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Gene Therapy. AQA A2 Biology (Unit 5) revision source.

Just an easier way to get your head around the importance and relevance of each section of the AQA syllabus on Gene Technology.
by

Charlotte Alsop

on 16 June 2013

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Transcript of Gene Therapy. AQA A2 Biology (Unit 5) revision source.

Gene Technology
Biology 5 -
Summary of Chapter 16 - DNA technology

An aid for A-level students studying the AQA course
DNA
Electrophoresis
DNA probes
DNA ligase enzyme
Vectors e.g. plasmids
Sticky ends
Restriction endonuclease ezymes
Reverse transcriptase enzyme
Polymerase chain reaction (PCR)
It uses these tools
and techniques....

Stages
1

Isolate required gene
2

Insert gene into vector
3

Introduce gene into host using vector
4

Cloning of host cells and mass production e.g. insulin
It involves these
stages....

Applications
Gene therapy e.g. for cystic fibrosis
Use of bacteria to produce human protein e.g. insulin
Transgenic plants and genetically modified food
e.g. long life tomatoes
Transgenic animals e.g. human proteins in sheep milk
Genetic fingerprinting
Potential benefits
Potential risks
It has these
commercial uses....

It raises these
ethical issues....

Effectively treat genetic diseases
Cheaper medical proteins e.g. hormones
Cheaper food to reduce worldwide shortages
'Designer' babies by genetic modification of humans
Escape of modified bacteria to infect humans
'Genetic Pollution' the transfer of modified genes into wild species
Separated by...
Identified by...
Joined by...
Carried by...
Cut by...
Produces...
Made from mRNA using...
Amplified by...
Electrophoresis is the way in which strands of DNA are separated according to size.
DNA fragments are placed in wells at the positive end of the gel plate and a voltage is applied across it. Due to the resistance of the gel, larger fragments move more slowly, and so move less distance in a given time than the smaller fragments, which move faster.
During electrophoresis, different size fragments move different distances producing invisible banding.
The pattern of DNA bands can be revealed by transferring the pattern onto a nylon membrane by Southern Blotting. The double strands are made single stranded by heating, and radioactive DNA probes are washed over the membrane. The probes attach to the DNA bands by specific base pairing. The position of the probes and therefore the bands can now be revealed by autoradiography. An X-ray film is place over the membrane and the film is fogged by the radioactive probes. This produced a 'fingerprint' - a visible pattern of dark bands.
A DNA probe is a short, single-stranded section of DNA that has some sort of label attached that makes it identifiable. Such as:
- Radioactively labelled probes
- Fluorescently labelled probes
DNA probes are used to identify particular genes in the following way:
- A DNA probes is made that has bases complementary to the portion of the DNA sequence that makes up part of the gene whose position we want to find.
- The DNA that is being tested is treated to separate its two strands.
- The separated DNA strands are mixed with the probe, which binds to the complementary bases on one of the strands. This is known as DNA hybridisation.
- The site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits.
DNA sequencing
One method of sequencing the exact order of nucleotides in a section of DNA is the Sanger method. This method uses modified nucleotides that cannot attach to the next base in the sequence when they are being joined together, They therefore act as terminators, ending the synthesis of the DNA strand. Four different terminator nucleotides are used, each with one of the four bases, adenine, thymine, guanine or cytosine.
The first stage is to set up four test tubes, each containing:
- Many single-stranded fragments of the DNA to be sequenced. This acts as a template for the synthesis of its complementary strand.
- A mixture of nucleotides with the bases adenine, thymine, guanine and cytosine.
- A small quantity of one of the four terminator nucleotides
- A primer to start to process of DNA synthesis. This primer is radioactively labelled.
- DNA polymerase
As the binding of the nucleotides to the template is a random process, the addition of a normal nucleotide or terminator nucleotide is equally likely. Depending upon exactly where the nucleotide binds to the DNA template, DNA synthesis may be terminated after only a few nucleotides or after a long fragment of DNA has been synthesised. As a result, the DNA fragments in the tube will be of varying lengths. One thing they have in common is that all the fragments of new DNA in any of the test tubes will end with a nucleotide that has the same base. These fragments can be identified because the primer attached to the other end of the DNA section is radioactively labelled.
Restriction mapping
Deciphered by....
Restriction mapping involves cutting DNA with a series of different restriction endonucleases. The fragments produced are then separated by gel electrophoresis. The distance between the recognition sites can be determined by the patterns of fragments that are produced.
DNA ligase is the enzyme used to join the phosphate-sugar framework of two sections of DNA and so unite them as one. It is used in the synthesis of recombinant DNA.

