AOS 4

Þ DNA Manipulation - Enzymes and DNA manipulation - Recombinant plasmids and Bacterial transformation - PCR - Gel electrophoresis

Þ Changes in biodiversity over time - DNA applications - Genetic modifications - Emergence of new diseases - Rational drug design

Enzymes & DNA manipulation What are KEY KNOWLEDGE: the use of enzymes including endonucleases (restriction enzymes), ligases and restriction polymerases. enzymes? Restriction enzymes = enzymes that cut DNA into fragments (note that it cuts not unwinds) by breaking the bonds between 2 .

Note - this doesn’t break the H bonds between base pairs but instead breaks the covalent bonds between adjacent nucleotides.

Can a The ’s cut DNA at certain recognition sites. These sites are complementary to the sticky end enzymes active site and are usually palindromic. bind to a blunt end? For a sequence to be palindromic, the complementary strand has to be read the same way backwards.

The enzyme can either cut the DNA so it has sticky ends or blunt ends

Where are the bonds Sticky ends can bond to other broken by complementary sticky ends via restriction enzymes? hydrogen bonds between complementary base pairs.

I.e. like only binds to like

Blunt ends don’t have to stick to complementary pairs, as they will be forming covalent bonds with other pieces of DNA not hydrogen What are Ligase? bonds with base pairs. Any sticky end can bond to any other sticky end through using a ligase enzyme.

an enzyme that sticks pieces of DNA Ligase = together (basically the opposite of restriction enzymes)

It is used for both sticky and blunt ends, and joins together segments of DNA by catalysing the formation of covalent bonds between nucleotides. What are Polymeras e? Polymerases = enzymes that synthesise nucleic acids (e.g. RNA polymerase)

⇒ Restriction enzymes cut DNA into pieces, by either sticky or blunt cuts ⇒ Restriction enzymes break the covalent bonds between nucleotides ⇒ Ligases are enzymes that join DNA segments together by catalysing the formation of covalent bonds between nucleotides. ⇒ Polymerases are enzymes that synthesise nucleic acids

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Recombinant plasmids What are KEY KNOWLEDGE: the use of recombinant plasmids as vectors to transform bacterial cells. plasmids? Recombinant DNA The alteration of genes (e.g. adding or removing genes to sections of DNA). This enables new genes to be introduced to things like bacterial cells. E.g. giving a bacterial cell a section of human DNA that codes for the Why are production of insulin. This insulin can then be used to give plasmids to people with diabetes. useful?

Plasmids Bacteria have chromosomal DNA but most also contain one ore more plasmids. Plasmids: Why do you ⇒ Are small circular pieces of double stranded DNA need to use the ⇒ They are good to use as a vector (something same that acts as a carrier for genes) restriction enzyme for Use as vectors cutting the You can transform bacterial cells by removing a gene vector & from one of its plasmid’s and inserting another gene gene? from somewhere else.

Making recombinant plasmids 1. The DNA of the plasmid is cut at a specific point using restriction enzymes that cut sticky ends. To e Why are 2. The foreign DNA is then cut using the same plasmids restriction enzyme so that the foreign DNA good vectors? has sticky ends that match those of the cut plasmid. (Complementary sticky ends) 3. The foreign DNA and plasmid are mixed and if successful the sticky ends of each will form weak hydrogen bonds. (Anneal with each) Note – other unsuccessful scenarios can

happen What is the process of 4. The enzyme ligase is added to permanently making join the foreign DNA and plasmid together by recombina catalysing the covalent bonds between nt nucleotides plasmids? Note - other pairings will also occur, such as cut

plasmids resealing themselves so that they are not

recombinant plasmids.

⇒ Plasmids are small circular pieces of DNA found in bacteria, that act as good vectors ⇒ Plasmids are good vectors due to their circular nature and can be used to introduce new genes into bacteria ⇒ The same restriction enzyme is used for both so that the foreign DNA has ends that match those of the cut plasmid. ⇒ Recombinant plasmids are plasmids that have had an alteration made to their genes.

