Nucleic Acid Extraction Notes

Nucleic Acid Extraction Overview

  • Instructor: Marcia A. Firmani, Ph.D., MSPH, MT(ASCP)

  • Course: Molecular Diagnostics

Learning Objectives

  • Compare and contrast organic and solid phase approaches for isolating nucleic acids.

  • List and describe functions of enzymes and reagents utilized for nucleic acid isolation.

  • Describe methods used to determine the quantity and quality of nucleic acid preparations, including gel-based, spectrophotometric, and fluorometric methods.

  • Calculate concentration and yield of DNA and RNA from a nucleic acid preparation.

  • Calculate quality of extracted DNA and RNA.

  • List advantages and disadvantages of commercial extraction kits.

Nucleic Acid Release and Contamination

  • The release of nucleic acids from cells must ensure complete removal of contaminants including:

    • Protein

    • Carbohydrates

    • Lipids

    • Other nucleic acids

Enzymes Utilized in Nucleic Acid Isolation

  • Proteinase K: A proteolytic enzyme used to digest proteins.

  • RNAse: Enzyme that degrades RNA. Utilized to remove non-DNA nucleic acids.

  • DNAse: Enzyme that degrades DNA. Utilized to remove non-RNA nucleic acids.

Methods for Cell Disruption

  • Breaking cell and nuclear membranes is necessary to release cellular material.

    • This must occur under conditions that only damage membranes, preserving nucleic acids.

Common Sample Types for Nucleic Acid Extraction

  • Nucleated cells

  • Tissue samples

  • Microorganisms

DNA Yield from Various Sample Types

  • Blood: 20–60 µg DNA/ml

  • Buffy coat: 100–250 µg/ml

  • Bone marrow: Specific yield not specified, typically high

  • Solid tissue: 2–25 µg DNA/mg

  • Lavage fluids: Yield may vary

  • Microorganisms: Variable yields depending on species

  • Organelles, mitochondria: 2–10 µg/10^7 cells

Techniques for Extracting DNA from Blood and Tissue

  • Blood/Bone Marrow:

    • Density Gradient Centrifugation: Separates components based on density.

    • Differential Osmolysis: Relies on cellular osmotic fragility.

    • Streptokinase: May assist in extraction methodologies.

  • Tissue Extraction:

    • Methods include freeze/crush, mincing, and enzymatic digestion.

Sample Preparation Techniques

  • Plants/Fungi:

    • Homogenization or vortexing with glass beads to break cell walls.

Techniques for Isolating White Blood Cells (WBCs)

  • Use differential density gradient centrifugation by mixing whole blood or bone marrow with saline, overlaid with Ficoll. This results in:

    • WBCs remain atop the Ficoll.

    • Erythrocytes settle below, with serum above WBCs.

  • Differential Lysis: Method based on osmotic fragility differences.

Ficoll Gradient Explanation

  • Ficoll is a highly branched sucrose polymer.

    • It does not penetrate biological membranes and allows WBCs to be easily separated during centrifugation.

Cell Lysis and Contaminant Removal

  • Whole blood or bone marrow can be incubated in water or hypotonic buffers, leading to:

    • Lysis of red blood cells (RBCs) prior to white blood cells (WBCs).

    • WBCs can be pelleted via centrifugation.

Preparation of Tissue Samples

  • In the presence of paraffin, removal is necessary for effective extraction.

  • Tissue must be dissociated by grinding, homogenizing, or mincing.

Cell Wall Polymer Digestion Methods

  • Enzymatic Digestion: Utilizing enzymes such as:

    • Lysozyme

    • Zymolase

    • Lysostaphin

  • Mechanical Methods: Including grinding or mixing with glass beads.

  • Chemical Methods: Utilization of detergents (e.g., sodium dodecyl sulfate) or strong bases (e.g., NaOH).

Purification of Extracted DNA

  • Essential to purify DNA post-release using organic solvents:

    • Phenol and Chloroform: Dissolves hydrophobic contaminants, collects debris, and strips away proteins.

  • This creates a biphasic emulsion, with:

    • Hydrophobic components settling at the bottom.

    • DNA remains in the upper aqueous phase, while debris forms a precipitate between layers.