The enzyme is used to join the cut ends of the DNA of the plasmid and the gene. The sugar-phosphate backbones are joined. Recombinant DNA has now been produced because the gene DNA has been joined to the plasmid DNA. The gene has been 'stitched' into the plasmid.
Vectors, such as plasmids are used during 'in vivo' gene cloning.
The vector is used to transport the DNA into the host cell. Plasmids are circular lengths of DNA, found in bacteria, which are separate from the main bacterial DNA. A restriction endonuclease cuts the plasmid at a specific recognition site, that produces 'sticky ends' in the broken plasmid allowing sections of foreign DNA (the desired gene) to incorporate itself when mixed with the plasmid and DNA ligase.

Once the DNA has been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells. This process is called transformation and involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions. The Calcium ions and changes in temperature make the bacteria permeable, allowing the plasmids to enter through the membrane into the cytoplasm. However not all the bacterial cells will possess the DNA fragments.
The first task is to identify which bacterial cells have taken up the plasmid.
Antibiotic-resistance markers
Fluorescent markers
A technique called replica plating is used to identify those cells with the plasmids that have taken up the new gene.
This process uses the other antibiotic-resistance gene in the plasmid: the gene that was cut in order to incorporate the required gene (in this example, the gene that coded for resistance to tetracycline). As this gene has been cut it will no longer be resistant to tetracycline.
-The bacterial cells that survived treatment with the first antibiotic (ampicillin) are known to have taken up the plasmid.
-These cells are cultures by spreading them very thinly on nutrient agar plates.
-Each separate cell on the plate will into a genetically identical colony.
-A tiny sample of each colony is transferred onto a secon plate in exactly the same position as the colonies on the original plate.
-This replica plate contains the second antibiotic (tetracycline)
- The colonies killed by the antibiotic must be the ones that have taken up the required gene.
-The colonies in exactly the same position on the original plate are the ones that possess the required gene.
Another method is the transference of a gene from a jellyfish into the plasmid. The gene in question produces a green fluorescent protein (GFP). The gene to be cloned is transplanted into the centre of the GFP gene. Any bacterial cell that has taken up the plasmid with the gene that is to be cloned will not be able to produce GFP. Unlike the cells that haven't taken up the gene, these cells will not fluoresce. Results can be obtained by simply viewing the cells under a microscope and retaining those that do not fluoresce.
Another gene marker is the gene that produces the enzyme lactase. Lactase will turn a particular colourless substrate blue. Again, the required gene is transplanted into the gene that makes lactase. If a plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase. Therefore, when these bacterial cells are grown on the colourless substrate they will be unable to change its colour. Where the gene has not transformed the bacteria, the colonies will turn the substrate blue.
Enzyme markers
Restriction endonucleases are a group of enzyme that cut DNA molecules at a specific sequence of bases called a recognition site.

Restriction endonucleases are obtained from bacteria.

There are many different types of restriction endonuclease. Each one cuts a DNA double strand at a specific sequence of bases called a recognition sequence. Sometimes the cut occurs between two opposite base pairs. This leaves two straight edges known as 'blunt ends' in the middle of the base recognition sequence.
Other restriction endonucleases cut DNA in a staggered fashion. This leaves an uneven cut in which each strand of DNA has exposed, unpaired bases. The two strands formed will have what are known as 'sticky ends'. These 'sticky ends' are often palindromic.
Sticky ends are important in producing recombinant DNA. If the same restriction endonuclease is used to cut the required gene out of DNA as well as to cut the plasmid then the ends of both types of DNA will have complementary 'sticky ends' and so can be combined to form a new piece of double-stranded DNA. Hydrogen bonds form between the complementary bases, and then DNA ligase is used to join the phosphate-sugar framework of the two sections of DNA.
Retroviruses are a group of viruses of which the best known is human immunodeficiency virus (HIV). The genetic information of retroviruses is in the form of RNA . However, they are able to synthesise DNA from their RNA using an enzyme called reverse transcriptase. The process of using reverse transcriptase to isolate a gene is as follows:
A cell that readily produces the desired protein is selected. (e.g. the beta-cells of the islets of Langerhans from the pancreas are used to produce insulin). These cells have large quantities of the relevant mRNA, which is then extracted. Reverse transcriptase is then used to make DNA from RNA. This DNA is known as complementary DNA (cDNA) because it is made up of the nucleotides that are complementary to the mRNA. To make the other strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand of DNA is the required gene.
PCR is an automated way of copying fragments of DNA, making both rapid and efficient.
PCR requires the following things:

- The DNA fragment - to be copied
- DNA polymerase - an enzyme capable of joining together tens of thousands of nucleotides in a matter of minutes. It is obtained from bacteria in hot springs and is therefore tolerant to heat and does not denature during the high temperatures of the process.
- Primers - short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.
- Thermocycler - a computer controlled machine that varies temperatures precisely over a period of time.
Stages of PCR:
1. Separation of the DNA strand

2. Addition (annealing) of the primers

3. Synthesis of DNA
Stage 1. Separation of DNA strand.

The DNA fragments, primers, and DNA polymerase are placed in a vessel in the thermocycler. The temperature is increased to 95°C, causing the two strands of the DNA to separate, as the hydrogen bonds break.
Stage 2. Addition (annealing) of the primers