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Bacterial transformation What is a KEY KNOWLEDGE: the use of recombinant plasmids as vectors to transform bacterial cells. transforme d When a plasmid has been given a foreign gene it is referred to as a recombinant plasmid. Once the bacterium? plasmid is inserted into bacteria, the bacteria is said to be transformed. i.e. Plasmid + foreign gene = recombinant plasmid Recombinant plasmid + bacteria = transformation

Equipment required for recombinant plasmids:

Plasmid, restriction enzyme, buffer solution, heat block, DNA sample.

What is a After a recombinant plasmid has been made, it must be inserted into recombina bacteria. In general, the success rate for the transfer of recombinant nt plasmids into bacterial cells is low, but the success rate can be increased plasmid? through various techniques.

Testing for success:

• Using gel electrophoresis (Recombinant plasmids) • Measuring gene expression, i.e. measuring if a protein has been produced as a result of the introduced gene. (Recombinant plasmids) • An antibiotic resistant gene may be inserted in the plasmid along with the gene of interest, and What is a the bacteria are tested to see whether or not they are antibiotic resistant. The ones that are will method of testing for successfully have the gene of interest inserted into their plasmid and the rest will die. successful (Transformation) bacterial transforma tion?

How does heat shock enable bacterial

transforma tion? Cloning Producing genetically identical copies of an organism and hence its genes. I.e. Insulin gene can be isolated and purified using PCR. The gene can then be inserted in bacterial cells that will produce copies of the gene as well as insulin.

What is the Bacteria transformation techniques: difference 1. Heat shock – Bacteria and vector are put in ice and then in heat. This heat shock makes the between conjunctio membrane more permeable and allows the vector to be adsorbed into the bacteria n and 2. Electroporation – electric shocks put temporary holes in the bacteria membrane and allows the transductio vector to be absorbed by the bacteria n? 3. Conjunction – using a bacteria to transfer it’s genetic material to another bacteria

Transformed bacterium4. Transduction = recombinant – putting plasmid the successfully gene in a viinsertedrus and into then the infecting bacterium the bacteria with that virus ⇒ ⇒ Recombinant plasmid = foreign gene successfully inserted into the plasmid ⇒ You can test for success through gel electrophoresis, measuring gene expression for recombinant plasmids ⇒ To test for successful transformation of bacteria, you can insert an antibiotic resistant gene along with the gene of interest and then testing for antibiotic resistant among the final sample ⇒ Heat shock and electroporation make the bacteria membrane more permeable which allows the recombinant plasmids to be adsorbed

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PCR What does KEY KNOWLEDGE: amplification of DNA using the polymerase chain reaction PCR stand for? PCR = polymerase chain reaction. DNA polymerase – synthesizes DNA in a 5’ to 3’ direction Overview This technique is used to make lots of copies of specific fragments of DNA, so they can be examined. You are essentially amplifying the amount of a specific sequence of DNA. What is the Primers purpose of PCR? It doesn’t amplify all the DNA, but rather a specific section. This is done by using primers that have complementary bases to the ends of the genes of interest. The amplification begins at the site of this primer binding. What are Steps: the steps in PCR? 1. Denaturation When heated, the template DNA strands will split apart and become single stranded 2. Annealing stage What are When cooled down again, primer pairs will bind/anneal to specific sections of the single DNA the required strands of interest. temps at 3. Elongation (extending) stage each When heated again, Polymerase will begin to add complementary bases to the template strand stage? starting at the primers. This will cause new strands of DNA to be synthesized. For this a supply of nucleotides must be available. 4. The process is then repeated many times to create large amounts of the specific DNA section

What do Note - The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant primers bacterium from which it was found in. do? Cycles After each cycle the amount of DNA pairs doubles. You need to go through a fair few cycles if you want to have enough of your gene of interest How are news so it can be analyzed. strands Reagents: Template DNA, Primer, synthesize Nucleotides, Taq polymerase d? Equipment: PCR rubes, thermal cylinder

The amount of double stranded DNA Does it need to be grows exponentially after each cycle. repeated E.g. if you have 4 strands of DNA that more then you put through 5 PCR cycles how many double strands will you have? once? =45 =1024

