Polymerase Chain Reaction
- The polymerase chain reaction can be used to make millions of copies of a fragment of DNA.
- A reaction mixture is set up that contains the DNA sample, free nucleotides, primers, and DNA polymerase.
- DNA polymerase is an enzyme which creates new strands of DNA.
- The DNA mixture is heated to 95oC to break the hydrogen bonds between the two strands of DNA.
- The mixture is then cooled to 50-65oC so that the primers can anneal to the strands.
- The reaction mixture is heated to 72oC so that DNA polymerase can work.
- The DNA polymerase lines up free DNA nucleotides alongside each template strand. complementary base pairing means new complementary strands are formed.
- Twon new copies of the fragment of DNA are formeed and one cycle of PCR is complete.
- The cycle startes again and the two strands are used as templates.
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- Restriction enzymes are used to remove DNA fragments from an organisms DNA.
- Some sections of DNA have palindromic sequences of nucleotides. These sequences consist of antiparallel base pairs - base pairs that read the same but in opposite directions.
- Restriction enzymes recognise specific palindromic sequences and digest the DNA in these places.
- Different restriciton enzymes cut at different specific recognition sequences, because thw shape of the recognition sequence is complementary to the enzyme's active site.
- If recognition sequences are present at either end of the DNA fragment wanted, restriction enzymes can be used to separate it from the rest of the DNA.
- The DNA sample is incubated with the specific restriction enzyme, which cuts the DNA out via a hydrolysis reaction.
- Sometimes the cut leaves sticky ends - small tails of unpaired bases at each end of the fragment. Sticky ends can be used to anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences.
- DNA ligase is used to catalyse a condensation reaction which joins the phosphate-sugar backbones of the DNA double helix together.
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- Electrophoresis separates DNA fragments.
- DNA samples are treated with restriction endonucleases to cut them into fragments.
- The DNA samples are placed in wells at the negative elctrode end of the gel.
- The gel is immersed in buffer solution, and an electric current in passed through the solution. Dna is negatively charged due to the many phosphate groups. The DNA fragments diffuse through the gel towards the positive electrode.
- Shorter lengths move faster than longer lengths and so move further through the gel.
- The position of the fragments in the gel can be shown by using a dye that stains DNA molecules.
- The sample is then blotted using southern blotting.
- The DNA can be shown up using radioactive or fluorescent tags/probes, or by being X-rayed.
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- Genetic engineering is the manipulation of an organisms DNA.
- Organisms that have had their DNA altered by genetic engineering are called transformed organisms.
- These organisms have recombinant DNA - DNA formed by joining together DNA from different sources.
- Genetic engineering usually involves extracting a gene from one organism and then inserting it into another organism.
- Genes can also be manufactured instead of extracted from an organism.
- The organism with the inserted gene will then produce the protein coded for by that gene.
- An organism which has been genetically engineered to include a gene froma different species is sometimes called a transgenic organism
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- The DNA fragment containing the desired gene is isolated using restriction enzymes.
- The isolated DNA is then inserted into a vector using restriction enzymes and DNA ligase.
- The DNA fragment is inserted into vector DNA - plasmid or bacteriophages.
- The Vector is cut open using the same restriction endonucleases as used to cut the DNA fragment containing the desired gene.
- The sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene.
- The vector and DNA fragment are mixed together with DNA ligase. DNA ligase joins up the sugar-phosphate backbones of the two pieces in ligation.
- The new combination of bases in the DNA is called recombinant DNA.
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- The vector with the recombinant DNA is used to transfer the gene into the bacterial cells.
- If a plasmid vector is used, the bacterial cells have to be persuaded to take in the plasmid vector and its DNA. They're placed into ice cold calcium chloride solution to make their cell walls more permeable. The plasmids are added and the mixture is heat-shocked, which encourages the cells to take in the plasmids.
