Biology unit five, DNA technology.

These forms of delivery are not always effective because:

- Adenoviruses may cause infections. - Patients may develop immunity to adenoviruses. - The liposome aerosol may not be fine enough to pass through tiny bronchioles in the lungs. - Even when the CFTR gene is successfully delivered to the epithelial cells, very few are actually expressed.

The process of genetic fingerprinting, Development:

- An x ray film is put over the nylon membrane. - Film is exposed by radiation from radioactive probes, if fluorescent probes are used the positions are located visually. - Because these points correspond to the position of the DNA fragments as separated during electrophoresis, a series of bars are revealed. - The pattern of bands is unique.

Cystic fibrosis.

- Caused by a mutant recessive allele. - Three DNA bases are missing (AAA). An example of a deletion mutation. - The normal gene, CFTR, produces a protein of 1480 amino acids. The deletion leads to a single amino acid being left out. - This is enough to make the protein unable to carry out its role of transporting chloride ions across epithelial membranes. - When chloride ions are transported out of epithelial cells, water naturally follows by osmosis. Epithelial membranes are kept moist. - If the protein is not made, or doesn't function properly, epithelial membranes are dry and the mucus is viscous and sticky.

Interpreting the results from genetic fingerprinting.

- DNA fingerprints from two samples are visually checked. If there appears to be a match, then the pattern of bars of each fingerprint is passed through an automated scanning machine to calculate the length of the DNA fragments from the bands. - It does this by using data obtained by measuring the distances travelled of known lengths of DNA. - The odds are then calculated of someone else having an identical fingerprint. - The closer the match of the two patterns, the greater the probability that the two sets of DNA came from the same person.

Gel electrophoresis.

- DNA fragments are placed onto an agar gel and voltage is applied across it. The resistance of the gel means that the larger the fragments, the more slowly that they move. - Over a fixed period, the smaller fragments move further than the larger ones. - DNA fragments of different lengths are separated. - A sheet of photographic film is placed over the agar gel for several hours. - The radioactivity from each DNA fragment exposes the film and shows where it is situated on the gel. - How to find out the whole sequence of bases: Read from the smallest to the largest, which terminator nucleotide was present. - Only DNA fragments around 500 bases can be sequenced in this way. - Larger genes and whole genomes must be cut into smaller fragments by restriction endonucleases for each fragment to be separated. The sequenced fragments must be put back together to work out the original gene/genome. This can be done using restriction mapping.

The process of genetic fingerprinting, Digestion:

- DNA is cut into fragments using restriction endonucleases. - The endonucleases are chosen for their ability to cut close to, but not within, groups of core sequences.

Effectiveness of gene therapy:

- Effect is short lived. Somatic cells, with a cloned gene added, are not passed to daughter cells so repeat treatments is needed for any effect. -Can induce an immune response. Both the gene being introduced and the vector/ liposome. Often rejected. Antibodies may be produced, which will remain to initiate a greater response in the future infection. - Using viral vectors can lead to problems, as the viruses can often lead to toxic, inflammatory and immune responses in the recipient. May recover the ability to create disease. - The genes are not always expressed. - Not effective in treating conditions that arise in more than one gene. Only works for disorders that are the result of a single mutation.

The process of genetic fingerprinting, Extraction:

- Even the tiniest sample of animal tissue, such as a drop of blood or hair root, is enough to give a genetic fingerprint. - First stage is extracting the DNA by separating it from the rest of the cell. As the amount of DNA is usually small, its quantity can be increased using PCR.

Sickle cell anaemia.

- First human disease to be understood on a molecular level. - Results in a gene mutation in the gene producing haemoglobin. - A is substituted by T. - mRNA produced has a different code, and codes for a different amino acid. - Produces a molecule of haemoglobin with a sticky patch. - When the haemoglobin is not carrying oxygen, they tend to adhere to one another by their sticky patches and become insoluble. Forms long fibres with red blood cells. - The fibres distort red blood cells making them inflexible and sickle. - Sickle cells are unable to carry oxygen and may block small capillaries as their diameter is greater than the capillaries.

