GENE MODIFICATION Flashcards
Totipotent Cells
cells that can mature into any type of body cell, i.e. they are undifferentiated
Where we get totipotent cells from
adult stem cells, embryonic stem cells (however they only occur for a limited amount of time), induced pluripotent stem cells
When a cell becomes specialised, we say it has lost its totipotency. Whilst it has totipotency, it can be used to treat genetic diseases.
Features of totipotent cells
Will replace themselves, i.e. they are immortal (except for multipotent cells which divide to form only a limited number)
They are undifferentiated
In plants some cells remain totipotent throughout life. These plants are used naturally and artificially in 3 ways
Vegetative Propagation
Cuttings
Tissue culture
Cells specialise themselves by controlling which genes are expressed in two ways
Oestrogen; only works in Target Cells, target cells are cells
SiRNA
Oestrogen
Transcription begins when the gene being transcribed is stimulated by Transcription Factors
Transcription Factors have an active site which binds to a specific sequence of DNA to start transcription, this specific sequence is called the PROMOTER SEQUENCE
This active site is blocked by an inhibitor molecule which is attached to the Transcription Factor and Receptor, this inhibitor molecule prevents transcription and thus prevents the gene from being expressed
Oestrogen diffuses into cell via LIPID DIFFUSION
Oestrogen then binds to the active site of the RECEPTOR
This changes the shape of the RECEPTOR molecules, therefore releasing the inhibitor molecule and thus leaving the active site of the Transcription Factor vacant
This allows Transcription Factor to ENTER THE NUCLEUS and attach itself to a specific base sequence on a DNA promoter
How Transcriptional Factors are important for synthesis of particular proteins
They bind to DNA at specific region called the PROMOTER SEQUENCE
They then stimulate RNA Polymerase to arrive and begin transcription
If we want a cell to stop expressing its genes, we can introduce a molecule to compete with Oestrogen for the active site of the RECEPTOR.
SiRNA
An enzyme cuts large double stranded RNA into smaller sections called Small Interfering RNA (SiRNA)
One of the strands of the SiRNA combines with an enzyme called RNA-induced silencing complex
SiRNA guides enzyme to mRNA and forms complimentary base pairs with a specific mRNA
The enzyme still attached to the SiRNA then breaks the mRNA into smaller pieces
mRNA no longer capable of translation
Therefore target gene can no longer make a specific protein as mRNA has been cut into pieces
SiRNA is used to block genes that cause diseases, i.e. can be used to stop genetic diseases by stopping the gene from producing proteins.
Genetic Modification
Isolation: finding DNA fragments that have the desired gene that produces the desired protein, then separating it
Insertion: putting the desired DNA fragments into a vector
Transformation: transferring the DNA into a suitable host cell
Identification: deducing which host cells have taken up the DNA and which haven’t
Growth: making more copies of the host cell
Isolation can be done in two ways
Use Reverse Transcriptase
Reverse Transcriptase catalyses the production of DNA FROM RNA
PROCESS:
Find cell that produces desired protein and remove mRNA from this cell
Add Reverse Transcriptase to make a strand of DNA from mRNA, this strand that is made is called Complementary DNA (cDNA)
To make the other strand of DNA, the enzyme DNA Polymerase is used to form complimentary base pairs with cDNA, therefore cDNA acts as a template for the second strand
Using Reverse Transcriptase is better than taking actual DNA from the organism because
DNA contains Introns too whereas mRNA will contain only activated desired genes
Makes stable copy of a gene since DNA is less readily broken down by enzymes than RNA
It makes genes easier to find, as there are thousands of genes but only a few types of mRNA exist
Isolation: Use Restriction Endonucleases
Restriction Endonucleases cut a DNA double strand VIA HYDROLYSIS at a specific sequence of bases called a recognition sequence in order to isolate the useful DNA sequence
Usually, the DNA sequence the Restriction Endonucleases will cut will be a PALINDROMIC SEQUENCE, which means: sequence of one strand is the reversal of the corresponding strand.
Restriction Endonucleases cuts DNA in two ways
Blunt Ends and Sticky Ends
The sticky ends/blunt ends can be attached to other sticky ends/blunt ends that have been cut by the SAME Restriction Endonucleases using DNA LIGASE to form a Recombinant DNA VIA CONDENSATION:
Sometimes, there are quite a few recognition sequences leading to Restriction Endonucleases causing many fragments, but scientists only need one specific fragment containing the desired DNA fragment, therefore they will use Gel Electrophoresis to separate the fragments. (Gel Electrophoresis will be explained shortly) Once the fragments are separated, they will use a DNA Probe (will be explained shortly) which will bind to a complimentary base sequence in the gene, the fluorescence/radioactivity of the DNA Probe will tell us which fragment contains the desired gene. Then we will movie onto insertion, etc.
