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M16. Human beings can manipulate DNA.


Student Outcome: M16.1

Know that the DNA can be extracted from cells.


There are two to three basic steps in DNA extraction. The cell must be lysed (broken open) to release the nucleus. The nucleus (if present) must also be opened to release the DNA. At this point the DNA must be protected from enzymes that will degrade it, causing shearing. Once the DNA is released, it must then be precipitated in alcohol.


Source: http://www.accessexcellence.org/AE/AEC/CC/DNA_extractions.html


This video shows how it might be done. Fairly quick and not much evidence but it was done by High School students.


This is a bit more professional



Student Outcome: M16.2

Describe how particular genes can be selected and removed, using probes and restriction enzymes.


  • Identifying a gene.

To identify the gene for a particular characteristic from the huge amount of DNA within an organism is a daunting task. Before you begin, you need to know something about the gene; for example what protein it contains instructions to make or its base sequence.


Initially, scientists used information about the protein, such as its amino acid sequence, to eventually isolate the DNA molecule that contained the instructions for that protein. More recently, scientists have determined the entire sequence of bases that make up the genome of single-celled organisms. Projects to sequence the genomes of more complex organisms are well advanced. These projects include those aimed at sequencing the human genome and the genome of a small plant called Arabidopsis, a relative of commercially important plants such as oil-seed rape.


Proteins with similar functions often have similar structures, and the genes coding for these proteins will have a related base sequence. So, as more and more genes are identified, from more and more organisms, the task of identifying a new gene for a particular characteristic becomes easier.


Source: http://www.science.org.au/nova/009/009print.htm#box%203


  • Gene probes


Every gene contains a unique sequence of the four bases: adenine (A), cytosine (C), guanine (G) and thymine (T). We can test to see if a specific gene is present in a person’s genetic make-up by searching for its unique base sequence.


The search uses a gene probe, which is a piece of single-stranded DNA. The design of a probe uses the fact that when DNA strands pair up, adenine only pairs with thymine and cytosine only pairs with guanine. The base sequence on the probe matches the unique sequence in the gene that the probe is designed for.


To test a DNA sample using a gene probe, the DNA is first treated so that each of the double-stranded DNA molecules unzips into single strands. The probe is then added to the solution. Because of the way the bases pair up, the probe will attach itself only to the section of DNA that contains a base sequence that matches the probe’s sequence.


Probes are constructed with a radioactive or a fluorescent section, or tag, in them, so that they can be detected after attaching to the DNA. Detecting the probe gives us information about which chromosome the gene is on, and where the gene is on that chromosome.


We know the base sequences in a number of disease-causing genes, and can find out if they are present using probes specifically designed for them.


Source: http://www.biotechnologyonline.gov.au/biotec/findgene.cfm


  • Restriction Enzymes

Restriction enzymes recognize specific sequences on DNA, then cut the DNA at those sequences. There are about 2500 known restriction enzymes which have different specificities for cutting the DNA. A complete list is available in the REBase web site (http://www.neb.com/rebase/). Most of the sequences that are recognized by restriction enzymes are palindromic, meaning they read the same backward and forward. For example, the restriction enzyme BamH1 recognizes the DNA sequence GGATCC. The complementary strand of DNA, reading in the opposite direction, would also read GGATCC. This palindromic quality makes it easier for the restriction enzyme to recognize the sequence, since it is present on both strands of DNA.


Sometimes the restriction enzyme cuts straight through the DNA, cutting both strands at the same location. Most of the time, however, restriction enzymes cleave the DNA in a staggered cut - leaving a few nucleotides of single stranded DNA extending from the cut site. For example, the BamH1 restriction enzyme cuts both strands of DNA between the adjacent G's, leaving "sticky ends" of DNA on each strand:


5'-----G GATCC------


-------CCTAG G-----5'


These sticky ends allow separate DNA molecules to get together. The short sticky ends actually can base pair between two different DNAs to align the two DNA molecules. Any two DNAs cut with the same restriction enzyme will have the same sticky ends and therefore can be joined. It is this ability provided by restriction enzymes that allows most of recombinant DNA techniques to work.


