Molecular techniques

Nucleic acid hybridisation

Radioactively labelled complementary ssDNA or ssRNAs form a duplex

biotin molecule is used to label non-radioactively

Detected via x-ray and assays

Northern blotting

Identify specific RNA sequences in a sample by size and sequence.

Separated by size in an agarose gel. Nucleic acids (-ve) move to negative pole.

Complementary target sequences stick to the radioactive probe

induces chaperon proteins by promoting protein refolding and assembly

however - not refined, needs lots of RNA

in situ hydridiation

Reveals the presence and location of specific nucleic acids in fixed tissues or cells.

Able to detect rare RNAS

However - less sensitive than PCR, and more complex

Gene expression changes

Dynamic - varies with development, environment, disease

Developmental - embryonic development

Environment - circadian rhythm

Disease conditions - can alter gene expression patterns, affecting cellular responses and behaviors.

PCR and qPCR

Amplifies specific DNA sequences with primers

RNA is converted into complementary DNA (RT)

DNA amplified with specific primer to the target gene

Fluorescent dyes track amplification

• highly sensitive and fast

• Not quantitative

What is so special about PCR

It can detect tiny amounts of DNA and RNA

Allows researchers to determine gene expression with high precision

Laser capture microdissection

Adv - precise extraction of specific cells/tissues using a laser. Very sensitive with PCR

can see intestinal villi

Disadv - limited amount of sample can be obtained at once

Microarrays

Analyse large sets of labelled genes at once. via specific probes .

Gene chips use photolithography to synthesise specific DNA sequences onto a silicon chip. Measures t

Scan across the whole chip: more transcript = more cDNA = higher intensity of the fluorescence

Application of microarrays

Gene profiling - Patients with different genetic expression patterns respond differently to treatment.

• Large-scale expression profiling helped identify distinct subtypes of lymphoma.

Limitations - noisy, and subjective due to variability in intensity

Sanger sequencing

chain terminators that block copying of DNA using dideoxynucleotides.

Gel electrophoresis determines sequence order by adding radioactivity to nucleotides

can read 10,000 bases a day.

Slow and labour intensive

High-throughput sequencing

Illumina sequencing - simultaneous sequencing of millions of DNA fragments,

Ion torrent & pyrosequencing -detects nucleotide incorporation based on pH

Nanopore - pass ssDNA through membranes. bases detected based on their affect on ionic current flow

What is a major application of single-cell RNA-Seq?

Identifying tumours, understanding developmental gene expression, and uncovering disease pathways

Summary of techniques

Technique	Use	Strengths	Limitations
PCR	Detects specific DNA/RNA sequences	High sensitivity, simple	Not quantitative
qPCR	Measures gene expression levels	Quantitative, real-time tracking	Limited to known targets
Microarrays	Large-scale gene expression profiling	Simultaneous analysis of thousands of genes	Fluorescence variability, limited to known sequences
RNA-Seq	Whole transcriptome sequencing	Digital quantification, novel transcript detection	Expensive, requires computational analysis

Post-translational modifications

Alter protein activity, stability, and location

ex. cleavage, GPI linkages phosphorylation, ubiquitination, methylation, acetylation

Cannot be studied by DNA/RNA analysis bc they aren’t encoded in the genome

What must be studied to understand protein function?

Location, interactions, function, and quantity.

Protein analysis methods

Dyes to stain proteins

Antibodies to recognise target proteins (immunofluorescence and western blots)

Electron microscopy reveals protein organisation

biochemical purification (isolated via synaptosomes, endosomes, protein complexes

SDS-PAGE Gel electrophoresis

RNA separated by size in polyacrylamide gel

Large proteins move slower than small

SDS page denatured proteins and coats them with a negative charge to strandardise protein charge

2D gel electrophoresis

proteins placed in pH gradient and move to their isoelectric point (no net charge) and stop.

Proteins then separate by size in SDS-PAGE

Each spot in the gel represents a protein. Each PTM appears as a different spot for the same protein

Types of antibodies

Feature	Polyclonal Antibodies	Monoclonal Antibodies
Source	Derived from multiple B-cell clones	Derived from a single B-cell clone fused with a cancer cell
Target Recognition	Recognize multiple epitopes (parts) of the same protein	Recognize one specific epitope on the protein
Production	Easier and faster to produce	More complex and time-consuming to produce
Specificity	Less specific	Highly specific
Batch Consistency	Varies between batches	High consistency across batches
Applications	General detection, research use	Diagnostic tools, therapeutic drugs, research

Western blotting

Proteins separated by SDS-PAGE

transferred to membrane for Ab incubation

primary Ab binds to target protein, secondary Ab binds to Fc region

Fluorescence/enzymes are used to detect

Specific Ab’s can detect phosphorylated proteins and ubiquitinated proteins

Protein-protein interactions

Use Abs to purify proteins and their partner

1. Ab binds to target proteins in mix

2. Ab is captured on beads, and complex is isolated

3. Western blot detects interacting proteins

Maps protein interaction networks

Mass Spectrometry

Identifies and quantifies proteins my measuring mass-to-charge ratio

1. digest proteins into peptides via trypsin

2. Analyse peptide masses via spectrometer

3. Compare to protein database to identify protein

used in proteomic analysis, tracking ptms, and studying protein interactions

What do chemical cross-linking and mass spectrometry reveal together

Which protein domains interact

How protein assemble into complexes

Used to build protein interaction network

Summary

SDS-PAGE → Separates proteins by size.

2D-PAGE → Separates proteins by charge + size.

Western Blot → Detects specific proteins using antibodies.

Co-IP → Identifies protein-protein interactions.

Mass Spectrometry → Identifies proteins and modifications without assumptions.

Immunofluorescence → Visualizes protein localization.

Cross-linking + Mass Spec → Reveals protein complex structure.