The mixture is cooled to 55°C, causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment. The primers provide the starting sequence for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain. Primers also prevent the two original strands from rejoining.
Stage 3. Synthesis of DNA

The temperature is increased to 72°C. This is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands. It begins at the primer on both ends and adds the nucleotides in sequence until it reaches the end of the chain
Are used in...
Genetic fingerprinting relies on the fact that the genome of any organism contains many repetitive, non-coding bases of DNA which are called introns. They contain repetitive sequences of DNA called core sequences. For every individual (except identical twins) the number and length of core sequences has a unique pattern. However, the more closely related two individuals are, the more similar their core sequences will be.The making of a genetic fingerprint involves 5 main stages:
1. Extraction
DNA is extracted from a suitable sample. Even the tiniest sample of animal tissue such as a drop of blood or a hair root is enough to give a genetic fingerprint. As the amount of DNA extracted is usually small, its quantity can be increased by using PCR.
2. Digestion
The DNA is then cut into fragments using restriction endonucleases. The endonucleases are chosen for their ability to cut close to, but not within, groups of core sequences.
3. Separation
The fragments of DNA are then separated according to size using gel electrophoresis under the influence of an electrical current. The gel is then immersed in alkali to separate the double strands into single strands. The single strands are then transferred onto a nylon membrane by a technique called Southern blotting.

Southern blotting:
- A thin nylon membrane is laid over the gel
- The membrane is covered with several sheets of absorbent paper, which draws up the liquid containing the DNA by capillary action
- This transfers the DNA fragments to the nylon membrane in precisely the same relative positions that they occupied on the gel
- The DNA fragments are then fixed to the membrane using ultraviolet light
4. Hybridisation
Radioactive (or fluorescent) DNA probes are used to bind with the core sequences. The probes have base sequences which are complementary to the core sequences, and bind to them under specific conditions, such as pH and temperature. The process is carried out with different probes, each of which binds with a different core sequence.
5. Development
Finally, an X-ray film is put over the nylon membrane. The film is exposed by the radiation from the radioactive probes. Because these points correspond to the position of the DNA fragments as separated during electrophoresis a series of bars is revealed.

One reason why we use transgenic animals is to produce rare and expensive proteins for use in human medicine. Domesticated milk-producing animals such as sheep can be used. The gene required for the protein is inserted alongside the gene that codes for proteins in sheep's milk. The gene can be inserted into the fertilised egg of a sheep, so that all the female offspring of that idividual will be capable of producing the protein in their milk.
Mature eggs are removed from female sheep and fertilised
The normal gene for the desired protein from a human is added to the fertilised eggs alongside the gene that codes for proteins in sheep's milk
These genetically transformed eggs are transplanted into the female sheep
Those resulting sheep with the protein gene are crossbred, to give a herd in which sheep produce milk rich in the protein.
The protein is extracted from the milk, purified and given to humans.
Genetically modified tomatoes have been developed using the insertion of a gene. This gene has a base sequence that is complementary to that of the gene producing the enzyme that causes tomatoes to soften. The mRNA transcribed from this inserted gene is therefore complementary to the mRNA of the original gene. The two therefore combine to form a double-strand. This prevents the mRNA of the original strand from being translated. The softening enzyme is therefore not produced. This allows the tomatoes to develop flavour without the problems associated with harvesting, transporting and storing soft fruit.
Advantages of PCR:
It is extremely rapid
It does not require living cells
Advantages of 'in vivo' cloning:
It is particularly useful where we wish to introduce a gene into anoth organism
It involves almost no risk of contamination
It is very accurate
It cuts out specific genes
It produces transformed bacteria that can be used to produce large quantities of gene products
Treatment of cystic fibrosis
There are two ways in which gene therapy can be used t0 treat cystic fibrosis patients:
Gene replacement, in which the defective gene is replaced with a healthy gene
Gene supplementation, in which one or more copies of the healthy gene are added alongside the defective gene
In addition, there are two different techniques of gene therapy that may be adopted, according to which type of cell is being treated:
Germ-line therapy, which involves replacing or supplementing the defective gene in the fertilised egg. This ensures that all the cells of the organism will develop normally, as will all the cells of their offspring.
Somatic-cell gene therapy, which targets just the affected tissues, such as the lungs, and the additional gene is therefore not present in the sperm or eggs cells, and so is not passed on to future generations.

Delivering cloned CFTR genes
Using a harmless virus.
Adenoviruses cause colds and other respiratory diseases by injecting their DNA into the epithelial cells of the lungs. The adenoviruses are made harmless by interfering with a gene involved in their replication. These adenoviruses are then grown in epithelial cells in the laboratory, along with plasmids that have had the normal CFTR gene inserted. The CFTR gene becomes incorporated into the DNA of the adenoviruses.
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