BamH1 will recognize this sequence and cut DNA approximately once in every 4096 basepairs (46). If a plasmid is cut by a restriction enzyme, it will also have sticky ends and could base pair with pieces of human DNA.


Source: http://www.dartmouth.edu/~cbbc/courses/bio4/bio4-1997/09-GenEngin2.html


Here is an animation showing how restriction enzymes work.


Student Outcome: M16.3

Describe how selected genes can be transferred between species, using bacterial plasmids, viruses, and micro injection.



Cloning DNA using bacteria. Scientists use a restriction enzyme to cut all the DNA of a donor organism into manageable fragments of a few thousand bases in length. They then splice each fragment into a bacterial plasmid, a small circular DNA molecule, to create a recombinant plasmid. Scientists reintroduce each recombinant plasmid into a separate bacterium, creating a bacterial 'library' of the donor DNA. To clone the DNA the bacteria are spread thinly on a nutrient agar plate so that each bacterium is well separated from the others. Each bacterium grows into a colony of millions of cells, each of which contains an identical recombinant plasmid with its DNA fragment from the donor organism. Since there are millions of cells, there are now millions of copies of each DNA fragment.


Source: http://www.science.org.au/nova/009/009print.htm#box%203


Viruses in gene therapy


Some of the different types of viruses used as gene therapy vectors:


  • Retroviruses - A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus.


  • Adenoviruses - A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus.


  • Adeno-associated viruses - A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19.


  • Herpes simplex viruses - A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.



Source: http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml



Other Methods:

Gene insertion involves the insertion of new genes into the cell's existing genetic material. Different methods are used to transfer genes into different living things.


In animals, the desired gene can be inserted by injecting the gene into a single-celled embryo. This embryo is then allowed to develop into an adult animal. This technique is called microinjection.


In plants, the gene of interest can be coated onto tiny metal particles which are then shot into the cell using a special gun. A second method uses bacteria, usually one from the Agrobacterium family as they have a natural ability to infect plant cells and incorporate the bacterial DNA into the plant cell. Scientists can add the desired gene to the DNA of the bacteria, which then enter the cells of the plant, transporting the gene in the process. The gene integrates into the DNA of the plant cell. This added or foreign gene is called a transgene.


Source: http://www.biotechnology.gov.au/index.cfm?event=object.showContent&objectID=D2E34694-BCD6-81AC-1FDA0F2FA29BCBB1


This video shows what plasmids are and how they can be used to transfer DNA





Student Outcome: M16.4

Discuss the social consequences of the manipulation of DNA.


Concerns about gene technology


While the potential benefits of gene technology are immense (eg, higher yields, resistance to pests and diseases, adaptation to particular environments, and increased convenience in harvesting and storage), many people have a number of concerns about genetically modified organisms.


  • Pesticide resistance


It is now possible to genetically engineer plants that produce their own pesticide. This exposes pests to the pesticide every day rather than as burst of pesticide application by the grower. If the pesticide-producing capability is introduced into a number of different plant species, it could accelerate the development of pesticide resistance among pests. (To reduce this concern the US Environmental Protection Agency has restricted the sales of pesticide-producing corn to states that do not grow pesticide-producing cotton.)


  • Increased use of herbicides


When farmers spray a herbicide to remove weeds growing among crops, the sprayed chemical often damages the crop plants. If the crop is engineered to be resistant to the chemical, the weeds will be killed but the crop plants will remain undamaged.


At first glance, this seems like a good thing. But it is likely to lead to greater use of the particular herbicide, which would have two negative effects:


    • The crop is likely to contain greater herbicide residues; and
    • the increased spraying will contaminate the rest of the environment.


Of course, not all herbicides are dangerous, but it seems safer to minimise rather than encourage their use.