The overall isolation using Restriction Endonucleases
Restriction enzymes cut DNA at specific base sequences VIA HYDROLYSIS forming a sticky end
Same restriction enzyme also cuts DNA into which gene is inserted forming another sticky end
DNA Ligase joins the two pieces of DNA together which happen to be complimentary VIA CONDENSATION to form recombinant DNA
Why DNA base sequences must be cut with the SAME Restriction Endonucleases
So that it cuts VIA HYDROLYSIS at the same base sequence therefore allowing pairing of bases when DNA Ligase is used
Why Restriction Endonucleases (enzyme) will cut DNA only at specific recognition sites
Different lengths of DNA have different base sequences
The enzyme’s active site has different shapes
Therefore only specific sequence will fit active site of an enzyme
One the DNA sequence has been isolated; we may continue the process of insertion, transformation, etc. in two ways
In Vivo: using living cells
In Vitro: using the Polymerase Chain Reaction
Before a plasmid with the desired gene is put into, the bacteria, the gene for conjugation is removed from the plasmid because
This prevents bacteria cells from conjugating
Therefore stops the transfer of DNA, thus reducing the risk of other organisms getting altered genes
Why biologists will often use plasmids which contain antibiotic resistant genes
It can act as a marker
This allows detection of the cells containing the desired DNA
If in the exam it asks for the definition of the term ‘sticky ends’, you will write
Cut ends of DNA
One strand longer than the other
Can attach to complimentary DNA
In Vivo
1) Isolation: This has been completed
2) Insertion: putting the desired DNA fragments into a vector
Using Restriction Endonucleases we can combine the DNA of one organism with that of another organism as long as the Restriction Endonucleases used to create the sticky ends in both was the same.
PROCESS OF INSERTION
DNA isolated from cell which manufactures desired protein
DNA and a plasmid both cut using the same Restriction Endonucleases, therefore creating DNA fragment with sticky end and plasmid with a hole that has a sticky end
DNA fragments and plasmid with holes are mixed together with DNA LIGASE, the DNA fragment fits into the hole in the plasmid like a jigsaw puzzle therefore forming recombinant DNA
The plasmids containing the DNA fragment will be the vectors, which are materials used to transport DNA into the HOST CELL
Recombinant DNA
Contains genes of 2 types of organisms (plasmid will become like this)
If asked in the exam, how a DNA fragment in inserted into a vector, you will write
Cut vector DNA with same Restriction Endonucleases used to cut DNA containing desired gene
Use DNA Ligase to join the sticky ends
Why plasmid is described as a vector
It can carry foreign DNA into a bacteria cell
Properties of a vector
Big enough to hold gene
Circular so that it is less likely to be broken down
Contains control sequences so that it can replicate the gene
After the DNA fragment is inserted into the plasmid, the plasmid will function differently, i.e. if you replaced a resistance gene with the desired gene, then the plasmid will no longer be resistant like it was before because:
The plasmid DNA base sequence has been altered
Therefore different proteins will be made
Transformation: putting the vector into the host cell
Transformation is obtained by mixing the plasmids and bacterial cells together in a medium of Ca2+ ions which causes the bacterial cells to become permeable to the plasmids
Identification: deducing which host cells (bacterial cells) have taken up the DNA and which haven’t
The method used for Identification is Gene Markers
Antibiotic-Resistance Markers
Remove resistance gene to a certain antibiotic from a plasmid and add the desired gene in its place, put this plasmid into bacterial cells
Put bacterial cells on nutrient agar plates
Transfer a small colony of the bacterial cells onto a second plate in exactly the same position as the original plate
Add the antibiotic to the second plate (the antibiotic for which the plasmid containing the desired gene no longer has resistance)
The cells that die are the ones that have taken up the desired gene as they have replaced their antibiotic resistance gene with the desired gene
The colonies in exactly the same position on the original plate are the ones that possess the required gene
Fluorescent Markers
Transfer gene of Jellyfish that produces a green light into the plasmid, put all plasmids into bacterial cells which will all begin to glow
Now transplant the desired gene into centre of the green light gene in plasmid
The bacterial cells which contain the plasmids that have taken up the desired gene will stop glowing as the desired gene has stopped the green light gene from working
Enzyme Markers
Lactase turn colourless substrate into blue
Therefore put the desired gene into the gene that makes lactase and insert into plasmid, then insert plasmid into bacterial cells
Therefore whichever bacterial cells have a plasmid with the desired gene, their lactase gene will not work
Therefore when the bacterial cells are put in a colourless substrate, the bacterial cells containing the plasmid with the required gene will not change to blue
Growth
The bacterial cells that have the plasmid with the desired gene are now replicated
PROCESS: Using a fermenter
Importance of Markers
Allows transformed bacteria to be separate from non-transformed bacteria
OVERALL, THE PROCESS OF GENETIC ENGINEERING/GENETIC MODIFICATION
Cut desired gene out of cell using Reverse Transcriptase/Restriction Endonucleases
Cut plasmid using the same Restriction Endonucleases
Insert the gene into the plasmid, thereby joining the sticky ends using DNA Ligase
Transfer the plasmid into a cell
Use gene markers to identify which cells have taken up the new gene
Cells which have taken up the new gene cloned
Sometimes, instead of a bacteria cell, a virus is used for genetic engineering.
