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.
How are pathogens used for research ?
Scientists carry out research into genetically engineered pathogens in order to find treatments for disease.
What are positive and negative ethical issues with genetically modified pathogens?
Positive - can help cure dieseases
Negative - some people are worried scientists researching pathogens could become infected with the pathogen and potentially cause an outbreak of the disease. Some people are worried that a genetically modified version of a pathogen will never return to its original form and cause an outbreak of the disease. Some people are worried that knowledge of how to genetically engineer dangerous pathogens could be used maliciously to create agents for biowarfare.
What is technology transfer?
Many scientists around the world are working on techniques to improve genetic engineering. Scientists working in these different institutions share their knowledge so that globally, beneficial GM products can be created at a faster rate. The sharing of knowledge, skills and technology like this is technology transfer.
How can scientists obtain legal protection?
They can get a patent. This means they can control who uses the product and how for a set period of time.
What are the positive and negative ethical issues with patents?
Positive- it means of the owner of the patent will get money generated from selling their product. This encourages scientists to compete to be the first to come up with a new beneficial genetic engineering idea, s we get genetically engineered products faster.
Negative- farmers in poorer countries will not be able to afford patented genetically modified seeds. Even if they can afford seeds for one year, some patents say they will not be able to grow the, again until they pay again. Many people think this is unfair and that big companies should be more lenient with the rules to help farmers in poorer countries.
What are genetic disorders?
Genetic disorders are inherited disorders caused by abnormal genes or chromosomes, eg cystic fibrosis.
What is gene therapy?
Gene therapy involves altering alleles inside cells to cure genetic disorders.
How does gene therapy work?
1) if the condition is caused by two mutated recessive alleles, you can add a dominant alleles to make up for them.
2) if it is caused by a mutated dominant allele you can “silence” the dominant allele by sticking a bit of DNA in the middle of the allele so it doesn’t work anymore.
3) to get the new allele inside the cell, the allele is inserted into cells using vectors.
What are two types of gene therapy?
1) somatic therapy
2) germ line therapy
What is somatic therapy?
This involves altering the alleles in body cells, particularly the cells that are most effected by the disorder. For example cystic fibrosis I’d a genetic disorder that damages the respiratory system, so somatic therapy for CF targets epithelial cells lining the lungs.
Somatic therapy doesn’t affect the individual’s sex cells though, so any offspring could still inherit the disease.
What is germ line therapy?
This involves altering the alleles in sex cells. This means that every cell of any offspring produced from these cells will be affected by the gene therapy and they won’t inherit the disease. Germ line therapy is currently illegal in humans though
What are the positive ethical issues of gene therapy?
1) gene therapy could prolong lives of people with life threatening genetic disorders.
2) gene therapy could give people with genetic disorders a better quality of life as it helps ease symptoms
3) germ line therapy would allow the carriers of genetic disorders to conceive a baby without that disorder.
4) germ line therapy could decrease the number of people that suffer from genetic disorders and cancer, which is beneficial for society as a whole as fewer people require treatment
What are the negative ethical issues of gene therapy?
1) the technology could potentially be used in ways other than for medical treatment, such as treating the cosmetic effects of ageing.
2) there’s the potential to do more harm than good by using the technology eg risk of overexpression of genes
3) there’s concern that gene therapy is expensive.
What are the disadvantages of gene therapy?
1) the body could identify vectors as foreign bodies and start an immune response against them.
2) an allele could be inserted into the wrong place in the dna, possibly causing more problems eg cancer
3) an inserted allele could be over expressed, producing too much of the missing protein and so causing other problems.
4) the effects of the treatment may be short lived in somatic therapy
5) the patient might have to undergo multiple treatment with somatic therapy.
6) it might be difficult to get the allele into specific body cells
What method is used to sequence DNA?
The chain termination method. It works by amplifying lots of copies of the DNA using PCR, but with varying lengths . The DNA a strands are put into size order and the terminator base is read to determine the overall sequence
Step 1 of the chain termination method?
The DNA sample, primers, DNA polymerase and free nucleotides are mixed in four seperate tubes. Modified nucleotides are also added which are attached to a fluorescent tag. In one test tube, fluorescently labelled adenine is added. To another tube, fluorescently labelled cytosine is added, and so on. Once these are incorporated into the newly synthesised DNA chain, no more bases can be added after it.
Step 2 of chain termination method?
1) PCR is carried out converting the original DNA copy into millions of DNA a strands. The strands vary in length due to the addition of fluorescent nucleotides at random positions along the chain.
Step 3 of the chain termination method?
Electrophoresis seperates DNA fragments according to size and are visualised under UV light. The complementary base sequence can be read off from the gel. The band furthest to the bottom represents the smallest DNA fragment. Reading the gel from the bottom to the top will give you the DNA a sequence.
What is whole genome sequencing?
Sequencing DNA using the chain termination method only works for short strands of dna. So, to sequence an entire organism, scientists fragment it, insert it into bacteria which replicate and amplify the DNA fragments. The DNA is sequenced and put back in order,
What is step 1 in genome sequencing?
Restriction enzymes cut the genome into fragments. DNA fragments are inserted into man made plasmids called bacterial artificial chromosomes (BACs)
What is step 2 in genome sequencing?
The BACs are inserted into bacteria so that each bacterium contains a BAC with a different DNA fragment. The bacteria divide to form a colony of identical cells. Each colony will contain the same BAC because they originate from the same bacterium. All the colonies together form a genomic DNA library.
Step 3 of genome sequencing?
DNA fragments are purified from each colony and further fragmented using restriction enzymes. This produces overlapping DNA fragments. The shorter DNA fragments are sequenced using the chain termination method. Computer software uses the overlapping sequences to put fragments back in order, generating a complete genomic sequence.
What are some advances in sequencing?
Advances in the field have led to an automatic version of the chain termination method, where all four fluorescent bases are added to the same tube and a machine reads off the sequence, generating a computer readout.
Other developments include high-throughout sequencing and pyrosequencing, where DNA can be sequenced cheaply and thousands of time faster.
How is sequencing linked to synthetic biology?
If you know the dna sequence within a gene, you can work out its amino acid sequence and predict the protein’s primary structure. Scientists have used DNA sequences to artificially build proteins from scratch. It also includes designing new biological systems and molecules that don’t exist in the natural world but could be beneficial for humans eg fuel and drugs
How can you use sequencing to compare genomes?
Gene sequences can be compared between organisms. It is made easier with the use of computational biology (using computers to study biology) and bioinformatics (developing and using computer software that can analyse organise and store biological data.
What are the three uses of comparing genomes?
1) epidemiology
2) evolutionary relationships
3) genotype-phenotype relationships
How can you study epidemiology?
Comparisons between healthy people and those with a disease can help identify genes linked to a certain condition. For example an allele called APOE4 is linked to an increased risk of developing Alzheimer’s disease.
How can you study evolutionary relationships?
Comparing DNA sequences of different organism can provide an insight of how closely related they are in terms of evolutions. For instance the genomes of humans and chimpanzees are 99% identical, suggesting we share a recent common ancestor.
How can you study genotype-phenotype relationships?
Information about an organism’s genotype can allow us to predict its phenotype. Genome sequencing can allow scientists to uncover whether someone will develop Huntington’s disease for instance, or an increased likelihood of developing breast cancer.
