNA.

Troubleshooting Purity Issues

  • Contamination Indicators:

    • <1.7 due to protein or phenol; >2.0 suggests RNA contamination.

    • Check 260/230 ratio for organic material contamination.

Alternative Quantitation Methods

  • Fluorescence Measurements:

    • Sensitive and selective detection of nucleic acids.

  • Quantitative Real-Time PCR (qPCR):

    • Used to assess the amount of amplifiable DNA from samples.

  • Integrity Gels:

    • For quality assurance of DNA or RNA.

Math and Calculations

  • Importance of being able to calculate concentrations and yields of nucleic acids correctly, with given formulas.

  • Utilizing absorbance readings to determine DNA/RNA yields and purity ratios.