Oligo Synthesis and Blotting Flashcards
1
Q
DNA synthesis - biochemical
A
- 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
2
Q
DNA synthesis - chemical
A
- 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
3
Q
Loading in solid phase synthesis
A
- Immobilize DMT-5’-OH protected nucleoside on solid support
- solid support: controlled pore glass (CPG) or macroporous polystyrene (MPPS)
4
Q
Synthesis cycle in solid phase synthesis
A
- 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
5
Q
What happens after synthesis cycle in solid phase synthesis?
A
- Repeat synthesis cycle x-times until desired length of correct sequence
- Cleave from solid support and deprotect
6
Q
Oligonucleotide synthesis
A
- 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
7
Q
Complete gene synthesis
A
- 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
8
Q
DNA synthesis - beyond the 4 bases
A
- 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
9
Q
Oligonucleotide synthesis: Beyond ATCG
A
- DNA / RNA
- Phosphorohioate
- 2’-fluoro
- 2’-fluoroarabino
- 2’-O-methyl
- 2’-O-methoxymethyl
- 2’-5’-bridged
- 2’5’-locked
10
Q
Oligonucleotide synthesis: Beyond DNA
A
- 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
11
Q
Denaturation/Hybridisation of DNA in Solution
A
- 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
12
Q
Hybridisation of DNA Probe
A
- 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
13
Q
Hybridisation: Key Parameters
A
- 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
14
Q
Types of probe
A
- 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
15
Q
Degenerate Oligonucleotide Probes
A
- 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