  • Herbicide-resistant weeds


Genetic engineers are producing crops that are herbicide-resistant and pesticide-resistant. If the genes for these characteristics were to end up in a weed species, the weed would thrive and be difficult to control. (Field trials in Denmark of a genetically engineered, herbicide-resistant rape showed that the gene for herbicide resistance had jumped into a closely related plant.)


  • To label or not to label


One of the main points of controversy surrounding the release of genetically modified organisms is the question of labelling food products. Supporters of labelling point to potential problems for people with food intolerances. An investigation carried out in the mid-1990s found that seven out of nine people allergic to brazil nuts were also allergic to soya beans that had been genetically modified to contain a protein usually found in the nuts. These people showed no reaction to unmodified soya beans, so the protein taken from the brazil nuts must have been responsible for their allergic reaction. Because serious food reactions can kill, people need to know when genetically modified products might cause allergic reactions.


Supporters of labelling also point to the principle of the consumer's right to know what is in their food. Opponents point out that we don't know exactly what is in our food at the moment anyway. Many plants contain natural toxins to protect them against insect attack. These toxins are not good for us, and yet when we eat a parsnip we are not told the concentration of the potentially carcinogenic chemical that occurs naturally.


  • Concerns about marker genes


More serious worries stem from the use of marker genes. These are genes that are inserted into the genetically modified organism along with the desired gene. The presence of marker genes, which are easy to spot, allows researchers to recognise organisms that contain the desired gene. The problem arises with those marker genes that give antibiotic resistance to the organism.


Some people believe it is risky to allow genetically modified plants with marker genes for antibiotic resistance into the environment. For example, the British government objected to a proposal to import genetically modified corn from the United States into Europe because the plants contained a marker gene for resistance to the commonly used antibiotic, ampicillin. The government feared that the gene for antibiotic resistance could spread to bacteria that inhabit the human gut. In turn, these could pass the gene on to more dangerous bacteria. Or the marker gene could move from the plant into soil bacteria and then into disease-causing bacteria.


After much debate, in late 1996 the European Commission decided to allow the corn to be sold in Europe.


  • The role of big business


It is expensive to develop the potential that gene technology offers and it requires a long-term financial commitment to research. While large and well-funded corporations are able to provide this amount of money, there is some concern that the results of this research will not be readily accessible to small companies or developing countries. Some people are also concerned about placing responsibility for the world's food supply into the hands of a few large companies.


  • Tampering with nature


Critics of gene technology suspect that we still know too little about the systems that we are tampering with. Could an inserted gene have effects that we are unaware of? Could it upset the balance of existing genes, causing the plant to produce greater quantities of natural toxins, or to change its nutritional content?


Most researchers argue that there is no evidence of such unexpected changes. They point out that gene technology is much less likely to have unwanted effects on a plant than traditional selective breeding methods. These traditional methods, which have been carried out for thousands of years, involve the movement of thousands of genes from one organism to another. Modern gene technology, on the other hand, moves only a few targeted genes.


  • Safeguards


Food Standards Australia New Zealand (formerly ANZFA) develops food standards for composition, labelling and contaminants that apply to all foods produced or imported for sale in Australia and New Zealand. Food standards are developed with advice from other government agencies, stakeholders and food regulatory policies, and apply to the entire food supply chain from ‘the gate to the plate’.


The Office of the Gene Technology Regulator (OGTR) is the government agency responsible for gene technology regulation in Australia. The OGTR provides administrative support to the Gene Technology Regulator, who is responsible for enforcing the Gene Technology Act 2000. According to the OGTR web site, the Act is ‘a national scheme for the regulation of genetically modified organisms in Australia, in order to protect the health and safety of Australians and the Australian environment by identifying risks posed by or as a result of gene technology, and to manage those risks by regulating certain dealings with genetically modified organisms’.


‘Dealings’ include research, production, manufacture, import, storage, transport and disposal of genetically modified organisms (GMOs). To release a GMO into the environment, the Regulator prepares a risk assessment and risk management plan by consulting scientific experts, stakeholders and the public. The Regulator then decides whether or not to issue a licence to allow the release of a GMO.