⇒ PCR = polymerase chain reaction, which helps amplify a specific segment of DNA so It can be analysed ⇒ The template strand is denatured (approx. 95°C), i.e. the strands separate, primers then anneal (bind at 55°C) to either side of the area of interest on the separated strands, then polymerase extends the primers (72°C ) synthesizing new DNA strands ⇒ The process must be repeated multiple times so you have enough of your specific DNA section to analyse

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Gel electrophoresis What is the KEY KNOWLEDGE: the use of gel electrophoresis in sorting DNA fragments, including interpretation of gel goal of gel runs electropho resis? It is used to separate fragments of DNA based on their size. Process: • DNA samples are loaded into wells and an electric current is applied • Because DNA is negatively charged it will begin to How does Move towards the positive electrode the

process • The smaller DNA fragments will move faster work? Through the porus gel and will be closer to the positive Electrode, whereas the larger fragments will move Slower and be closer to the negative electrode • A sample of DNA that has been cut to known lengths (molecular weight ladder/marker) will also be run through the gel and you will be able to Why is the charge of compare the unknown samples to this. DNA important? Staining: The samples are stained with a “tracking” dye so it is easy to See the samples as they move through the gel

DNA charge:

DNA has a negatively charged phosphate group in each Why is a , which gives it an overall negative charge. DNA ladder used?

What is the importance of the gel? Gel: Generally agarose gel is used for large samples. It provides a medium for the DNA to be separated in. It is porus.

What is the Ethidium bromide: role of the It illuminates the DNA so the location of buffer? the separated fragments can be seen

Buffer: It contains ions that allow the

flow of electricity in the gel

⇒ Gel electrophoresis separates DNA fragments by length, by applying an electric field causing the natively charged DNA to move through gel towards the positive electrode, causing the fragments to separate as they move through the gel at different speeds based on their size. ⇒ A DNA ladder/marker is a sample of known DNA lengths used as a reference to compare the sizes of the unknown sample to ⇒ The gel allows the DNA to move through with different sized fragments moving through at different speeds

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Application of DNA knowledge What KEY KNOWLEDGE: techniques that apply DNA knowledge (specifically gene cloning, genetic screening and occurs DNA profiling) including social and ethical implications and issues during DNA DNA profiling profiling? • It involves identifying individuals using their DNA. Everybody’s DNA is unique (except for in identical twins) and Some parts of our DNA is ‘more unique’ than others and we can use these to identify people. • These unique segments we use for DNA profiling are called short tandem repeats (STRs). • Short tandem repeat = repetitions of 2-5 base pairs

What is a • Different individuals will have a different number of ‘repeats’ at a particular locus short • Individuals can also have a different number of repeats on each chromosome tandem repeat?

• PCR and gel electrophoresis is then used to determine the source of the DNA Genetic screening It involves conducting tests to detect genetic abnormalities or risk of developing certain inherited conditions. Why can DNA’s For genetic screening for a particular condition to be viable it must: source be 1. The genetic condition being screened for needs to be significant identified by looking 2. There needs to be a cost effective test available that is reliable (minimal false positives and at various negatives). STR’s? 3. A treatment or prevention for the condition should be available Reasons for testing: Screening for disease causing alleles • If individuals suspect that they may be at a greater risk of developing a certain disease or

What condition that can undergo genetic testing to have this risk evaluated occurs • The individual’s DNA is screened to identify if they have a variant of a gene that may make then during predisposed to a certain disease of condition. genetic Screening to see if you are a carrier screening? Individual can also have testing done if they are unaffected by a disease but have a higher risk of being carriers. It allows parents to identify wether their offspring could be affected by a certain condition.

Ethical considerations • Gene pool of future generations may be altered and the allele frequency of certain alleles may be What are 3 decreased due to parents deciding to terminate the pregnancy or not procreating due to the risk ethical of their offspring developing a certain disease concerns • Information can be burdensome – e.g. parents knowing that their unborn child has a serious of genetic genetic condition (having to decide whether to continue the pregnancy or not). screening? • People may experience distress if their test has positive results but may never develop the disease in question. • Some diseases (e.g. Huntington’s disease) for which genetic testing exists have no cure. • Who has a right to this information? Other family members, insurance agencies??

