Producing DNA Fragments
The process of making a protein using the DNA technology of gene transfer and clonging involves a number of stages:
1. Isolation of the DNA fragments that have the gene for the desired protein.
2. Insertion of the DNA fragments into a vector
3. Transformation, the transfer of DNA into suitable host cells
4. Identification of the host cells that have been successfully taken up the gene by the use of gene markers
5. Growth/cloning of the population of host cells
Before a gene can be transplanted, it must be identified and isolated from the rest of the DNA. Two of the methods empolyed use enzymes that have important roles in microoragnisms: reverse transcriptase and restriction endonucleases.
Producing DNA Fragments 2
Reverse Transcriptase is used to catalyse the production of DNA from RNA, which is the reverse of the more usual transcription of RNA from DNA.
- A cell that readily produces the protein is selected.
- These cells have large quantities of the relevant mRNA, which is therefore extracted.
- Reverse transcriptase is then used to make DNA from RNA. This is known as complementry DNA 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 in the cDNA template. This double strand of DNA is the required gene.
Restriction Endonucleases: each one cuts a DNA double strand at a specific sequence of bases called a recognition sequences. Sometimes, this cut occurs between two opposite base pairs, this leaves blunt ends. Others cut the DNA in a staggered fashion. This leaves an uneven cut in which teach strand of the DNA has exposed, unpaired bases, these are known as sticky ends.
In Vivo Gene Cloning - The Use Of Vectors
Once the fragments of DNA have been obtained, the next stage is to clone them to provide sufficient quatities, one way of this is...
In vivo; transferring the fragments to a host cell using a vector.
The sequences of DNA that are cut by restriction endonucleases are called recognition sites. If the recognition site is cute in a staggered fashion, the cut ends of the DNA strand are left with a single strand which is a few nucleotide bases long. If the same restriction endonuclease is used to cut DNA, then all the fragments will have the same ends and be complementry to each other. Once complementry bases have paired up, an enzyme called DNA Ligase is used to join the phosphate-sugar framework of the two sections of DNA and so unite them as one.
Insertion of DNA Fragments into a Vector.
Once the DNA has been cut it must then be joined with a carrying unit, a vector. The most comonly used is the plasmid. Plasmids are circular length of DNA, found in bacteria. Plasmids most commonly contain genes for antibiotic resistance, and restriction endonucleases are used at one of these antibiotic-resistance genes to break the plasmid loop.
The restiction endonuclease used is the same as the one that cut out the DNA fragment. This ensures the sticky ends are complementry. When DNA fragments are mixed with the opened-up plasmids, they may become encorporated into them.The join is made permanent using DNA ligase.
In Vivo Gene Cloning - The Use Of Vectors 2
Introduction of DNA into host cells.
Once the DNA has been incorporated into the plasmids, it must then be reintroduced into bacterial cells. This process is called transformation and involves the plasmid and bacterial cells being mixed together in a medium containing calcium ions.The Calcium ions and a change in temperature make the bacteria permeable, allowing the plasmids to pass through the membrane into the cytoplasm.Not all the bacterial cells will contain the DNA fragments. There are two main reasons for this:
- Only a few bacterial cells take up the plasmids when the two are mixed together.
- Some plasmids will have closed up again without incorporating the DNA fragment.
To find out which bacterial cells have taken up the plasmids the gene for antibiotic resitance is used:
- All the bacterial cells are grown on a medium that contains the antibiotic ampicillin.
- Bacterial cells that have taken up the plasmids eill have aquired the gene for ampicilin reistance.
- These bacterial cells are able to break down the ampicillin and therefore survive. The bacterial cells that have not taken up the plasmid will not be resitant to ampicilin and therfore die.
- However some of those that contain the plasmid may not contain the new gene, these are identified using gene markers...
In Vivo Gene Cloning - The Use Of Vectors 3
Gene markers: There are a number of different ways of using gene markers, they all involve using a second, seperate gene on the plasmid: 1. It may be resitant to an antibiotic 2. It may make a fluorescent protein that is easily seen. 3. It may produce an enzyme whose action can be identified.
