Lecture 4 - Electrophoresis and Synthesis

Question 1

what affects the decision on which type of gel to use in electrophoresis

Answer 1

the number of base pairs/length of DNA you are interested in

Question 2

what are the wells filled with before the sample is added

Answer 2

a buffer solution

Question 3

where is a source of contamination in electrophoresis

Answer 3

samples entering neighbouring wells when loading

Question 4

DNA absorbs UV light well, so why are UV visualisation methods not used when visualising gels in electrophoresis

Answer 4

UV light damages DNA very quickly

Question 5

how can phosphorus 32 labelling be used in the visualisation of gels

but why was this method not the best

Answer 5

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

Question 6

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

Answer 6

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)

Question 7

why are the stains used in visualising gels only used in small amounts

Answer 7

they can be carcinogenic so be careful

Question 8

other than electrophoresis in solution or in a gel medium what other way can electrophoresis be done

how does this method work

Answer 8

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

Question 9

what is a benefit of gels over capillary systems (6)

Answer 9

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.

Question 10

where can nucleic acids be used once they have been synthesised into polynucleotides (genes)
(give 3 uses)

Answer 10

in vaccines e.g Covid-19 to trigger an immune response

protein expression (genes can be used to create our own proteins)

biotechnology

Question 11

where can nucleic acids be used once they have been synthesised into oligonucleotides
(give 4 uses)

Answer 11

PCR
DNA profiling
Diagnosis (match a particular trait with a DNA strand)
Sequencing

Question 12

how are oligonucleotides and polynucleotides synthesised

Answer 12

start off with nucleosides (base+sugar)

these undergo a chemical synthesis to form oligonucleotides

these undergo an enzymatic synthesis to form polynucleotides

Question 13

what are polynucleotides also known as

Answer 13

genes

Question 14

how many bases are needed for something to be classed as an oligonucleotide

Answer 14

less than 200 bases

Question 15

how many bases are needed for something to be classed as a polynucleotides

Answer 15

1000-4000 bases

Question 16

To control the synthetic pathway of polynucleotides, what it is essential to have knowledge on

Answer 16

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

Question 17

however, what is the issue with targeting one phosphate group at a time in the chemical synthesis

therefore what method has been employed

Answer 17

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

Question 18

how does the solid phase synthesis method work

Answer 18

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

Question 19

what column is solid phase synthesis normally performed using

Answer 19

using CPG (controlled pore glass) columns

Question 20

what makes solid phase synthesis easy to do

Answer 20

it can be automated using a benchtop method to programme whatever residue we want

Question 21

what are the 4 stages in the synthetic cycle involved in solid phase synthesis of polynucleotides

Answer 21

1 = coupling then wash
2 = cap then wash
3 = oxidise then wash
4 = deprotect then wash

repeat as many times as you need

Question 22

explain the coupling process in the synthetic cycle involved in solid phase synthesis of polynucleotides

Answer 22

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

Question 23

what happens to the efficiency of the coupling as the DNA strand length increases

Answer 23

as the number of bases increases the efficiency decreases = why we can’t use this method to synthesise an entire genome

Question 24

why do we not achieve a 100% efficient coupling yield

Answer 24

as one couple failure (wrong base) is seen for every 200 strands

Question 25

explain the capping process in the synthetic cycle involved in solid phase synthesis of polynucleotides

Answer 25

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

Question 26

what can the lack of capping or failure to cap result in

Answer 26

deletion mutations = errors in the DNA sequence compared to the desired sequence set out to synthesise

Question 27

if any errors in the synthesised DNA sequence are not identified what does this result in (3)

Answer 27

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

Question 28

explain the oxidation process in the synthetic cycle involved in solid phase synthesis of polynucleotides

Answer 28

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

Question 29

explain the deprotection process in the synthetic cycle involved in solid phase synthesis of polynucleotides

Answer 29

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

Question 30

once the polynucleotide or oligonucleotide has been cleaved from the solid support and deprotected what happens

Answer 30

it is precipitated from solution in ethanol using salts to stabilise the DNA

the failed strands need to be removed = purification

Question 31

what two methods are used to purify the synthesised poly/oligo nucleotide

briefly describe each

Answer 31

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

Question 32

What purification method should be used to separate failed and finished strands using DMT to help

Answer 32

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

Question 33

following the purification process of the newly synthesised nucleotide sequence what happens

Answer 33

it is held at a stable pH in solution and then freeze dried for storage as a solid

Question 34

what is the issue when synthesising oligonucleotides when we want to produced genes e.g vaccines

Answer 34

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

Question 35

do vaccines tend to use RNA or DNA

Answer 35

RNA as they are designed to reprogramme our cells

Question 36

so how are full length genes made from multiple chemically synthesised oligonucleotides

Answer 36

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

Question 37

what type of genes is this synthesis (solid phase) suitable for and why

Answer 37

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

Question 38

how can a gene be synthesised if it is made of more than 2000 base pairs

Answer 38

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

Question 39

what is a method used when creating vaccines to scale up the target gene you have synthesised (6)

Answer 39

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

Question 40

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

Answer 40

by encasing it in lipid nanoparticles which are then incorporated into the vaccine solution