Electrophoresis & Synthesis Flashcards
1
Q
Synthesis of oligonucleotides
A
- Take a residue and attach it to the surface of the OH bond (coupling)
- Wash
- Cap to stop further interactions of that sample
- Wash
- Oxidise the surface
- Wash
- Deprotect the sample to allow the process to repeat until we’re happy with the length
2
Q
Synthetic cycle of oglionucleotides
DMT
A
- Dimethoxytrityl (DMT) is used to terminate further reactions
- It is also an indicator of how well the coupling has been on the surface
- This approach can’t be used to build an entire gene as it has limits
- The theoretical limit is 200 base pairs
- There is a lot of wastage using this methog
3
Q
Capping
A
Killing unreacted strands
4
Q
Synthetic cycle of oligonucleotides
Capping
A
- One coupling failure every 200 strands so there is a naximum best yield of 99.5%
- Leads to deletion mutations (missing certain bases)
- Primer may not bind
- Error will be carried froward to protein synthesis
- Other biochemistry can be altered
- Capping is done to delete the failures
5
Q
Synthetic cycle of oligonucleotides
Oxidation
A
- Iodine is used as the oxidising agent
- Water and pyrimidine are also required to obtain P=O bond
- THF solvent is used
- Pyridium iodide side product
6
Q
Synthetic cycle of oglionucleotides
Deprotection
A
- Dichloroacetic acid in dichloromethane is used
- Trityl cation is stabilised by resonance making it easy to remove
- Deep yellow/orange colour used to measure coupling efficiency
7
Q
Purification of oligonucleotides
A
- Once finished through final steps of cleavage and deprotection and then precipitated from solution in ethanol with salt to stabilise
- Failed strands have to be removed
8
Q
Purification of oligonucleotides
PAGE
A
- PAGE gel is often used in the purification step
- Run a gel and we find the length we want and cut them out
- Freeze down to extract them and then they’re squeezed out
- This method isn’t done now as we’ve moved towards the HPLC / capillary purification
9
Q
HPLC Purification of oglionucleotides
Columns
A
- Choice of column allows us to select what we want.
- Silica Column - polar (e.g. H-bond forming) molecules will stick to the column
- C-18 Silica Column - ‘Reverse phase’ - non-polar stick to column
10
Q
HPLC Purification of Oglionucleotides
A
- Keep the DMT on until the final product as it is very hydrophobic so it is easy to separate full length strands from failed strands that have to be removed
- DMT group we want to remove in final product so if anything is on there is an easy target as it is very hydrophobic.
- We can separate out anything thing we don’t want as it will be attached to the DMT group.
- Once separated - held at a stable pH in solution and then freeze dried for storage as a solid.
- DNA absorbs at 260nm
11
Q
Maximum and typical length of chemically synthesised oglionucleotides
A
- Maximum length of chemically synthesised oligonucleotide: 200 bases
- Typical length of chemically synthesised oligonucleotide: 50 bases
12
Q
Gene synthesis
Ligations
A
- Use ligase to bridge between the pairs to stick them together into a single gene
- Chemically stick them together
- Ligase bridges between phosphates rgroup to close the two sugars
- We have a few errors as all bases of the final gene have been chemically synthesised
- This method is suitable when the taget gene is <2000 bases as it is limited by chemial synthesis
- PCR amplication is performed next
13
Q
Gene synthesis
Polymerase cycling assembly
A
- We only synthesis some of the fragments chemically
- We do this in an offset pattern and we leave base in between that aren’t artificially synthesised by us and then polymerase is used to polymerase across the strands and fill the gaps.
- We have slightly less control so there is errors, for example an incorrect base pair
- That error site is then cut off enzymatically
- It is then taken and melted to break it down using stability of the DNA and other factors.
- It is then heated back up and stuck back on at the top to allow for polymerase to fix the gaps again.
- Eventually this results in a target gene that is >2000 bases
- As this involves less chemical synthesis is allows us to get longer genes however this approach allows for more potential errors compared to ligation.
14
Q
Gene synthesis
Polymerase cycling assembly basic steps
A
- Only synthesis minimal fragments chemically (the important ones)
- Polymerase
- Enzymatically cut off error site
- Melt, anneal and polymerase
15
Q
Purification & Scale up
A
- Further scale up after the polymerase cycling assembly
- We take the chemically synthesised chains from PCA and enzymatically insert that into bacteria
- It is then swabbed on a plate of agar and allow them to grow up
- The bacteria will make more of it efficiently
- We sequence the ones that have managed to grow up without lots of defects as the bacteria is good at producing it
- The relevant DNA is extracted from the DNA in order to get the target DNA
- This the has to be turned into linear DNA from plasmid DNA