Collection and Rehydration of DNA

  • DNA collected from the upper aqueous layer and precipitated using ethanol or isopropanol in the presence of salt.

  • The DNA pellet is rinsed in 70% ethanol to remove excess salt and rehydrated in:

    • 10 mM Tris, 1 mM EDTA (TE), or water.

Organic vs. Solid Phase DNA Extraction

  • Organic Isolation Process:

    • Lysis utilizing NaOH and SDS, followed by acidification with acetic acid and salt, extraction using phenol and chloroform, and finally DNA precipitation using ethanol.

  • Solid Phase Isolation:

    • Utilizes silica-based spin columns for DNA adsorption from cell lysate, followed by washing and elution.

  • Major methods are suited for different lab environments and sample types.

Advantages and Disadvantages of Commercial Extraction Kits

  • Advantages:

    • No caustic solvents (e.g., phenol).

    • Faster processes with estimates of DNA concentration.

  • Disadvantages:

    • Higher costs associated with kits.

    • Potentially lower yields compared to classic methods.

    • Possible impurities in DNA post-extraction.

Limitations and Rapid Extraction Methods

  • Some specimens may limit the efficacy of typical extraction techniques (e.g., fixed tissue, dried blood).

  • Rapid extraction methods available for certain low-quality samples, including use of specific lysis solutions.

DNA Extraction from Fixed Tissue

  • Deparaffinization: Using xylene and ethanol washes, followed by digestion with proteinase K and Tris buffer in low-micron sections.

Isolation of Mitochondrial DNA

  • Isolated by centrifugation, with sample subjected to two rounds of centrifugation at varying speeds to pellet intact cells, nuclei, and debris, followed by high-speed centrifugation for mitochondrial DNA.

Methods to Assess Quantity and Quality of Nucleic Acids

  • Gel Electrophoresis: Used with known standards for visual assessment of size and integrity.

  • Spectrophotometry: Measures absorbance at 260 nm (1 OD260 = 50 μg/ml for dsDNA) and evaluates purity using OD260/OD280 ratios (ideal range 1.6-2.0).

  • Fluorometry: Uses fluorescent dyes like RiboGreen, which can provide higher sensitivity against DNA samples.

RNA Extraction and Its Challenges

  • RNA extraction is more sensitive due to susceptibility to degradation by RNAse. All operations must occur in RNAse-free environments.

  • Key procedures include:

    • Use of acid citrate dextrose and heparin for blood samples.

    • Immediate processing of fresh tissue or storage in appropriate conditions (-70°C, nitrogen).

RNA Isolation Techniques

  • Organic Extraction: Similar to DNA, but using guanidine isothiocyanate for lysis.

    • Followed by phenol-chloroform extraction and ethanol precipitation.

  • Solid Phase Isolation: Involves RNA adsorption at low pH, with washing and elution processes defined.

Types of RNA

  • Common forms of RNA include:

    • Messenger RNA (mRNA)

    • Ribosomal RNA (rRNA)

    • Transfer RNA (tRNA)

    • Heteronuclear RNA (hnRNA)

    • Small nuclear RNA (snRNA)

    • Double-stranded RNA (dsRNA)

    • Various small/micro RNAs

Composition of RNA Yield

  • Approximately 80–90% of total RNA is rRNA.

  • mRNA contributes only about 2.5–5% of total RNA yield.

  • mRNA can be isolated using oligomers that bind to its polyA tail, allowing other RNA types to be washed away.

Measuring RNA Quality and Quantity

  • Utilizes gel electrophoresis, spectrophotometry (OD260 = 40 μg/ml for RNA), and fluorometry.

  • RNA samples assessed for quality based on band appearance and brightness.

Visual Analysis of Nucleic Acids and their Types

  • Gel electrophoresis results indicating different types:

    • Plasmid DNA shows bright supercoiled bands, while genomic DNA shows bands with less mobility.

    • RNA exhibits distinct rRNA bands during electrophoretic analysis.

Summary of Key Extraction Methods

  • Comprehensive overview of DNA and RNA extraction techniques from organic, inorganic, and solid phase methods.

  • Acknowledgement of rapid methods for extracting nucleic acids and evaluating their concentration, yield, and purity using various quantification techniques.