The process of genetic fingerprinting, Separation:

- Fragments of DNA are separated according to size using gel electrophoresis, under the influence of an electrical voltage. - The gel is then immersed in alkali to separate the double strands into single strands. - The single strands are then transferred to a nylon membrane, in a technique called southern blotting, involving a series of stages.

Delivering cloned CFTR genes in somatic gene therapy. Wrapping the gene in lipid molecules.

- Genes are easily wrapped in lipid molecules because they can easily ass through the phospholipid portion of the cell surface membranes. 1) CFTR genes are isolated from healthy human tissues and inserted into bacterial plasmid vectors. 2) The plasmid vectors are reintroduced into their bacterial host cells and gene markers are used to detect which bacteria have successfully taken up plasmids with the CFTR gene. 3) These bacteria are cloned to produce many plasmids with the CFTR gene. 4) The plasmids are extracted from the bacteria and wrapped in lipid molecules to form a liposome. 5) Liposomes containing the CFTR gene are sprayed through the nostrils of the patient as an aerosol and are drawn into the lungs through inhalation. 6) The liposomes pass across the phospholipid portion of the cell surface membrane of the lung epithelial cells.

The risks of recombinant DNA technology.

- Impossible to predict with complete accuracy the ecological consequences are of releasing genetically engineered organisms into the environment. Irreversible damage. - Recombinant genes may pass from the organism it was placed into a completely different one. - Manipulation of DNA will have consequences for the metabolic pathways. - GM bacteria have the antibiotic resistance marker added to them, may be harmful. - All genes mutate. What are the consequences of engineered gene mutation. May turn the organism into a pathogen that we cannot control. - Economic consequences of developing plants and animals to grow in a new region. - How far can we take gene therapy.

Benefits of recombinant DNA technology:

- Microorganisms can be modified to produce a range of substances to treat diseases and disorders. -Microorganisms can be used to control pollution, break up and digest oil sticks or destroy harmful gases released from factories. Make sure they don't destroy oil in places where it is required. A suicide gene can be incorporated that causes the bacteria to destroy themselves after the oil slick has been digested. - GM plants can be transformed to produce a specific substance in a particular organ of the plant. Organ can be harvested and desired substance extracted. Plant pharming. - Plants can be modified to manufacture antigens. - GM crops can be engineered to have economic and environmental advantages. Making plants tolerant to environmental extremes. - Plants can help prevent disease by adding vitamins. Rice with vitamin A added. - Gene therapy and genetic fingerprinting.

Implications of genetic screening.

- More information, power and opportunity. - Who decides who should be screened, it is expensive and decisions have to be made about who has priority. - Who has the right to access test results. - What are the responsibilities of someone who carries the gene for an inherited disease. - Should we preserve genetic diversity. Evolution depends on genetic diversity.

Advantages of in vivo gene cloning:

- Particularly useful when introducing a gene into another organism. Once the gene has been introduced into a plasmid, it can deliver the gene into another organism. Gene therapy. - Involves almost no risk of contamination. Gene is cut by the same restriction endonuclease which match the sticky ends of the opened up plasmid. -Very accurate, DNA copied has few if any errors. Although mutations arise, this is very rare. - Cuts out specific genes. Very precise procedure as the culturing of transformed bacteria produces many copies of a specific gene and not just the whole DNA sample. - Produces transformed bacteria that can be used to produce large quantities of gene products such as proteins for commercial or medical use (hormones).

The process of genetic fingerprinting, Hybridisation:

- Radioactive or fluorescent DNA probes are now used to bind with the core sequences. - The probes have base sequences complementary to the core sequences. - Bind to them under certain conditions such as temperature and pH. - The process is carried out with different probes, each of which binds with a different core sequence.

Finding out which cells have taken up the plasmids, using the gene for antibiotic resistance.