Source: http://www.science.org.au/nova/009/009print.htm#box%203


The science of gene technology: benefits and risks


Everything in life has its benefits — and risks. Gene technology is no exception. For the last few decades, scientists have been working on genes, the basic building blocks, to find out what might be possible for the human race and the essential materials for its existence.


Here we give a few examples of the two sides of gene technology, not an exhaustive list. Much has been said about possible risks of gene technology, but there is little actual evidence so far from scientific studies.


  • Crops — the benefits:


The Cotton Example - Cotton is one of many crops being genetically modified or made transgenic to make them insect resistant, so reducing the need to spread large amounts of insecticide.


Many farmers in the US and Australia now grow transgenic cotton varieties that carry inbuilt protection against major pests like Helicoverpa caterpillars. They contain a gene (the Bt gene) that makes a natural insecticide targeting the leaf-chewing larvae of certain species of moth.


As a result Australian cotton farmers have been able to reduce their use of synthetic pesticides by 50 per cent where the GM cotton Ingard® is used. A new variety, Bollgard II®, commercially available in 2003 has shown a 75 per cent pesticide reduction in trials.


  • Crops — the risks:


Some laboratory studies in Europe and the US have shown that a toxin produced in transgenic crops by the "insect resistance" Bt gene has the potential to affect at least two insect species apart from the pest it was designed to target.


Larvae of the ladybird beetle, which attack leaf-chewing and sap-sucking insect pests, can be killed by eating caterpillars that have ingested a lethal dose of the toxin. And caterpillars of the migratory monarch butterfly could be killed if, in the act of eating their favorite plant, milkweed, they accidentally ingest wind-blown pollen from maize crops protected by the Bt gene.


Preliminary results of follow-up studies suggest that most Bt corn does not have a significant effect on monarch mortality. A consortium of US scientists has been formed to determine if Bt corn truly presents a threat to monarch butterfly populations under actual environmental conditions, including refining our understanding of how monarchs and corn pollen interact in the field.


There is some evidence that crops modified for herbicide tolerance could cross-breed with nearby weeds of the same family. A study by the CRC for Weed Management found that pollen from herbicide-tolerant canola could travel up to 2.6 kilometres but in very minute amounts. Any herbicide tolerance gained from gene transfer does not confer any special advantage on weeds unless they are in an area regularly sprayed by the relevant herbicide.


A UK farm study reported in 2003 found that growing some GM crops could potentially reduce biodiversity in fields (e.g. fewer insects and birds). However, the same environmental conditions do not exist in Australia for this to happen.


  • Medicine — the benefits:


Scientists can now locate and study the genes that cause genetic diseases, or those making some individuals prone to cardiovascular disease, degenerative brain disorders like Alzheimer’s disease and motor neuron disease, certain forms of cancer, diabetes and other auto-immune disorders like rheumatoid arthritis and lupus.


Here is a video showing how flourescent genes from a jelly fish have been used in mice.


Gene technology has provided a host of precise new tests for rapid diagnosis of infectious diseases in humans and livestock, and new vaccines to protect against diseases where conventional vaccines have been unsuccessful.


Scientists are now mapping and cataloguing the complete genetic blueprints of the world’s most common infectious bacteria, to identify new targets for "designer" antibiotics.


Gene technologists have also made promising progress towards understanding two of the world’s biggest killers — the malaria parasite, and the human immunodeficiency virus that causes AIDS — and developing vaccines to prevent them.


  • Medicine – the risks:


In medicine, gene technology has raised difficult ethical questions, particularly around issues such as


    • human cloning


    • so-called germline gene therapy that would introduce permanent, inheritable changes into the human gene pool
    • the privacy of genetic information.


Source: http://www.biotechnologyonline.gov.au/biotec/moreinfo.cfm


Check this example of a super cow:



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