Advantages of using a virus to introduce genes into cells
Virus can enter cells and replicate inside cells
Viruses target specific cells
Disadvantages of using a virus to introduce genes into cells
May cause disease
Immunity may develop to the virus
DNA polymerase
An enzyme that joins free nucleotides with their complimentary bases on the DNA, but it does work for the start and end sequence of the DNA strand
Primers
have base sequence complimentary to that of the start and end sequence of the DNA to be copied
PROCESS (POLYMERASE CHAIN REACTION)
The desired DNA fragments, primers and DNA polymerase are placed in a vessel in a thermocycler
The temperature is increased to 95oC, causing the two strands of the desired DNA fragments to separate
Mixture is cooled to 55oC, causing primers to join with complimentary bases on the start and end of the two desired DNA strands, therefore original DNA strands are unable to rejoin
Now DNA Polymerase begins to join free nucleotides to the middle sections of the two desired DNA strands
The temperature is increased to 72oC, this is the optimum temperature for DNA polymerase to work
We then have two copies of the original DNA fragment, so the cycle is repeated
In the exam, you describe PCR as
Heat DNA to 95oC to allow strands to separate
Add primers and nucleotides
Cool so that primers bind to DNA and nucleotides attach by complementary base pairing
Temperature then taken to 72oC to allow DNA Polymerase to form a new strand
How a Polymerase Chain Reaction may stop by itself
Free nucleotides get used up therefore nothing to make complementary chains
Primers get used up there cannot start complementary chains
Enzymes lose activity so no polymerisation of complementary strands
As a recap, the reasons for the following are
Heating sample of DNA above 90oC: separate stranded DNA
Adding primers: attaches to start of gene and replication of base sequence starts from here
DNA Polymerase used at high temperatures: DNA polymerase not denatured by high temperatures thus allowing rapid replication of DNA
How PCR differs from DNA Replication
PCR uses heat to separate strands
PCR replicates pieces of DNA because DNA has been cut
Primer is added in PCR to initiate replication
How PCR differs from transcription
Transcription uses RNA Polymerase, PCR uses DNA Polymerase
In transcription, the template is one strand, in PCR, the template is two strands
Advantages of In Vivo
1) Useful when we wish to introduce a gene into another organism
2) No risk of contamination
3) Very accurate
4) Cuts out specific genes
5) transformed bacteria can be used to produce large quantities of gene products
Advantages of In Vitro
1) Extremely rapid
2) Does not require living cells
3) Very little purification of final sample needed
4) Very sensitive, can clone DNA molecule up to 1kpb long
Disadvantages of In Vivo
1) Relatively slow
2) complex purification
Disadvantages of In Vitro
1) Mistakes in copying in bases take place
Cystic Fibrosis
caused by a mutation in the gene for the protein CFTR, which is a chloride ion channel
Effect of Cystic Fibrosis: too much sticky mucus produced by epithelial cells
Gene Therapy
replacing defective genes with healthy genes
Gene Replacement: defective gene replaced with healthy gene
Gene Supplementation: healthy copy of gene added alongside defective gene, the healthy genes will be dominant
There are two ways in gene therapy may be adopted
Germ-line Therapy: replacing or supplementing the defective gene in the fertilised egg; prohibited due to ethics
Somatic-cell Therapy: targets just the affected areas, but the treatment needs to be repeated periodically
Somatic-cell Therapy is carried out in two ways
Using a virus called Adenoviruses as vectors for the healthy gene
Wrapping the plasmids containing the healthy gene in lipid molecules to form a liposome which can pass through phospholipid bilayer
Somatic-cell Therapy is not always effective because:
Adenoviruses may cause infection
Patients may develop immunity from Adenoviruses
Liposome may not be able to pass through phospholipid bilayer
Healthy gene may not be expressed
Healthy gene may enter genome of another gene such as tumour suppressor genes, thus causing cancer
How a person may become cured through Gene Therapy
Healthy gene is expressed
Healthy genes replicated with cells
Why Gene Therapy may still result in children with the genetic disease
Gamete cells do not take up the healthy gene
Therefore the person is able to pass on defective gene
Effectiveness of gene therapy
Effect is short lived
It can induce an immune response
Using viral vectors to deliver the gene present problems
The genes are not always expressed
Not effective in treating conditions that arise in more than one gene
Gene Therapy can also be used to replace the defective gene in a gamete, however this is illegal in the UK because:
Changes to genetic make-up of individual may affect normal development
Concerns regarding Gene Therapy
High cost compared with conventional treatments
Unknown side-effects
Use of animals in preliminary testing
Other genes introduced which may have damaging effects