⇒ DNA profiling involves finding the source of a DNA sample by comparing it’s short tandem repeats to other sources ⇒ Short tandem repeat = repetitions of 2-5 base pairs

⇒ People have different number of STR’s t particular locus

⇒ Genetic screening involves testing for an genetic abnormalities present in a foetus or person ⇒ Genetic screening may affect the gene pool and lead elimination of certain alleles.

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Genetic modification What is the KEY KNOWLEDGE: The distinction between genetically modified and transgenic organisms, their use in difference agriculture to increase crop productivity and to provide resistance to insect predation and/or disease, and the between a biological, social and ethical implications that are raised by their use GMO and transgenic Genetically modified organism (GMO) = an organism that has had its DNA changed in some way organism? • Can be though the addition of a gene/piece of DNA • Can be the loss of function of a gene

Transgenic organism = an organism that has had genetic information from another species inserted into its genome.

Are all GMO’s Note – all transgenic organisms are also genetically modified organisms, but not all genetically modified transgenic organisms are transgenic. ? Improving crops Crops can be genetically altered to: • Increase their size

How can • Make them resistant to certain pests genetic • Make them resistant to certain diseases modificatio • Making them more suited to certain environmental conditions including pesticides n improve • Increasing the nutrients that they contain crops?

Considerations

• Genetically modifying organisms reduces the genetic variation in the populations being altered, and this decreased genetic diversity makes them more susceptible to being wiped out. As a whole they won’t be able to adapt to selection pressures over time. What are 4 • Not everyone likes GMO’s because they think they may be harmful to their health, even though potential there are no scientific links to this benefits of Potential benefits genetic modificatio • The ability to grow crops in areas that would otherwise not support crop growth. This may help n? provide enough food for our growing population. • Larger yield and size/quality of the crop. • The ability to make foods more nutritious

• Increase shelf life

• Produce crops all year round

What are 4 Potential issues/ethical/social problems potential • Reduces the biodiversity – crops could all be wiped out by change in environmental conditions issues with • Contaminating non GMO with GMO genetic

modificatio • Large companies could take over as they have more money to buy GMO crops then smaller n? farmers • Economic impact – reduced price from decreased demand and increased supply

⇒ A GMO is an organism that has had its DNA altered in some way, and a transgenic organism is an organism that has had genetic information from another species inserted into its genome. ⇒ Genetic modification can increase the yield and quality of a crop and enable it to grow in an environment that was previously unsuitable ⇒ Genetic modification can reduce biodiversity and could cause crops to be wiped out by a change in environmental conditions

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Emergence of new diseases What is the KEY KNOWLEDGE: strategies that deal with the emergence of new diseases in a globally connected world, difference including the distinction between epidemics and pandemics, the use of scientific knowledge to identify the between an pathogen, and the types of treatments epidemic and a pandemic? Outbreak = when a disease suddenly appears in a restricted location or population

Epidemic = when an increased incidence of a certain condition occurs in a population in a certain area. A high percentage of the population will be affected.

Pandemic = An epidemic occurring at a much greater scale. The disease is seen across multiple countries What are 4 or continents different techniques used for Outbreak management: outbreak • Preventing people from traveling to certain countries management • Prevention ( hygiene, disinfection, sterilisation, PPE) ? • Isolation/quarantine (At home or in hospital)

• Control carriers (Cull infected herds, Destroy infected crops, Eliminate vectors) • Eradication of vectors (Vectors or pathogens) • Vaccination • Education How could a • Response plans person be tested to identify if they Pathogen identification: have been • Test that identify pathogen’s Protein and DNA (PCR, Sequencing etc) infected by a • Test that look at antibodies ( Enzyme-Linked Immunosorbent Essay, ELISA) specific pathogen?

What is the difference between an antibiotic and antiviral?

How do antibiotics work? Antibiotics – Antibiotics work by impacting on cell components that bacteria have but human cells don’t have. Bacteria becoming resistant, allergies to antibiotics and killing good bacteria, are disadvantages of their use

Þ An epidemic is when an increased incidence of a certain condition occurs in a population in a certain area whereas a pandemic occurs when the disease has spread across countries and continents and a large proportion of the population has been infected Þ Antibiotics impact parts of bacteria that only they have, so human cells are not damaged, like their cell walls

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