Molecular investigative techniques

Study Notes

Recombinant DNA Technology in Microbiology

Gene Cloning: The process of creating multiple copies of a specific gene.
- Steps:
  - Isolation of the gene of interest: Using restriction enzymes and vectors.
  - Insertion into a vector: Such as a plasmid or phage.
  - Transformation into a host cell: Eg., bacteria or yeast.
  - Selection of transformed cells: Identify cells that have successfully taken up the recombinant DNA.
  - Growth of transformed host cells: Which will make copies of the gene of interest.
  - Extraction and purification of the cloned gene: Or product made by its expression.
- Applications: Creating new proteins, improving existing ones, gene therapy, and agricultural engineering.
Gene Probes: Short, labeled pieces of DNA or RNA used to identify and isolate specific genes.
- Types:
  - Oligonucleotide probes: Short sequences that are complementary to a specific target sequence.
  - cDNA probes: Single-stranded DNA copies of mRNA, complementary to target RNA sequences.
  - Riboprobes: RNA probes synthesized by a polymerase enzyme, complementary to target RNA sequences.
- Uses: Diagnosing genetic disorders, pathogen detection, and research.
Polymerase Chain Reaction (PCR): A technique used to amplify specific DNA sequences.
- Components:
  - DNA Template: The target DNA to be amplified.
  - Primers: Short, single-stranded DNA sequences complementary to the target sequence.
  - DNA Polymerase: An enzyme that synthesizes new DNA strands.
  - Nucleotides: (dTNPs) Building blocks of DNA needed for replication.
- Method:
  - Denaturation: Separating the DNA strands.
  - Annealing: Primers bind to the target sequence.
  - Extension: Polymerase extends the primers to create new DNA strands.
- Types:
  - Real-time PCR: Quantifies the amount of DNA present before and after amplification.
  - Nested PCR: Enhances sensitivity by performing two rounds of PCR with nested primers.
  - Reverse transcription PCR (RT-PCR): Transcribes RNA into DNA, allowing for the amplification of RNA targets.
- Uses: Diagnosing infections, genetic testing, forensic science, and research.
Restriction Enzyme Analysis: The use of restriction enzymes to cut DNA at specific sequences.
- Uses: Mapping DNA, detecting mutations, identifying pathogens, and DNA fingerprinting.
Restriction Fragment Length Polymorphism (RFLP): Variations in DNA fragment lengths resulting from differences in restriction enzyme cut sites.
- Uses: Genetic mapping, paternity testing, forensics, and diagnostics.
Pulsed Field Gel Electrophoresis (PFGE): Used to separate large DNA molecules by applying alternating electric fields.
- Uses: Analyzing large genomes, tracking bacterial strains, and outbreak investigation.
Pyrosequencing: A sequencing method that detects the release of pyrophosphate during DNA synthesis.
- Uses: Clinical diagnostics, microbiome analysis, and research.
Next Generation Sequencing (NGS): High-throughput sequencing technologies that can sequence millions or billions of DNA fragments simultaneously.
- Uses: Genome sequencing, variant detection, exome sequencing, and cancer research.

Nucleic Acid Hybridization Techniques

The binding of two single-stranded nucleic acids (like DNA or RNA) that have complimentary sequences.
- Uses: Diagnosing genetic disorders, pathogen identification, and research.

Immunoassay

Techniques that use antibody-antigen interactions to detect and quantify specific molecules.
- Uses: Diagnostics, drug discovery, and research.

Immunohistochemistry

A technique that uses antibodies to visualize specific antigens in tissue samples.
- Uses: Diagnosing diseases, researching tissue structure, and developing new biomarkers.