Anti-biotic Resistant markers - Replica Plating
- The bacterial cells that survived treatment with the first antibiotic are known to have taken up the plasmid.
- These cells are cultured by spreading them thinly on nutrient agar plates.
- A tiny sample from each colony is transferred onto a second (replica) plate in exactly the same position as the original plate.
- The replica plate contains the second antibiotic (tetracycline), against which the antibiotic-resistance gene will have been made useless if the new gene was taken up.
- The colonies killed by the antibiotic must be the ones that have taken up the required gene.
- The colonies in exaactly the same position on the original plate are the ones that possess the required gene.
Flourescent Markers: Taking a plasmid that produces GFP and then removing this gene to implant the wanted one, will mean that any bacteria that does not have the wanted gene will be floursecent.
Enzyme Markers: The enzyme Lactase will turn a clourless substrate blue.
In Vitro Gene Cloning - The Polymerase Chain React
PCR is a method of copying fragments of DNA. The process is automated, making it both rapid and efficient. The process requires:
- The DNA Fragments to be copied.
- DNA Polymerase - Joins together nucleotides, it does not denature a high temperatures as is obtained from bacteria that come from hot water springs.
- Primers - short sequences of nucleotides that have a set of bases complementary to thise at one end of each of the two DNA fragments.
- Nucleotides - which contain eacch of the four bases found in DNA.
PCR is carried out in three stages:
1. Separationof the DNA strand. The DNA fragments, primers and DNA polymerase are placed in the thermocycler. The temperature is increased to 95'C, causing the two DNA strands of fragments to seperate. 2. Addtion of the primers. The mixture is cooled to 55'C, causing the primers to join to their complementry bases. Primers provide a starting sequence and prevent the two strands from rejoining. 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 seperated DNA strands. It begins at the primer and then adds the nucleotides in sequence until it reaches the end.
Advantages Of In Vitro and In Vivo Gene Cloning
The advantages of in vitro gene cloning are:
- It is extremely rapid. This is particularly useful when on a crime scene and there are only small amounts of DNA. In the in vivo technique it would take days or weeks to produce what in vitro can produce in a few hours.
- It does not require living cells. No complex culturing techniques are required.
The advantages of in vivo gene cloning are:
- It is particularly useful where we wish to introduce a gene into another organism.
- It involves almost no risk of contamination. This is because the gene that has been cut by the same restriction endonuclease can match the sticky ends of the opened-up plasmid. In the in vitro technique are very pure sample is required as any contaminant DNA will also be amplified.
- It is very accurate. The DNA copied has few, if any, errors. Within in vitro any errors will then be amplified in future cycles.
- It cuts out specific genes. It is therfore a very precise procedure as the culturing of transformed bacteria produces many copies of a specific gene and not just copies of the whole DNA sample.
- It produces transformed bacteria that can be used to produce large quantities of gene products. The transformed bacteria can produce proteins for commercial or medical use.
- 1 in 20,000 have it
- Recessive allele
- 1480 amino acids long
- Epithelial cells line organs eg. lungs and gut
- In the membrane of epithelial cells there is a channel protein called CFTR
- Chloride ions move out and are followed by water through osmosis.
- This maintains a loose non-sticky mucas.
- If the channelis faulty the chloride ions do not move out, so the water does not follow
- This causes the mucas to become thick and viscous
- Lungs mucus is not removed, so it stays in the lungs causing bacterial infections.
- Thick mucus makes gas exchange very difficult; resulting in symptoms such as tiredness
- Pancreatic duct becomes blocked; this means that food cannot be digested properly, resulting in sufferers becoming underweight.
- Sperm ducts and fallopian tubes become blocked; this causes the sufferer to become infertile
Gene Therapy 2
Ways to treat Cystic Fibrosis...
- Gene replacement - Healthy gene takes place of defective gene
- Gene supplementation - Healthy genes are inserted next to defective gene which masks the effect of the defective gene.
