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Questions and Answers
The Hybrid Capture Assay is used for the detection of human immunodeficiency virus.
False
The target nucleic acid is released from cells in the first step of the Hybrid Capture Assay.
True
The Invader assay is a type of signal amplification method based on cleavage activity.
True
The Hybrid Capture Assay uses a chemiluminescent signal to detect the target nucleic acid.
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The target nucleic acid binds to capture probes fixed to solid support in the third step of the Hybrid Capture Assay.
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The extender probes have sequences that are complementary to sequences in the target molecules and to sequences in the amplifier molecules.
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The reporter molecules are labeled with alkaline phosphatase conjugated anti-DNA/RNA hybrid antibodies.
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The DNA is denatured in the second step of the Hybrid Capture Assay.
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The Hybrid Capture Assay detects target nucleic acid by a series of probes that bind to the target and overlap.
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The Cleavage-based amplification method is used in the Hybrid Capture Assay.
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Study Notes
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.
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Description
This quiz covers the different methods of nucleic acid isolation, including the use of isopropyl alcohol and silica-based products. Learn about the steps involved in solid phase extraction and how to obtain pure DNA or RNA samples. Test your knowledge of nucleic acid isolation techniques and protocols.