- Some plasmids carry the gene for more than one resistance. The R plasmid carries the gene for resistance for ampicillin and tetracycline. 1) Bacterial cells are grown on a medium contained ampicillin. 2) Bacterial cells that have taken up the plasmid will have the gene for resistance, will break down the ampicillin and survive. 3) Those who have not taken up the plasmid will die.

Examples of genetically modified animals.

- Transferring genes from an animal that has natural resistance to a disease into a totally different animal. The second animal is then also made resistant. Domesticated animals can be more economic to rear and reduce the price of food production. - Growth hormones added to produce fast growing animals. - Production of rare and expensive proteins for use in human medicine. Domesticated milk producing goats can be used. Gene for required protein is inserted alongside the gene that that codes for the proteins in goats milk. Required protein is produced in the goats milk. Gene can be inserted into fertilised egg of a goat, so that all the female offspring of that individual will be capable of producing this protein in their milk. Anti thrombin protein. - Individuals who have an inherited disorder that affects the alleles coding for the protein anti thrombin are not able to produce sufficient quantities of this protein. Risk of blood clots. Small amounts of the protein can be extracted from human blood, but far more can be produced in the milk of genetically transformed goats. - Domesticated chickens have also had human genes for medicinal proteins added to their DNA. The eggs laid by these transgenic chickens contain the proteins in the white portion from which they can easily be extracted. The human genes are passed from generation to generation. Large flocks can be formed with a potentially unlimited supply of cheap medicinal proteins. A form of interferon used to produce MS and an antibody with the potential to treat skin cancer and arthritis has been formed in this way.

Uses of genetic fingerprinting:

- Used extensively in forensic science, indicate whether or not an individual is connected with a crime. - Help to resolve questions of paternity, each band on a DNA fingerprint of an individual should have a corresponding band in the parents DNA fingerprint. - Useful for determining the genetic variability within a population. More closely related two individuals are, the closer the resemblance of their genetic fingerprints. Population with greater variety of genetic fingerprints have a greater genetic diversity. - Can identify the nature of a microbial infection by comparing the fingerprint of microbes found in patient with that of known pathogens. - Prevent undesirable interbreeding during breeding programmes at farms or zoos. Can also identify organisms with a desirable gene, can be selected for breeding to increase chance of offspring having this gene.

The sanger method.

- Uses modified nucleotides which cannot attach to the next base in the sequence when they are joined together. Act as terminators. Ends the synthesis of the DNA strand. Thymine. 1) Four test tubes are set up, each containing many single stranded fragments of the DNA to be sequenced. Acts as a template for the synthesis of its complementary strand. A mixture of nucleotides with the bases A,T,G and C. A small quantity of one of the four terminator nucleotides, (1 with adenine terminator nucleotide etc). A primer to start the process of DNA synthesis, which is radioactively labelled and DNA polymerase to catalyse the synthesis of DNA. 2) The binding of nucleotides to the template is random, the addition of a normal or terminator nucleotide is equally likely. Depending on where the terminator nucleotide binds to the DNA template, DNA synthesis may be terminated after only a few nucleotides or after a long fragment has been synthesised. 3) DNA fragments produced will be of varying length. All the fragments produced will end with a nucleotide with the same base. 4) These fragments can be identified because of the primer attached to the other end. 5) The different length fragments of DNA then have to be separated.

Risks and benefits of gene therapy.

- Who decides what is normal and what is a disability. Some diseases are clearly disabling. - Do disabilities need to be cured or prevented or are they just part of the genetic variety that makes up species. - Gene therapy research and treatment is expensive, money may be better spent on proven treatments. - Germ line therapy could be effective but there may be long term consequences of introducing inheritable genes into the population.

Advantages of in vitro gene cloning:

-Extremely rapid, within the matter of hours 100 billion copies of a gene can be made. Valuable when only a tiny amount of DNA is available. No loss of time before forensic analysis and matching can take place. - Does not require living cells, only a base sequence of DNA that requires amplification, no complex culturing techniques requiring time and effort are required. - But, requires a very pure sample as contaminated DNA will also be multiplied, leading to a false result. - High risk of errors.