- With a bacteriphage vector, the bacteriophage will infect the becterium by injecting its DNA into it. The phage DNA with the desired gene in it then intergrates into the bacterial DNA.
- Cells that take up vectors containing desired genes are transformed.
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Identifying Transformed Bacteria
- Marker genes can be inserted into the vectors at the same time as the desired gene. This means that any transformed bacteria cells will contain the desired gene and the marker gene.
- The bacteria are grown on agar plates, and each cell divides and replicates its DNA, producing a colony of cells.
- Transformed cells will produce colonies where all the cells contain the desired gene and the marker gene.
- The marker gene can code for antibiotic resistance - the bacteria are grown on agar plates containing the antibiotic, so only cells that have the marker gene will survive and grow.
- The marker gene can code for fluorescesnce- when the agar plast is placed under UV light only transformed cells will fluoresce.
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- The gene for human insulin is identified and isolated using restriction enzymes.
- A plasmid is cut ope using the same restriction enzymes used to isolate the gene.
- The insulin gene is inserted into the plasmid forming recombinant DNA.
- The plasmid is taken up by bacteria and any transformed bacteria are identified using marker genes.
- The bacteria are grown in a fermenter - human insulin is produced as the bacteria grow and divide.
- The human insulin is extracted and purified so it can be used in humans.
- This process produces insulin identical to human insulin, so these is less risk of allergic reaction or rejection.
- It's cheaper and faster to produce than animal insulin, providing a more reliable and larger supply of insulin.
- Using genetically engineered insulin overcomes any ethical or religious issues arising from using animal insulin.
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- Golden rice is a type of genetically engineered rice. the rice is genetically engineered to contain a gene from a maize plant and a gene from a soil bacterium, which enable the rice to produce Beta-Carotene.
- The Beta-Carotene can then be used by our bodies to produce vitamin A, and can be used where there's a shortage of dietary vitamin A.
- The psy gene from maize, and the crtl gene from the soil bacterium are isolated using restriction enzymes.
- A plasmid is removed from the Agrobacterium tumefaciens bacterium and cut open with the same restriction enzymes.
- The psy and crtl genes and a marker gene are inserted into the plasmid.
- The recombinant DNA is put back into the bacterium.
- Rice plant cells are incubated with the transformed A. tumefaciens bacteria, which infect the rice cells.
- A. tumefaciens inserts the genes into the plant cells' DNA, creating transformed rice plant cells.
- The rice plant cells are then grown on a selective medium - only transformed rice plants will be able to grow because they contain the marker gene that's needed to grow on this medium.
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- Organ failure may be treated with an organ transplant.
- Xenotransplantation is the transfer of cells, tissues, or organs from on species to another.
- Rejection is a great issue with xenotransplantation because the geentic differences between organisms of different species are even greater than between organisms of the same species.
- Genes for human cell surface proteins are inserted into the animals DNA;
- Genes for human cell surface protein are injected into a newly fertilised animal embryo.
- The genes integrate into the animal's DNA.
- The animal then produces human cell surface proteins, which reduces the risk of transplant rejction.
- Genes for NIMl cell surface proteins are removed or inactivated;
- Animal genes involved in making cell surface proteins are removed or inactivated in the nucleus of an animal cell.
- The nucleus is then transferred into an unfertilised animal egg cell.
- The egg cell is stimulated to divide into an embryo, and the animal produced doesn't have cell surface proteins, whih reduces the risk of rejection.
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Ethics and Genetic Engineering
- Some people are worried that using antibiotic-resistance genes as marker genes may increase the number or antibiotic-resistant, pathogenic microorganisms in our environment.
- Environmentalists are worried that GM crops may encourage farmers to carry out monoculture. Monoculture decreases biodiversity, and could leave the whole crop vulnerable to disease, as all the plants are genetically identical.
- Some people are worried that genetically engineering animals for xenotransplantation may cause them suffereing.
