Molecular Cell Biology in Veterinary Science

What is molecular biology?

The study of biological processes at a molecular level (DNA, RNA, proteins)

What is the importance of molecular biology?

• Crucial for understanding cellular function, genetics and disease mechanism

• Applications in diagnostics, disease treatment and animal care

What are the 3 key biomolecules?

• DNA (blueprint of life)

• RNA (mediates gene expression)

• Proteins (workhorses that perform cellular functions)

What is the concept of central dogma?

(DNA replication) —> DNA —> (transcription) —> mRNA —> (translation) —> Protein

What is omics sciences?

Share the aim of identifying, describing and quantifying the biomolecules and molecular processes that contribute to the form and function of cells and tissues

What is genomics?

The identification of genetic variant in DNA sequence including coding or non-coding regions

What is epigenomics?

Evaluation of non-DNA sequence changes such as methylation

What are transcriptomics? #ff7700

Evaluation of quantity and quality of RNA

What are proteomics?

Evaluation of protein map network, production and modification

What are metabolomics?

Evaluation of endogenous/exogenous metabolites

Name the 9 molecular techniques used in veterinary diagnostics

• Polymerase chain reaction (PCR)

• DNA sequencing

• FTIR

• Next generation sequencing

• ELISA

• Flow cytometry

• Southern/Northern/Western blotting

• IFA

• Mass spectrometry

Polymerase Chain Reaction (PCR) #ea00ff

• What

• Importance

• Uses

• Focuses on which aspect of central dogma

• Why is it difficult to find evidence of disease in earliest stages of infection?

• 4 Steps

What: Laboratory technique used to make multiple copies of a segment of DNA

Importance:

• Detecting bacterial and viral infections

• Quantification of pathogen load in infectious diseases

Uses: The genome that is stored inside DNA

Focuses on: DNA replication and connected to central dogma as it amplifies DNA

Why is it difficult to find evidence of disease in earliest stages of infection: Not enough pathogens and insufficient time to develop antibody response

4 steps:

• Denaturation

• Annealing

• Extension

• Amplification

PCR: Denaturation

• Temperature

• What occurs

• Central dogma step does it relate to

Temperature: 95 degrees

What occurs: Separation of dsDNA template into 2 single strands by breaking hydrogen bonds between complementary bases

Central dogma step it relates to: DNA replication

PCR: Annealing

• Temperature

• What occurs

• Central dogma step does it relate to

Temperature: 68 degrees

What occurs: Allows primers to bind to complementary sequences on single stranded DNA template

Central dogma step it relates to: Transcription

• Why: Because in transcription, RNA primers bind to DNA template and in annealing, primers bind to single stranded DNA template

PCR: Extension

• Temperature

• What occurs

• Central dogma step does it relate to

Temperature: 72 degrees

What occurs: Taq polymerase enzyme adds nucleotides to the 3’ end of primers to synthesise new strands of DNA complementary to template strand

Central dogma step it relates to: DNA replication

• Why: DNA polymerase in PCR extends primers and synthesise new DNA strands, mirroring replication process of DNA replication

PCR: Amplification

• What

The 3 steps (D A E) are repeated multiple times where each cycle, DNA target is doubled

So which steps in central dogma is not mirrored in PCR and why?

mRNA synthesis and translation because PCR doesn’t involve RNA

ELISA #b600ff

• Stands for

• What

• Relies on

• Used in

• 4 key components

• 4 types

Stands for: Enzyme-linked immunosorbent assay

What: Technique used to detect and quantify substances such as peptides, proteins, antibodies and hormones

Relies on: Antigen-antibody interactions coupled with an enzyme that produces a detectable signal

Used in: Diagnostic applications, research and veterinary medicine

4 key components:

• Antigen: Target substance to detect

• Antibody: Specific to antigen

• Enzyme: Linked to antibody and facilitates detection signal

• Substrate: Reacts with enzyme to produce detectable signal

4 types:

• Direct

• Indirect

• Sandwich

• Competitive

Direct ELISA

• Antigen immobilised where

• Antigen detected with

• Detection of

#b600ff

Antigen immobilised: On surface

Antigen detected with: Specific primary antibody that is directly conjugated to an enzyme (detection molecule)

Detection of: Pathogens (parvovirus in feces)

Indirect ELISA

• Antigen immobilised where

• 2 step process

• Detection of

#b600ff

Antigen immobilised: On the surface of multi-well plate

2 step process:

• Primary antibody specific for antigen binds to target

• Labeled secondary antibody binds to primary antibody binds for detection

Detection of: Immune responses or infections (FIV antibodies in cats)

Sandwich ELISA

• Requires

  • AKA

• Process

• Detection of

#b600ff

Requires: 2 antibodies specific to different epitopes of antigen

• AKA: Matched antibody pairs

Process:

• 1 antibody coat the surface of the multi-well plate to facilitate the immobilisation of antigen

• The other antibody conjugated and facilitates detection of antigen

Detection of: Antigens, highly specific (Dirofilaria immitis antigen in blood)

Competitive ELISA

• Principle

• Measure antigen concentration by

• Process

• Detection of

#b600ff

Principle: Inhibition ELISA

Measure antigen concentration by: Detecting signal interference

Process:

• Sample antigen competes with a reference antigen for binding to a specific amount of labeled antibody

• Multi-well plate is pre-coated with reference antigen

• Sample is added with labeled antibody

Detection of: Antibodies to Leptospira species or other small antigens

ELISA: Pros and Cons for each Type

Table

Immunofluoresence Assay (IFA)

• To detect

• Relies on

• Types

• Importance

• Key components

#02d319

To detect: Specific antigens in cells or tissue sections using antibodies tagged with fluorescent dyes

Relies on: Antigen-antibody binding and visualised using fluorescence microscope

Types:

• Direct immunofluorescence

• Indirect immunofluorescence

Importance:

• Localisation of proteins within cells

• Study of cell signaling pathways

• Diagnosis of autoimmune diseases

• Detection of pathogens

Key components:

• Sample

• Fixation reagent

• Permeabilisation reagent

• Blocking reagent

• Antibodies (primary and secondary)

• Mounting medium

Fourier Transform Infrared (FTIR)

• Measures

• Based on

• Instrumentation

#009aff

Measures: The absorption of infrared radiation by a sample to identify chemical bonds and molecular structures

Based on: The principal that different functional groups absorb IR radiation at characteristic frequencies

Instrumentation:

• Transmission FTIR

• ATR-FTIR

• FTIR imaging