Benefits to genetic modification:

-Increases yield from animals or crops. - Improving nutrient content of foods. - Resistance to disease and pests. - Crop plants tolerant to herbicides. - Tolerance to environmental conditions. - Making vaccines. - Producing medicines to treat disease.

How are DNA probes used to identify particular genes:

1) A DNA probe is made that has the bases complementary to the portion of the DNA sequence that makes up part of the gene whose position we want to find. 2) The DNA that is being tested is treated to separate its two strands. 3) The separated DNA strands are mixed with the probe, which bind to the complementary base on one of the strands. DNA hybridisation. 4) The site at which the probe binds can be identified by the radioactivity or the fluorescence that it emits.

Isolation of a gene:

1) A cell that readily produces the protein is selected. 2) The cells have large quantities of relevant mRNA which is extracted. 3) Reverse transcriptase is then used to make DNA from RNA, this DNA is known as complementary DNA. 4) DNA polymerase is used to build up complementary nucleotides on the cDNA template. 5) Double strand of DNA produced of the required gene.

Genetically modified microorganisms.

1) Antibiotics produced naturally by bacteria, increased quantity of antibiotics and the rate at which they are made. 2) Hormones. Insulin. Previously extracted from cows/pigs but could be rejected by the immune system. Bacterial cells have the human gene incorporated, insulin is identical to human insulin with no adverse effects on the patient. Human growth hormones, sex hormones. 3) Enzymes. Food industry uses enzymes manufactured by genetically modified bacteria. Amylases to break down starch in beer production. Lipases improve the flavour of cheese and proteases to tenderise meat.

Antibiotic resistance markers.

1) Bacterial cells that survived treatment with the first antibiotic are known to have taken up the plasmid. 2) These cells are cultured by spreading them very thinly on nutrient agar plates. 3) Each separate cell on the plate will grow into a genetically identical colony. 4) A tiny sample of each colony is transferred onto a replica plate in exactly the same position. 5) Replica plate contains the second antibiotic, against which the resistance gene will be useless if it took up the new gene. 6) Colonies killed must have taken up the required gene. 7) Colonies are in the exact same position, and can be compared to the previous plate to see which ones have been transformed.

The process of genetic screening:

1) Fix hundreds of different DNA probes in an array on a glass slide. 2) A sample of DNA is added to the array, any complimentary DNA sequences in the donor DNA will bind to one or more probes. 3) Can test simultaneously for many different genetic disorders.

Two types of gene therapy which could be used to treat CF:

1) Gene replacement, the defective gene is replaced with a healthy gene. 2) Gene supplementation, one or more copies of the healthy gene are added alongside the defective gene. The added genes are dominant, so the effects of the recessive gene are masked.

Genetically modified plants.

1) Genetically modified tomatoes. Developed using the insertion of a gene. This gene has the base sequence that is complementary to that of the gene that produces the enzyme which causes tomatoes to soften. The mRNA transcribed from this inserted gene is therefore complementary to the original gene. The two combine to form a double strand. This prevents the mRNA of the original gene from being translated. The softening enzyme is not produced. Allows tomatoes to develop flavour without the problems associated with harvesting, transporting or storing fruit. 2) Herbicide resistant crops. Gene introduced which makes them resistant to a specific herbicide. When the herbicide is sprayed on the crop, the weeds that are competing with the crop plant for water, light and minerals are killed. The crop plants are resistant and unaffected. 3) Disease resistant crops. Genes introduced to give them resistance to particular diseases. 4) Pest resistant crops. Maize can have a gene added that allows the plant to make a toxin. This toxin kills insects that eat the maize, but is harmful to other animals and humans. 5) Plants that produce plastics are currently being explored. Hopes that we can genetically engineer plants that have the metabolic pathways necessary to make the raw materials for plastic production.

