Topic 8B: Genome projects and Gene technologies

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  • Created by: DBaruch
  • Created on: 22-02-17 09:54

Sequencing Genomes

  • Improvements in technology have allowed us to sequence the geneomes of a variety of organisms, from bacteria to humans. Gene sequencing methods only work on fragments of DNA, so if you want to sequence the entire genome of an organism, you need to break it into smaller pieces and then the smaller pieces are sequenced and put back in order to show the sequence of the whole genome
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Sequencing proteomes

  • The proteome of an organism is all the proteins that are made by it. Simple organisms such as bacteria, dont have much non-coding DNA. This menas that is it easy to determine their proteome from the DNA sequence of their genone. This can be useful in medicine for example, identifying protein antigens on the surface of diseaase-causing bacteria and viruses can help with vaccine development. This can allow pathogens to be monitored during outbreaks of disease, which can lead to management of the spread of infection.
  • More complex organisms have large sections of non-coding DNA and they also contain regulatory genes, which determine when the genes that for a protein should be switched off or on. This makes it harder to translate their genome into their proteome, because its hard to fine the coding parts among the non-coding and regulatory DNA.
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Developing new sequencing methods

  • In the past, sequencing methods were labour-intensive, expensive and could only be done on a small scale. During the 1970's Frederick Sanger developed a technique in which a sample of DNA was tagged with radioactive bases, separated into 4 lanes on a gel and allowed to mirgrate. The result was photographed by x-ray. As each lane represented one of the 4 bases the sequence of the DNa could be worked out by combining the results in each lane. Only one sample could be processed at one time. 
  • Pyrosequencing is a newer technique that can sequence around 400 million bases per hour in a 10 hour period.
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Recombinant DNA technology

  • Recombinant DNA technology involved transferring a fragment of DNA from 1 organism to another. Because the genetic code is universal and because transcription and translation mechanisms are similar too, the transferred DNA can be used to produce a protein in the cells of the recipient organism. The recipient and donor organisms dont even have to be from the same species. Organisms that contain transferred DNA are known as transgenic organisms.
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Using reverse transcriptase

  • Most cells only contain 2 copies of each gene, making it difficult to obtain a DNA fragment containing the target gene. But cells that produce the protein coded for by the target gene will contain many mRNA molecules that are complementary to the gene- so the mRNA is often easier to obtain. The mRNa molecules can be used as templates to make lots of DNA. The enzyme reverese transcriptase, makes DNA from an RNA template. The DNA produced is called complementary(cDNA).
  • To make cDNA, mRNA is first isolated from cells. Then its mixed with free DNA nucleotides and reverse transcriptase. The reverse transcriptase uses the mRNA as a template to synthesis the new strands of cDNA
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Using restriction endonuclease enzymes

  • Some sections of DNA have palindromic sequences of nucleotides. These sequences consist of antiparallel base pairs. Restriction endonucleases are enzymes that recognise specific palindromic sequences and cut the DNA at these places. Different restriction endonucleases cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to the enzyme's active site. If recognition sequences are present on either side of the DNA fragment you want, you can use restriction endonucleases to separate it from the rest of the DNA. The DNA with the specific restriction endonucleases are incubated and cut out via hydrolysis. Sometimes the cut leaves sticky end (small tails of unpaired bases at each end of the fragment). Sticky ends can be used to bind the DNA fragment to another piece of DNA that has sticky ends with complementary sequences

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Using a gene machine

  • More recently, technology has been developed so that fragments of DNA can be synthesised from scrath, without the need for pre-existing DNA template. Instead, a database contains the information to produce a DNA fragment, meaning the fragment doesn't have to exist naturally
  • The sequence that is required is designed
  • The first nucleotide in the sequence is fixed to some sort of support e.g. a bead
  • Nucleotides are added step by step in the correct order, in a cycle of processes that includes adding protecting groups. Protecting groups make sure that the nucleotides are joined at the right points, to prevent unwanted branching
  • Short sections of DNA called oligonucleotides, roughly 20 nucleotides long, are produced. Once these are complete they are broken off from the support and all the protecting groups are removed. The oligonulceotides can then be joined together to make long DNA fragments
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In vivo and in vitro cloning

  • In vivo- where the gene copies are made within a living organism. As the organism grows and divides it replicates the DNA, creating multiple copies of the gene
  • In vitro- where the gene copies are made outside of a living organism using the polymerase chain reaction (PCR)
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In vivo steps

