Lecture 4 - Electrophoresis and Synthesis Flashcards
what affects the decision on which type of gel to use in electrophoresis
the number of base pairs/length of DNA you are interested in
what are the wells filled with before the sample is added
a buffer solution
where is a source of contamination in electrophoresis
samples entering neighbouring wells when loading
DNA absorbs UV light well, so why are UV visualisation methods not used when visualising gels in electrophoresis
UV light damages DNA very quickly
how can phosphorus 32 labelling be used in the visualisation of gels
but why was this method not the best
phosphorus 32 is a beta emitter with a half life of 14days, can be used to label the samples and when exposed to photographic film they can be visualised
due to the isotopic release of radiation, bands on the gel were often seen to be smeared making interpretation difficult
what two methods are currently used to visualise gels and give an example of each
what are these molelcules also called and how do they bind to the DNA
most common are fluorescent stains = ethidium bromide (flash with light and it fluoresces)
visible stains= StainsAll
also called intercalators, bind into the major groove of DNA
(this does slowly degrade DNA overtime as it breaks hydrogen bonds between bases)
why are the stains used in visualising gels only used in small amounts
they can be carcinogenic so be careful
other than electrophoresis in solution or in a gel medium what other way can electrophoresis be done
how does this method work
via a capillary
the DNA is in one buffer solution
a fine capillary bridges
between two buffer solutions and is filled with a small amount of acrylamide gel
a detector lies around a certain part of the capillary and visualises anything that passes through it
we obtain a graph with y axis = detector intensity and x axis = time
what is a benefit of gels over capillary systems (6)
1 = gel are simpler
2 = gel are less expensive
3 = gel can handle greater sample volume
5 = easier to visualise gels using stains than interpreting the graph from the detector in capillary
6 = gels can be multiplexed whereas capillaries can’t
but gels are slower, can’t be automated and have lower sensitivity.
where can nucleic acids be used once they have been synthesised into polynucleotides (genes)
(give 3 uses)
in vaccines e.g Covid-19 to trigger an immune response
protein expression (genes can be used to create our own proteins)
biotechnology
where can nucleic acids be used once they have been synthesised into oligonucleotides
(give 4 uses)
PCR
DNA profiling
Diagnosis (match a particular trait with a DNA strand)
Sequencing
how are oligonucleotides and polynucleotides synthesised
start off with nucleosides (base+sugar)
these undergo a chemical synthesis to form oligonucleotides
these undergo an enzymatic synthesis to form polynucleotides
what are polynucleotides also known as
genes
how many bases are needed for something to be classed as an oligonucleotide
less than 200 bases
how many bases are needed for something to be classed as a polynucleotides
1000-4000 bases
To control the synthetic pathway of polynucleotides, what it is essential to have knowledge on
the phosphorus chemistry as these are how connections between nucleosides are formed - the phosphate group (PO4) acts as the linker between them
so targeting these linkages in synthetic pathways is probably the best method as the bases you want can be added one by one
however, what is the issue with targeting one phosphate group at a time in the chemical synthesis
therefore what method has been employed
as this is in solution we would need to clean and purify the product each time = too time consuming
so solid phase synthesis is used
how does the solid phase synthesis method work
one end of the strand being synthesised is attached to a solid support e.g a glass or polystyrene bead
the first based is normally already attached to the bead
the excess reagents and side products can be washed away
when the strand is complete tit can be cleaved from the solid support
multiple beads can be used if needed
what column is solid phase synthesis normally performed using
using CPG (controlled pore glass) columns
what makes solid phase synthesis easy to do
it can be automated using a benchtop method to programme whatever residue we want
what are the 4 stages in the synthetic cycle involved in solid phase synthesis of polynucleotides
1 = coupling then wash
2 = cap then wash
3 = oxidise then wash
4 = deprotect then wash
repeat as many times as you need
explain the coupling process in the synthetic cycle involved in solid phase synthesis of polynucleotides
the addition of a new nucleotide monomer to the growing chain at the 3’ of the new monomer
a protecting group is on the 5’ end of the new monomer to stop further reactions than the intended monomer being added
an example of a protecting group is DMT = dimethoxytrityl = a hydroxy protecting group that attaches to the 5’ end of the base
what happens to the efficiency of the coupling as the DNA strand length increases
as the number of bases increases the efficiency decreases = why we can’t use this method to synthesise an entire genome
why do we not achieve a 100% efficient coupling yield
as one couple failure (wrong base) is seen for every 200 strands
