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Oligo synthesis + blotting - Coggle Diagram
Oligo synthesis + blotting
DNA synthesis in vitro - oligonucleotide synthesis
No template needed
Phosphoramidite nucleoside building blocks
Requires protective groups
DMT-protected phosphoramidite nucleosides
Free amino groups protected too
3'-5' direction
Solid phase chemical synthesis
Process
Loading
Synthesis cycle
Deprotect 5'-OH on 5'end of growing primer
Add protected 2'-deoxynucleoside phosphoramidite
Cap excess unreacted 5'-OH to minimise oligo's with single deletions
Oxidise phosphite triester into phosphotriester
Repeat synthesis cycle x times until desired length
Cleave from solid support + deprotect
5'-3' primer produced
Immobilise DMT-5'-OH protected nucleoside on solid support
Purification after removes shorter/incomplete oligos
Highly reproducible
Can be done de novo
Defined reaction steps
Amenable to automation
Oligonucleotide synthesisers
Automated oligonucleotide synthesis
Complete gene synthesis
Solid phase synthesis limited in primer length
~200bp
Genes are at least ~1000bp
Base-pair driven assembly of partially overlapping oligonucleotides
Gaps sealed by enzymatic methods
Error prone
Post-assembly quality control expensive
Allows for cusomtisation of codon usage
Introduction of several simultaneous mutations
Beyond 4 bases
Phosphoramidite versions of labels
Incorporated on 5' end of oligo as final stage of synthesis
i.e. before cleavage from solid support
Beyond DNA
Very strong binding to RNA or DNA with only short stretch of bases
Mainly used to block particular regions
Down regulation of transcription + translation
DNA hybridisation
DNA can denature in solution
Solvents
Salts
Chaotropic agents
Heat
When denatured
Label added
Measurable signal from label
Key parameters
Sequence composition
SEquence length
Ionic strength
pH
Temperature
Backbone chemistry
Non-natural nucleotides
Mismatches
Calculations
[ssDNA1] and [ssDNA2] determine how much dsDNA forms
Basic
Each A-T pair contributes 2\(^o\)C
Each G-C pair contributes 4\(^o\)C
T\(_m\) = 2 x (A+T) + 4 x (G+C)
Works well for short probes
Advanced
Nearest neighbour modelling
Works for longer probes - up to 120bp
T\(_m\) = \(\frac{\Sigma \Delta H}{A+\Sigma \Delta S+R x ln(\frac{C}{4})}\) - 273.15 + 16.6log[Na\(^+\)]
Types of probe
Genomic (gDNA)
Isolated/cloned segment
Oligonucleotide
Synthesised known sequence
Complementary (cDNA)
Synthesised from mRNA by reverse transcription
Heterologous probe
From another organism
PCR-generated fragments
Conserved 'flanking' regions can be used to generate probes of varying regions
Degenerate oligonucleotide probes
Allows for variations at certain positions
More than one phosphoramidite nucleotide added during oligo synthesis
Regions with high proportion of amino acids coded by single or two possible codons are suited
Labelling probes
Types of label
Radiolabelling
\(^{32}\)P, \(^{33}\)P or \(^{35}\)S
Film exposure
Enzymatic methods
Substrate detection
Chromogenic/colorimetric
Fluorescent
Light-responsive colour measurement
Chemiluminescent
Light producing reaction
Film exposure
Methods of label incorporation
Post-DNA synthesis
In vivo + in vitro
Typically free 5' end
Labelled oligonucleotide
Expensive
Use of labelled nucleotides that compete with regular nucleotides
\(^{32}\)P-labelled nucleotides
dUTP-labelled nucleotides
Incorporate in replacement of dTTP by DNApols
Nick translation labelling
DNase I creates single-stranded nick
Exonuclease activity of DNApol
Feed with labelled nucleotides
Random hexamer labelling
Heat
Hexamers anneal at random
Then PCR with no exonuclease activity
Ligase zips together
PCR labelling
Primers anneal to region of known/homologous sequence that flank the probe region
Labelling can be done via incorporation of modified nucleotides or labelled primers
Enzyme/fluorochrome probes
Use modified nucleotides
Labelling of probe with molecule that binds strongly to protein coupled with an enzyme
Optical signal generated by converting a substrate
Enzyme
Typically alkaline phosphatase or horseradish peroxidase
Binding part of complex
Ab specific to label on probe or streptavidin (specific for biotin label)
Direct labelling with fluorochrome
No enzymes/substrates
Simpler
Requires fluorimeters (light source, wavelength specific sensor)
Background risk
Autofluoresence
Both methods rely on DNApol ability to incorporate labelled nucleotides
labelled dUTP instead of dTTP
Standard kits available to buy
Protein detection
Total protein
Stains
Coomassie blue
Ponceau S
Silver stain
Specific proteins detected using Ab labelled with enzyme, fluorophore, biotin, chemilumiphore etc
Blotting
Transfer of molecules to thin membrane for analysis
e.g. DNA, RNA, protein
Usually separated
Electrophoresis
Nitrocellulose/PVDF (protein) or nylon (NA) membranes
Capillary or electrophoretic transfer
Immobilise or fix on membrane by heat or UV
Store for long periods
Target molecules easily accessed by detection agents
Can be re-used
Stripping + re-probing
Why?
Find one target molecule in complex biomatrix
Highly specific under correct (stringent) conditions
Extremely sensitive
Terminology
DNA
Southern
RNA
northern
Protein
western
Southern blotting
Blotting
Transfer of DNA from gel to membrane
Gel on support
Membrane on top of gel
Weighted with paper towels on top of membrane
Buffer soaks through to paper towels
DNA bands on membrane in same position as gel
Probing
Wash DNA probe over membrane
Probe anneals to complementary DNA
Detection of relative position (size) and intensity (amount) of hydridisation
Excess washed off
Can be sped up by electroblotting
Reducing background
Membrane supports non-specific binding
Probe sticks indiscriminately
Signal to noise ratio (SNR)
Signal can't be increased
Background can be decreased