  • Insertion- The vector is isolatied. The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene. This produces sticky ends of the vector DNA which are complementary to the sticky ends of the DNA fragment containing the gene. The vector DNA and the DNA fragment are mixed with together with DNA ligase. This joins the sticky ends together. This process is called ligation. The new combination of bases in the DNA (vector DNa & DNA fragment) is called recombinant DNA
  • Transformation- is when the host cell takes up the plasmid and the DNA
  • Identification- only around 5% of host cells will take up the vector and its DNA, so its important to be able to identify which ones have. Marker genes show which ones have transformed. Marker genes can be inserted into vectors at the same time as the gene wanting to be cloned so they transform at the same time. Those which have the gene and the anitobiotic marker gene will grow whereas the other bacteria will die.
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In vitro steps

  • Step 1- a reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase. Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want. DNA polymerase is an enzyme that creates new strands.
  • Step 2- the DNA mixture is heated to 95 degrees to break the hydrogen bonds between the 2 strands of DNA. The mixture is then cooled to between 50 and 65 degrees so that the primers can bind to the strands
  • Step 3- the reaction mixture is heated to 72 degrees, so the DNA polymerase can work. The DNA polyemerase lines up free DNA nulceotides alongside each template strand. Specific base pairing means new complementary strands are formed.
  • Step 4- 2 new copies of the fragment DNA are formed and 1 cycle of PCR is complete. The cycle starts again- the mixture is heated 95 degrees and this time all 4 strands (2 new 2 original) are used as templates
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Transformed organisms- Recombinant

  • Microorganisms, plants and animals can all be transformed using recombinant DNA technolog. This is called genetic engineering. Transformed microoganisms can be made using the same technology as in vivo cloning.
  • In plants a gene that codes for a desireable protein is inserted into a plasmid the plasmid is then added to a bacterium and the bacterium is used a vector to get the gene into the plant. If the right promoter region has been added the transformed cells will be able to produce the desired protein.
  • In animals the gene can be inserted into an embryo or an egg cell. It is is in an embyro then all the body cells will contain the gene, inserting it into the gene means that all offspring from the female will contain the gene
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Benefits of transformed organisms

  • Crops can be transformed to give higher yield or are more nutritious(golden rice and vitamin A). They can also be transformed to have resistance to pests or droughts.
  • In industry enzymes are often used so the enzymes can be produced from transformed organisms meaning you can get larger quantities for less money reducing costs.
  • Many drugs and vaccines are produced by transformed organisms, for example insulin is used to treat type 1 diabetes and used to come from animals. However human insulin is now made from transformed microorganisms, using a cloned human insulin gene.
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Concerns of transformed organisms

  • Some people have ethical, financial and social concerns about the use of recombinant DNA technology.
  • Farmers who only plant one crop (monoculture) may be vulnerable to the same disease because they are genitically identical. Environmentalists are concerned about the reduction in biodiversity. Organic farmers can have their crops contaminated by wind-blown seeds from nearby GM crops.
  • Some people are worried about the process used to purify proteins and also if a few biotechnology companies control the genetic engineering then they could become more powerful and force smaller companies out of business
  • The companies that own the technology may limit the use that could save lives. Some people worry that it could be used to make desinger babies and recombinant DNA technology also creates ownership issues
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How does gene therapy work

  • Gene therapy involves altering the defective genes inside cells to treat genetic disorders and cancer. How this is done depends on whether the issue is caused by dominant alleles or 2 recessive alleles
  • If it caused by 2 mutated recessive alleles you can add a working dominant allele to make up for them, you "supplement" the faulty ones
  • If its caused by a mutated dominant alleles you can"silence" the dominant allele.
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2 types of gene therapy

  • Somatic therapy- this involves altering the alleles in body cells, particularly the cells that are most affected by the disorder. Somatic therapy doesn't affect the individuals sex cells though, so any 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 from these cells will be affected by gene therapy and they won't suffer from the disease. Germ line therapy in humans is currently illegal though.
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Ethical issues surrounding gene therapy

  • There are also many ethical issues associated with gene therapy. For example, some people are worried that the technology could be used in ways other than for medical treatment, such as for treating cosmetic effects of aging. Other people worry that there's the potential to do more harm than good for example risk of overexpression of genes- gene produces too much of the missing protein
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Locating alleles using DNA rpobes

  • DNA probes can be used to locate specific alleles of genes or to see if a persons DNA contains a mutated allele that causes a genetic disorder. DNA probes are short strands of DNA
  • Step 1- a sample of DNA is digested into fragments using restiction eznymes and separated  using electrophoresis.
  • Step 2- the separated DNA fragments are then transferred to a nylon membrane and incubated with a fluorescently labelled DNA probe. If the allele is present, the DNA will bind (hybridise) to it
  • Step 3- the membrane is then exposed to UV light and if the gene is present there will be a flourescent band.
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Screening for multiple genes