- Germ-line Replacement - Taking a fertilised egg and replacing the gene
- The person would have healthy genes in all cells
- There is no need for reapplication
- Off-spring would be healthy
- Ethical and moral issues
- Very difficult to do
- Somantic Line Therapy - Replace genes in effected tissues
- Cells constantly die and therfore requires constant reappliactions
- Low success rate of gene uptake
- Not a permanent solution, off-spring do not benefit
- Fewer ethical and moral issues
- One solution is that stem cells are targeted, it will then last a lifetime, but the off-spring will still not benefit
Gene Therapy 3
Severe Combined Immunodeficiency (SCID)
- People who suffer do not show a cell-mediated immune response as they are not able to produce antibodies.
- Previously, it was only found in children because they died soon after getting the infection
- The infection occurs when there is a defect in the gene of the enzyme ADA; this enzyme destroys toxins that would otherwise kill white blood cells.
- Survival hasdepended on patients being raised in a sterile environment and giving thema bone marrow transport and/or injections of ADA.
- Recent attempts to treat the disorder are as follows:
- The normal ADA gene is isolated from healthy human tissue using restriction endonucleases
- The ADA gene is inserted into a retrovirus
- The retroviruses are grown with host cells in the lab to increase their number and hence the number of copies of the ADA gene
- The retroviruses are mixed with the patients T cells
- The retroviruses inject a copy of normal ADA gene into the T cells
- The T cells are reintroduced into the patient's blood to provide the genetic code needed to make ADA.
Gene Therapy 4
Effectiveness of Gene Therapy
- The effect is short lived - The somatc cells, which have a cloned gene added, are not passed onto the daughter cells. Repeat treatments are necessary for the therapy to have any effect
- It can induce an immune respons - Both the gene that is being introduced and the vector can induce an immune response in the recipient. This means that it is often rejected. This is made worse by the fact that the immune system typically responds to foreign material by making antibodies, some of which remain to inititiate an even greater secondary response to infection.
- Using viral vectors to deliver the gene presents problems - Viruses are the usual means of getting genes to their target cells.
- The genes are not always expressed - Even if successfully delivered to their target.
Locating and Sequencing Genes
- Break down the DNA into single strands
- Gene probe - Small section of DNA of certain base sequence, labelled radioactive florescent.
- Primer is used to: 1. identify starting part of DNA to be sequenced. 2. To stop the DNA strands re-coupling
- 4 tubes, in each there will be: a single stranded DNA fragment that needs to be sequenced, nucleotides ATGC, DNA Polymerase, Adapted nucleotides of one type A*T*G*C*
- Altered/adapted nucleotides cannot join to the next nucleotide when DNA polymmerase tries to join them.
- Fragments each end in an altered nucleotide.
- DNA fragments are placed onto an agar gel and a voltage is applied accross it.
- The larger the fragments the slower they move.
- DNA fragments of different lengths are of different lengths are separated.
- A sheet of photographic film is placed over the agar gel for several hours.
- The radioactivity from each DNA fragment exposes the fil and shows where it is situated on the gel.
- Only DNA up to around 500 bases long can be sequenced in this way.
- Larger genes must be cut into smaller fragments by restriction endonucleases.
Locating and Sequencing Genes 2
- Is used to put genes back together which have been cut using restriction endonucleases.
- Involves cutting DNA with a series of different restriction endonucleases.
- The fragments produced are then separated by gel electrophoresis.
- The distance between recognition sites can be determinedby the patternsof fragments that are produced.
- If the two do not add up, it is likely that two fragments of the same size have been formed.
Genetic fingerprinting is a diagnostic tool used widely in forensic science. It is based on the fact that the DNA of every individual, except identical twins, is unique.
The process of genetic fingerprinting is as follows:
- Extract DNA - very small samples can be used, and then amplified using PCR
- Digest - Using restriction endonucleases, know where they are cutting. Eg, the DNA from the crime scene, from DNA of a person being tested.
- Electrophoresis - Seperate DNA fragments of sample from the crime scene.
- Southern Blot - Nylon sheet pressed onto electrophoresis gel. Absorbant paper is placed over this to **** DNA onto the nylon sheet. The DNA is fixed onto the nylon sheet using UV light.
- DNA of person - Wash it over the DNA on nylon sheet, if the conditions are correct, complementary DNA will hybridise.