Oligo Synthesis and Blotting

• In vivo DNA/RNA synthesis is templated, i.e. it needs a complementary strand to template the correct order • DNA polymerases can only add each new dNTP to the 3’-OH group of the proceeding nucleotide - synthesis occurs exclusively in the 5’ to 3’ direction • A primer is required • PCR has the same requirements

• Multiple repeated solid phase chemical synthesis steps, no need for a template • Growing DNA fragment is immobilized on solid support, which allows for easy removal of residual material after a synthesis step • Phosphoramidite nucleosides as building blocks • Requires protective groups • Proceeds in 3’ to 5’ direction (reverse of DNA polymerases) • Completely de novo, i.e. no priming needed

• Immobilize DMT-5’-OH protected nucleoside on solid support - solid support: controlled pore glass (CPG) or macroporous polystyrene (MPPS)

• wash away reagents from previous cycle in between every step • Deprotect 5’-OH on 5’-end of growing primer • Add protected 2’-deoxynucleoside phosphoramidite • Cap excess unreacted 5’-OH to minimize oligo’s with single deletions • Oxidize phosphite triester into phosphotriester

• Repeat synthesis cycle x-times until desired length of correct sequence • Cleave from solid support and deprotect

• Phosphoramidite synthesis: 3’ to 5’ direction (opposite to ‘natural’ 5’ to 3’ direction), can be done ‘de novo’ (i.e. without a template) • Defined reaction steps • Highly reproducible • Repeats of the same reaction: E.g. ATCGATCAGGCAGT…, Same as playing a tune with four notes • Amenable to automation • “Play” the oligo: four letter keyboard • Oligonucleotide synthesisers

• Solid phase synthesis is limited in terms of maximum fragment length (constant progress pushes limit further) • Base-pair driven assembly of partially overlapping oligonucleotides, gaps can be sealed by various enzymatic methods: Error-prone: post-assembly quality control is a costly affair • Price of synthetic genes has dropped from 20 USD/base in late 90’s to 0.07 USD/base in 2021 • Allows for customization of codon usage, simultaneous introduction of multiple mutations

• Phosphoramidite version of certain labels can be incorporated on 5’-end of the oligo as last step of solid phase synthesis, i.e. before cleavage from solid support and deprotection

• DNA / RNA • Phosphorohioate • 2’-fluoro • 2’-fluoroarabino • 2’-O-methyl • 2’-O-methoxymethyl • 2’-5’-bridged • 2’5’-locked

• phosphorodiamidate Morpholino oligomer (PMO) • PMOplus • Peptide Nucleic Acid (PNA) • Very strong binding to RNA (all) or DNA (PNA) with only a short stretch of bases • Mainly used to ‘block’ particular regions of DNA and RNA (antisense binding), knocking down transcription and translation

• dsDNA: denaturing agents such as solvent, salts, high pH or chaotropic agents (8 M urea or 6 M guanidine), heat • becomes denatured DNA: removal of denaturing agents, cool • dsDNA (re)forms (hybridises) : complementary base pairs re-formed

• re-formed dsDNA • denature by heat or chemicals (or combination thereof) • complementary oligonucleotide and label attached to denatured DNA • many identical DNA molecules → measurable ‘signal’ from the label

• Sequence composition: A/T vs C/G: global effect, local effect. • Sequence length • Ionic strength (Na+, K+, Mg2+, Mn2+, Ca2+, Cl-, F-, etc.) • pH (influences ionisation states) • Temperature • Backbone chemistry • Non-natural nucleotides • Mismatches

• Genomic DNA (gDNA): isolated/cloned segment • Oligonucleotide: synthesized known sequence: specific fragment based on known DNA/RNA sequence, degenerate probe based on peptide fragment • Complementary DNA (cDNA): synthesised from mRNA using reverse transcription • heterologous probe from another organism: based on a homologous region expected to be present in related organisms • PCR-generated fragments: degenerate primers based on peptide fragments can be used to amplify region with otherwise unknown sequence, conserved ‘flanking’ regions can be used to generate probes of variable regions

• Allows for variations at certain positions • More than one phosphoramidite nucleotide added during oligonucleotide synthesis • Regions with high proportion of aa’s coded by only a single (Met, Trp) or two (Asn, Asp, Cys, Gln, Glu, His, Lys, Phe, Tyr) possible codons are most suited

• Radiolabelling with 32P, 33P or 35S: (film exposure) • Enzymatic methods: substrate detection e.g. chromogenic / colorimetric • Fluorescent label: light-responsive colour measurement • Chemiluminescent label: light-producing reaction (film exposure)

• Post-DNA synthesis (both in vivo and in vitro, typically on the free 5’-end) • Labelled oligonucleotides (expensive) • Use of labelled nucleotides that compete with regular nucleotides: 32P-labelled nucleotides, dUTP-labeled nucleotides that can incorporate in replacement for dTTP by DNA polymerases

• Primers anneal to region of known/homologous sequence (respectively using a specific or degenerate oligonucleotide) that flank the region from which you want to generate a probe (for which the exact sequence does not need to be known) • Labelling can either be done via incorporation of modified nucleotides (dNTPs) during the PCR or by using labelled primers

• Labelling of probe with specific molecule that binds strongly to protein coupled with enzyme that can generate an optical signal by converting a substrate • Enzyme: typically alkaline phosphatase or horseradish peroxidase • Binding part of the complex: antibody specific to the label on the probe or streptavidin (specific for biotin label on probe)

• Direct labelling of the probe with a fluorochrome label • No enzymes, substrates, etc.: Faster, simpler • Require Fluorimeters (light source, wavelength specific sensor) • Background risk (autofluorescence)

• Signal generated by conversion of substrate by enzyme (Alkaline phosphatase (AP) or Horse Radish Peroxidase (HRP)) coupled to an antibody. Reaction product is chemiluminescent • antibody selectively binds to a molecule that is covalently connected to the probe • Labelled dUTP incorporated during labelling reaction instead of dTTP

• Signal originates from fluorophore directly connected to probe: fluorescein, Cy3 and Cy5 (often used in combination in microarrays) • Labelled dUTP is incorporated during labelling reaction instead of dTTP

• Total protein detection: stains eg. coomassie blue, ponceau S, Silver stain, etc • Specific proteins (Protein of Interest (POI)) detected using antibodies labelled with enzyme, biotin, fluorophore, chemilumiphor, etc., similar to ELISA

• Transfer of molecules (DNA, RNA or protein) to a thin membrane for further analysis: usually separated (e.g. by electrophoresis), nitrocellulose/PVDF (protein) or nylon (NA) membranes, capillary or electrophoretic transfer • Immobilise and fix on the membrane by heat or UV • Store for long periods • Target molecules readily accessed by detection agents • Can be re-used (stripping and re-probing)