Module 6 Section 4 - Manipulating Genomes Flashcards
What techniques are used to study genes?
1) the polymerase chain reaction (PCR)
2) cutting out dna fragments using restriction enzymes
3) gel electrophoresis
What are primers?
Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want
How does PCR work?
1) a reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.
2) the DNA mixture is heated to 95 degrees Celsius to break hydrogen bonds between the dna strands. DNA polymerase does not denature. The mixture is then cooled to 50-65 degrees Celsius so that primers can anneal to the DNA strands.
3) the reaction mixture is then heated to 72 degrees Celsius so DNA polymerase can work. The DNA polymerase lines up free DNA nucleotides alongside each template strand. Complementary base pairings mean new complementary strands are formed.
4) two new copies of the fragment are formed, and one cycle of PCR is complete. The cycle starts again, the mixture is heated to 95 degrees Celsius and this time all 4 strands are used as templates.
What are palindromic sequences?
They read the same forwards and backwards
How do restriction enzymes work?
1) restriction enzymes recognise specific palindromic sequences (recognition sequences) and cut the DNA at these places. Different restriction enzymes have different recognition sequences, due to the enzymes active site having to be complementary to the shape of the recognition sequence.
2) the restriction can cut the DNA straight down the middle forming “blunt ends”, or in a zig zag fashion, creating sticky ends. They cut the DNA by hydrolysing the phosphodiester backbone.
3) sticky ends are unpaired bases at the end of the fragment. Scientists will first analyse the DNA to determine which recognition sites are present either side of the gene of interest. Then they use correcposing enzymes to extract the gene from the longer section of DNA.
What is electrophoresis?
A process that uses an electrical current to seperate DNA fragments, RNA Fragments or proteins depending on their size.
How does stage 1 of electrophoresis work?
An agarose gel is prepared which contains a row of wells at the top of the gel. The gel is placed into a tank containing buffer solution which is able to conduct electricity. The end of the tray with the wells has to be closer to the negative electrode
How does stage 2 of electrophoresis work?
Using a micropipette, add the same volume of loading dye to each well. This turns the DNA a dark colour and makes them easier to see. A fixed volume of the DNA samples are pipettes into the wells.
How does stage 3 of electrophoresis work?
An electrical current is passed through the gel and the DNA will begin to move towards the bottom of the gel. Once the dye has reached the bottom, the electricity is turned off and the banding pattern is visualised under UV light.
What is done to DNA before electrophoresis?
So that the DMA can be visualised, we add a fluorescent molecule which binds to the DNA and makes it visible when exposed to UV light. A common fluorescent tag is ethidium bromide.
What is DNA profiling + why is it possible?
A technique which can be used to analyse a sample of DNA and compare it to DNA samples taken from suspects.
This is possible because individuals have different lengths of variable number tandem repeats ( short non-coding regions of DNA) which is inherited from your parents. The more closely related you are, the more similar the VNTRs are.
Process of DNA profiling?
1) the DNA sample will be collected and isolated. Then it will be amplified using PCR.
2) use restriction endonucleuases to cut the amplified DNA into fragments.
3) the fragments are seperated using gel electrophoresis, which seperates the DNA fragments according to length.
4) the gel is visualised using UV light and the banding patterns from the suspect’s dna can be compared to where it was collected from.
What are the uses of DNA profiling?
1) forensic scientists use dna profiling to compare samples of DNA collected from crime scenes to samples of DNA from possible suspects, to link them to crime scenes.
2) in medical diagnosis, a DNA profile can refer to a unique pattern of several alleles. It can be used to analyse the risk of genetic disorders.
What is genetic engineering?
Genetic engineering is the manipulation of an organism’s 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)
What is part 1 of genetic engineering?
The first step is to get a hold of a dna fragment that contains the desired gene. The fragment can be isolated from another organism using restriction endonucleases to cut at recognition sites. Converting the mRNA of the desired gene to cDNA, using reverse transcriptase. Double stranded DNA is then synthesised using DNA polymerase.
What is part 2 of genetic engineering?
1) have to insert DNA fragment into vector DNA. Vectors can be plasmids or bacteriophages. The plasmid is also cut with restriction enzymes. DNA ligase joins complementary sticky ends to form the recombinant DNA.
2) The recombinant plasmid is mixed with bacteria and placed in a machine called an electroporator. This creates an electric field to make the bacteria membranes more permeable.
How to check if bacteria has been correctly transformed.
1) use a marker gene. The gene should be for fluorescence. Examine bacteria under UV light.
2) use an antibiotic resistant gene and grow on agar containing the antibiotic.
How does it work if the vector for genetic engineering is bacteriophage?
The bacteriophage will infect the host bacterium by injecting its dna into it. The phage DNA then integrates into the bacterial DNA.
How are insect-resistant soya plants made from genetic engineering?
1) to genetically modify a soybean plant, the desired gene is isolated with restriction enzymes and inserted into a plasmid taken from the bacterium A.T. This forms recombinant DNA
2) the plasmid is taken back into A.T.
3) The soybean plants are then deliberately infected with the transformed bacteria. The desired gene gets inserted into the soybean’s plant cells’ dna, creating a gm plant.
What are the positive ethical issues with genetically modified plants?
1) reduce the number of chemical pesticides that farmers use on their crops, which can harm the environment.
2) GM plants can also be more nutritious.
What are the negative ethical issues with genetically modified plants?
1) GM plants may encourage monoculture. This decreases biodiversity and could leave the whole crop vulnerable to disease.
2) there is also a risk that GM plants could interbreed with wild plants creating “super weeds”, which are resistant to herbicides.
What is “pharming”?
Many Pharmaceuticals (medicinal drugs) are produced using genetically modified organisms, such as animals.
Example of pharming?
Hereditary antithrombin deficiency is a disorder that increases the risk of blood clots. This risk can be reduced with the protein antithrombin.
1) DNA fragments that code for production of human antithrombin in the mammary glands are extracted.
2) the DNA fragments are injected into a goat embryo.
3) the embryo is implanted into a female goat.
4) when the offspring is born, it is tested to see it it can produce the antithrombin protein.
5) if it does, selective breeding is used to produce a herd of goats that produce antithrombin in their milk.
6) the protein is extracted from the milk to make a drug to help people with the disorder.
What are the positive and negative ethical issues with pharming?
Positive - drugs made this way can be made in large quantities compared to other methods of production. This can make them more available to more people.
Negative- there is concern that manipulating an animal’s genes could cause harmful side effects for the animal, and using animals in this way enforced the idea that animals are just “assets” can be used however humans choose.
