Molecular and immunology investigation techniques 2.4

Nucleic Acids

• Techniques used to investigate nucleic acids include PCR, RT-qPCR, in situ hybridization, and blotting techniques like Southern blotting
• PCR: Uses a polymerase that amplifies a targeted sequence of DNA
• RT-qPCR: Measures the quantity of a specific RNA transcript
• In situ hybridization: Identifies the presence of specific DNA or RNA sequences within intact cells and tissues
• Southern blotting: Detects specific DNA sequences in a sample by using labelled probes

Protein

• Techniques used to investigate proteins include immunostaining, western blotting, electrophoresis, and mass spectrometry
• Immunostaining: Uses antibodies to stain cells, identifying specific proteins
• Western blotting: Detects and quantifies specific proteins in a sample through electrophoresis and blotting
• Electrophoresis: Separates proteins based on their size, charge, or other properties
• Mass spectrometry: Identifies, measures, and quantifies molecules using their mass-to-charge ratio

Immunostaining

• Uses antibodies to stain cells and identify specific proteins
• Direct immunostaining: uses a primary antibody with fluorophores, offering high specificity but lower detection sensitivity
• Indirect immunostaining: Less specific but easier to detect, uses a secondary antibody that binds to the primary antibody, often labeled with a visible tag

Immunocytochemistry

• Immunostaining technique used to detect proteins in cultured cells

Immunohistochemistry

• Immunostaining technique used to detect proteins in tissue sections

Immunofluorescence

• Immunostaining technique that uses fluorescent dyes to visualize the location of specific molecules in tissues or cells

Antibodies

• Biological molecules that specifically bind to and neutralize antigens
• Monoclonal antibodies: Produced by a clone of a single B cell, highly specific and uniform
• Polyclonal antibodies: Produced by multiple B cells, less specific but more readily available

Flow Cytometry

• Uses lasers and detectors to analyze physical and chemical characteristics of single cells as they pass through a laser beam
• Measures cell size, granularity, and fluorescence properties (based on antibody labeling)
• Can be used to analyze different cell populations within a sample, based on size, granularity, and specific markers
• Limitations: Limited to identifying cell populations based on predetermined markers and does not provide information about the cell's internal structure

Fluorescence Activated Cell Sorting (FACS)

• Specialized type of flow cytometry that physically sorts cells based on their characteristics

ELISA

• Enzyme-linked immunosorbent assay, a plate-based assay that uses antibodies to detect and quantify specific proteins or other molecules
• Direct ELISA: Uses an antibody directly conjugated to an enzyme, simplifying the process but reducing sensitivity
• Indirect ELISA: Uses a secondary antibody, which amplifies the signal and increases sensitivity
• Sandwich ELISA: Uses two antibodies, one to capture and another to detect the target molecule
• Competitive ELISA: Uses a target molecule to compete with an antibody for binding to the plate, providing a negative correlation between signal and target
• Multiplex ELISA: Simultaneously measure multiple analytes in a single sample, increasing efficiency and throughput

In situ hybridization

• Detects specific DNA or RNA sequences within intact cells or tissues
• Used to study gene expression and localization of specific transcripts
• Uses probes (labeled single-stranded DNA or RNA molecules) that recognize and bind to complementary sequences in the target nucleic acid

PCR

• Polymerase chain reaction, amplifies a specific DNA sequence, allowing researchers to study and analyze genes and genetic variations
• Utilizes a heat-tolerant polymerase enzyme to replicate the DNA sequence, generating copies of the target sequence
• Enables detection and quantification of specific DNA sequences in a sample

Reverse Transcriptase - Quantitative Polymerase Chain Reaction (RT-qPCR)

• Quantifies specific RNA transcripts in a sample, providing a method for studying gene expression
• Reverse transcriptase converts mRNA to cDNA, which is then amplified through qPCR
• Provides information about the abundance of specific transcripts

PCR Limitations

• PCR reactions can be prone to errors, leading to inaccurate results
• Primers used in PCR might bind to unintended sequences, resulting in false-positive data
• PCR is sensitive to contamination, which can lead to erroneous results

Electrophoresis

• Separates molecules based on their size and charge
• Agarose gel electrophoresis: Used primarily for separating DNA fragments based on their size
• Polyacrylamide gel electrophoresis: Separates proteins based on their size and charge

Post-Gel Analysis

• Visualizing and quantifying the separated molecules after electrophoresis
• Ethidium bromide: A fluorescent dye used to visualize DNA bands in agarose gels
• Coomassie blue, silver nitrate, and spyro ruby: Dyes used to visualize protein bands in polyacrylamide gels

Blotting Techniques

• Techniques for transferring biological molecules from a gel matrix onto different membranes
• Western blotting: Quantifies and identifies specific proteins in a sample
• Southern blotting: Identifies specific DNA sequences using labelled probes
• Northern blotting: Identifies specific RNA sequences using labelled probes

Mass Spectrometry

• Identifies, measures, and quantifies molecules based on their mass-to-charge ratio
• Used to analyze proteins, peptides, and other biomolecules
• Widely used in proteomics, metabolomics, and drug discovery

Mass Spectrometry Methods

• Matrix-assisted laser desorption/ionization (MALDI)
• Electrospray ionization (ESI)
• Gas chromatography-mass spectrometry (GC-MS)
• Liquid chromatography-mass spectrometry (LC-MS)

Explore various techniques used to investigate nucleic acids and proteins in this quiz. Learn about PCR, RT-qPCR, immunostaining, and mass spectrometry among others. This quiz provides a comprehensive overview of laboratory methods essential for molecular biology.