explain the capping process in the synthetic cycle involved in solid phase synthesis of polynucleotides
the killing of unreacted strands
preventing the failures that can happen in the coupling step by preventing subsequent reactions occurring that are not intended
crucial for minimising sequence errors
what can the lack of capping or failure to cap result in
deletion mutations = errors in the DNA sequence compared to the desired sequence set out to synthesise
if any errors in the synthesised DNA sequence are not identified what does this result in (3)
1 = failure of primer binding in DNA replication
2 = error in sequence being carried forward to the protein synthesise = could result in wrong amino acids
3 = other biochemistry of the protein function being altered
explain the oxidation process in the synthetic cycle involved in solid phase synthesis of polynucleotides
the phosphate linkage is converted into a more stable form
start with PO3 and a lone pair on the phosphorus and the result is the PO4 group with one double bonded oxygen between the nucleosides
usually achieved using a mild oxidising agent such as iodine
water and a pyridine are also needed and a THF solvent
side product = pyridium iodide
explain the deprotection process in the synthetic cycle involved in solid phase synthesis of polynucleotides
the removal of the protecting group (DMT)
using dichloroacetic acid in dichloromethane
the trityl cation is stabilised by resonance so it is easy to remove
the coupling efficiency is measured by a deep yellow/orange colour
once the polynucleotide or oligonucleotide has been cleaved from the solid support and deprotected what happens
it is precipitated from solution in ethanol using salts to stabilise the DNA
the failed strands need to be removed = purification
what two methods are used to purify the synthesised poly/oligo nucleotide
briefly describe each
PAGE (the gel) purification
- we know the base pair length of he desired strand so run on the electrophoresis gel and cut out the portion that contains the target strand using the ‘freeze+squeeze’ method
or
HPLC purification (more common now)
- using a silica column polar molecules with stick to the column
- or reversed phase using a C-18 silica column so non polar species stick to the column
What purification method should be used to separate failed and finished strands using DMT to help
HPLC reversed phase = using a C-18 silica column
DMT is highly hydrophobic so with stick to the column separating the full strands from the failed ones
following the purification process of the newly synthesised nucleotide sequence what happens
it is held at a stable pH in solution and then freeze dried for storage as a solid
what is the issue when synthesising oligonucleotides when we want to produced genes e.g vaccines
oligonucleotides max length to be chemically synthesised is 200 bases
the typical length of synthesised oligonucleotides is 50 bases
and vaccines - genes here tend to be 1,273 bases or longer!!
the efficiency goes down as the base length increases so it would be useless trying to make one this size
do vaccines tend to use RNA or DNA
RNA as they are designed to reprogramme our cells
so how are full length genes made from multiple chemically synthesised oligonucleotides
the oligonucleotide sequences can be bridged together and stuck to form the full length gene
this is done using ligase to bridge between the phosphate groups
the full gene can then go on to be amplified using a PCR where it can be put into things like vaccines
what type of genes is this synthesis (solid phase) suitable for and why
genes with less than 2000 base pairs
due to the controlled chemical synthesis performed there re likely to be few errors in the final gene (better than what our body does)
but the length of the gene this can be done for is also limited by this chemical synthesis
how can a gene be synthesised if it is made of more than 2000 base pairs
a method called polymerase cycle assembly
only synthesise some of the fragments chemically that have a 20-30 base overlap with the next fragment
then use polymerase to fill in the gaps for you
more errors are likely to be seen as there is less control here due to the whole thing not being chemically synthesised like before (with less than 2000 base pairs)
if a base pair is known to be faulty enzymes can be used to remove them, melt the strand anneal it and allow polymerase to fill the gap correctly
allows for longer nucleic acids/genes to be synthesised
what is a method used when creating vaccines to scale up the target gene you have synthesised (6)
1 = insert the gene into a plasmid using enzymes
2 = transform this into a bacteria cell = effective at taking in something and reproducing it
3 = put on an agar plate and allow the bacteria to multiply
4 = the colonies can identified for which contain the target gene and then the DNA can be extracted from the plasmids
5 = the DNA is linearized and trimmed
6 = it can then undergo transcription to form the RNA for the vaccine
how is the RNA then protected when it is put into vaccines so the body doesn’t reject it and allows it to be incorporated into the vaccine itself
by encasing it in lipid nanoparticles which are then incorporated into the vaccine solution