  • The probe can be used as part of a DNA microarray, which can screen for lots of different genes at the same time. A DNA microarray is a glass slide with microscopic sports of different DNA proes attached to it in rows
  • A sample of fluorescently labelled human DNA is washed over the array. If the labelled huamn DNA contains any DNA sequences that match any of the probes, it will stick to the array. So this means you can screen the DNA for lots of different mutated genes at the same time. The array is washed, to remove any fluorescently labelled DNA that hasn't stuck to it, and then visualised under UV light. Any labelled DNA attached to a probe will show up. Any spot fluoresces means that the persons DNA contains that specific allele for example if the probe is for a mutated allele that causes a genetic disorder, the person has the allele
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Uses of screening with DNA probe

  • It can be used to help identify inherited conditions for example the NHS offers to screen all newborn babies for the inherited disorder cystic firbrosis so that treatment for the condition can begin as soon as possible.
  • It can be used to help determine how a patient will respond to specific drugs for example breast cancer can be caused by a mutation in the HER2 proto-oncogene and treated with the drug herceptin. Herceptin is only effective against type of breast cancer because it targets a specific receptor. Screeing for this particular mutaiton helps determine whether herceptin will be useful or not
  • It can also be used to help identify health risks for example inheriting particular mutated alleles increase your risk of developing certain types of cancer might help them to make choices that could reduce the risk of the disease developing
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Genetic counselling

  • The results of screening can be used for genetic conselling. Genetic counselling is advising patients and their relatives about the risks of genetic disorders. It involves advising people about screeing and explaning the results of a screening. Screening can help to identify if someone is the carrier of a mutated allele, the type of a mutated they're carrying and the most effective treatment. If the results of a screening are positive then genetic counselling is used to advice the patient on the options available
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Producing genetic fingerprints

  • Step 1- PCR is used to make DNA fragments, a sample of DNA is obtained, PCR is then used to make many copies. Primers are used that bind either side of the repeats. Different primers are used for each position under investigation. You end up with DNA fragments where the length corresponds to the number of repeats the person has at the each specific position.
  • Step 2- To separate out DNA fragments, the DNA mixture is placed into a well in a slab of gel and covered in a buffer solution that conducts electricity. An electrical current is passed through the gel- DNA fragments are negatively charged, so they move towards the positive electrode at the far end of the gel. Shorter DNA fragments move faster and travel further through the gel, so the DNA fragments separate according to length and produces a pattern of bands
  • Step 3- After the gel has been running long enough, the equipment is turned off and the gel is placed under a UV light. Under the UV light DNA fragments can be seen as bands. These bands make up the genetic fingerprint. A DNA ladder may have been added to one well - this is a mixture of DNA fragments of known length that allows you to work out the length of the other bands on the gel. Two genetic fingerprints can be compared. If both fingerprints have a band at the same location on the gel it means they have the same number of nucleotides
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Uses of genetic fingerprinting

  • Determining genetic relationships-  we inherit variable number tandem repeat base sequences from out parents. Half comes from each parent. The more bands means that they are closely related. Genetic fingerprinting can also be used to look at much wider ranging genetic relationships. Too look at a female line of descent you need to look at DNA in mitochondria. If you want to look at the male side, you need to look at Y chromosome DNA.
  • Determining genetic variability within a population- the greater the number of bands that don't match on genetic fingerprint, the more genetically different individuals are. This means you can compare the number of repeats at several places in the genome for a population to find out how genetically varied that population.
  • In forensic science- The DNA is isolated from all the collected samples. Each sample is replicated using PCR. The PCR products are run on an electrophoresis gel and the genetic fingerprints produced are compared to see if any match.
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Uses continued

  • For medical diagnosis- in medical diagnosis, a genetic fingerprint can refer to a unique patter of sevel alleles, it can be used to diagnose genetic disorders and cancer. It's useful when the specific mutation isn't known or where several mutations could have caused the disorder, because it identifies a broader, altered genetic pattern.
  • In animal and plant breeding- genetic fingerprinting can be used on animals and plants to prevent inbreeding, which decreases the gene pool. Inbreeding can lead to an increased risk of genetic disorders, leading to health, productivity reproductive problems. Since genetic fingerprinting can be used to identify how closely related individuals are, it can be used to identify the least related individuals in a population so that we can breed them together. It can also be used by animal breeders to prove pedigree. Animals with a good pedigree will sell for more money 
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