- Some people are concerned about the possibility of 'superweeds' - weeds that are resistant to herbicides because they've bred with genetically engineered herbicide-resistant crops.
- Some people are concerned that large biotechnology companies may use GM crops to exploit farmers in poor countries.
- Some people worry that humans will be genetically engineered, producing a genetic underclass.
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- Gene therapy could be used to cure genetic disorders.
- Gene therapy involves altering alleles inside cells to cure genetic disorders.
- How this is done depends on whether the genetic disorder is caused by a dominant allele or two recessive alleles.
- If it's caused by recessive alleles, a working dominant allele can be added to make up for them.
- If it's caused by a dominant allele the dominant allele can be silenced.
- The allele is inserted into cells using vectors.
- Different vectors can be used for different purposes.
- Somatic Therapy - This involves altering the alleles in body cells, particularly the cells most affected by the disorder. Somatic therapy doesn't affect the individuals sex cells, so the offspring could still inherit the disease.
- Germ Line Therapy - This involves altering the alleles in the sex cells. This means that every cell of any offspring produced will be affected by the gene therapy and they won't suffer from the disease.
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Advantage and Disadvantages - Gene Therapy
- It could prolong the lives of those with genetic disorders.
- It could give people with genetic disorders a better quality of life.
- Carriers of genetic disorders might be able to concieve a baby without the disorders or risk of cancer.
- It could decrease the number of people affected by genetic disorders.
- The effects of the treatment my be short lived.
- The patient may have to undergo several treatments.
- It might be difficult to get the allele into specific body cells.
- The body could identify vectors as foreign bodies and start an immune response against them.
- An allele could be inserted into the wrong place in the DNA, possibly causing more problems.
- An inserted allele could be overexpressed, producing too much of the missing protein.
- Disorders caused by multiple genes would be difficult to treat with this technique.
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- DNA probes can be used to identify DNA fragments that contain specific sequences of bases.
- DNA probes are short strands of DNA. They have a specific base sequence that's complementary to the target sequence.
- This means that the DNA probe will bind/hybridise to the target sequence if it's present in the sample.
- A DNA probe has a lable attached so that it can be detected. This can include radioactive or fluorescent tagging.
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- CTM is used to determine the order of bases in a section of DNA/gene.
- The following mixture is added to four seperate tubes;
- A single stranded DNA template - the DNA to sequence.
- DNA primer
- DNA polymerase
- Free nucleotides
- Fluorescently labelled modified nucleotide
- The tubes undergo PCR, which produces many strands of DNA. The strands are different lengths because each one terminates at a different point depending on where the modified nucleotide was added.
- The DNA fragments in each tube are separated by electrophoresis and visualised under UV light.
- The complementary base sequence can be read from the gel. The smallest nucleotide is at the bottom of the gel. Each band after this represents one more base added. By reading the bands from the bottom of the gel to the top, the DNA sequence can be built up one base at a time.
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Sequencing Using BAC's
- This process allows a whole genome to be sequenced.
- A genome is cut into smaller fragments using restriction enzymes.
- The fragments are inserted into the Bacterial Artificial Chromosomes, BACs, - man made plasmids. Each fragment is inserted into a different BAC.
- The BACs are then inserted into bacteria - each bacterium contains a BAC with a different DNA fragment.
- The bacteria divide, creating colonies of cloned cells that all contain copies of the specific DNA fragment. Together the different colonies make a complete genomic library.
- DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping pieces of DNA.
- Each piece of DNA is sequenced, using the chain-termination method, and the pieces are put back in order to give the full sequence from the BAC.
- Finally, the DNA fragments are put back in order to complete the entire genome.
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- Understanding the evolutionary relationship between different species. DNA can tell us how closely related species are.
- Understand the way in which genes interact during development, and how they're controlled.
- Carry out medical research.
- Trace early human migration.
- Study the genetics of human diseases. Comparisons can be made to identify mutations.
- Develop medical treatments for particulr genotypes.
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