Nucleic Acid Isolation and Extraction Methods

Nucleic Acid Isolation

• Target nucleic acid (either RNA or DNA) is released from cells.
• Solid-phase silica-based products bind DNA or RNA in high-salt concentration.
• Silica-based products are commonly used due to ease of use, fewer safety concerns, and ability for high throughput and automation.
• Examples of silica-based products include column filters and magnetic beads.

Spin Column Method

• Patient samples containing intact virus particles are aliquoted into a tube.
• Lysis buffer containing detergent and denaturing materials is added to allow cell and viral lysis to occur.
• RNA is released in the solution and binding buffer and alcohol are added to facilitate the binding of RNA.
• The mixture is transferred into a spin column, which has a visible white layer composed of a silica matrix.
• Centrifugation allows the viral RNA and human RNA to bind to the silica matrix, while impurities are filtered out.
• The spin column is washed to remove impurities, and elution buffer or nuclease-free water is added to release the bound RNA.

Magnetic Beads Method

• Magnetic beads are used to isolate nucleic acids.
• The process involves binding of nucleic acids to the magnetic beads, washing, and elution.

Nucleic Acid Analysis

• DNA or RNA quantity, quality, and molecular size are characterized by:
  • UV spectrophotometry, which measures absorbance at 260nm and 280nm.
  • Agarose gel electrophoresis.
  • Fluorometry, which is specific to dsDNA.

Electrophoresis

• A laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge.
• An electric current is used to move the molecules through a gel or other matrix.
• A DNA ladder is a solution of DNA molecules of different lengths used in agarose or acrylamide gel electrophoresis.

Nucleic Acid Sequence Identification by Hybridization

• A technique used to identify specific DNA sequences.
• The principle of hybridization is the addition of a probe to a complex mixture of target DNA.
• The mixture is incubated under conditions that promote the formation of hydrogen bonds between complementary strands.
• Hybridization can be done in all combinations: DNA-RNA, DNA-DNA, or RNA-RNA.
• In situ hybridization and FISH analysis are types of hybridization.

Signal Amplification Methods

• Hybrid Capture Assay is used for detection of human papillomavirus, hepatitis B virus, and cytomegalovirus.
• The assay involves the binding of target nucleic acid to capture probes, and the binding of extender probes to amplifier molecules.
• Reporter molecules labeled with alkaline phosphatase bind to amplifier probes, and a chemiluminescent signal is emitted.
• Cleavage-based amplification is based on the activity of cleavase enzyme used in the Invader assay.
• Branched-chain DNA is frequently used for quantification of target sequences in clinical samples, especially viral load determinations.

Strand Displacement Amplification (SDA)

• A process that involves the binding of a probe to target DNA, and the simultaneous displacement of the opposite strand.
• The displaced strand is copied by the primers, and the process is iterated at 52°C without temperature cycling.
• The addition of a fluorogenic probe yields a fluorescent signal directly proportional to the amount of amplified probe/target.