Molecular and immunology investigation techniques 2.4

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

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

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

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

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

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

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

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

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

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

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 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

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

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

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

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