Two different techniques of gene therapy which could be adopted, according to the cell being treated:

1) Germ line therapy. Involves replacing or supplementing the defective gene in the fertilised egg. All cells of the organism will develop normally, as will cells of their offspring. Permanent solution which affects future generations. Moral and ethical issues of manipulating such long term genetic change, currently prohibited. 2) Somatic cell gene therapy. Targets the affected tissues such as the lungs, additional gene is not present in sperm or egg cells and is not passed onto future generations. Cells of the lung are continuously dying, so have to be replaced and treatment is repeated periodically, as often as a few days. Limited success. Long term aim is to target undifferentiated stem cells that give rise to mature tissues, treatment effective for the entire lifespan.

The process of making a protein:

1) Isolation of the DNA fragments that have the gene for the desired protein. 2) Insertion of the DNA fragment into a vector. 3) Transformation, the transfer of DNA into suitable host cells. 4) Identification of the host cells that have successfully taken up the gene by the use of gene markers. 5) Growth/ Cloning of the population of host cells.

Inserting the gene for anti thrombin into goats.

1) Mature eggs are removed from female goats and fertilised by sperm. 2) The normal gene for anti thrombin production from a human is added to the fertilised eggs alongside the gene that codes for protein production in cows milk. 3) These genetically transformed eggs are implanted into female goats. 4) Those resulting goats with anti thrombin gene are crossbred to give a herd in which they produce milk rich in the anti thrombin gene. 5) Anti thrombin is extracted from the milk, purified and given to humans.

Commonly used probes:

1) Radioactively labelled probes. Made up of nucleotides with the isotope P32. Probe is identified using a photographic plate exposed to radioactivity. 2) Fluorescently labelled probes. Emit light under certain conditions.

The insertion of DNA fragments into a vector.

1) Same restriction endonuclease used that was used to cut the DNA fragment. 2) This ensures complementary sticky ends. 3) DNA fragments are mixed with the opened up plasmids and become incorporated into them. 4) DNA ligase is added.

Treating SCID with gene therapy.

1) The normal ADA gene is isolated from healthy human tissue using restriction endonuclease. 2) ADA gene inserted into retrovirus. 3) Retrovirus is grown in host cells in the lab to increase their number and copies of the ADA gene. 4) The retroviruses are mixed with patients T cells. 5) The retroviruses inject a copy of the normal ADA gene into the T cells. 6) The T cells are reintroduced into the patients blood to provide the genetic code to make ADA. - Effectiveness is limited because T cells only live for 6-12 months. -Treatment repeated at intervals. - More recent treatment involves using bone marrow stem cells to divide to produce T cells. Constant supply of ADA gene and the ADA enzyme.

Summary of genetic screening:

1) The order of nucleotides on the mutated gene is determined by DNA sequencing. Genetic libraries now store DNA sequences of many genes responsible for common genetic diseases. 2) Fragment of DNA with complementary bases to the mutant portion is produced. 3) DNA probe is formed by radioactively labelling. 4) PCR techniques are used to produce multiple copies of the DNA probe. 5) Probe is added to single stranded DNA fragments from the person being screened. 6) If the donor has the mutated gene, some donor fragments will have a nucleotide sequence complementary to the probe and will bind to the complementary bases. 7) These DNA fragments will be labelled with the probe and can be distinguished from the rest using X ray film. 8) If complementary fragments are present, the DNA probe will be taken up and the X ray film exposed. 9) If no complementary fragments are present, tun the DNA probe will not be taken up and the X ray film will be unexposed.

Separation (Southern blotting).

1) Thin nylon membrane is laid over the gel. 2) Membrane is covered with several sheets of absorbent paper, this draws up the liquid containing the DNA by capillary action. 3) This transfers the DNA fragments to the nylon membrane in precisely the same relative positions that they occupied the gel. 4) The DNA fragments are then fixed to the membrane using UV light.

Vector.

A carrying unit. Most commonly used is the plasmid.

Thermocycler.

A computer controlled machine that varies the temperature precisely over a period of time.

Genetic fingerprinting.

A diagnostic tool used widely in forensic science. Based on the fact that the DNA of every individual, except identical twins, is unique. Relies on the fact that the genome contains many introns which contains many receptive sequences of DNA called core sequences.

Retroviruses.

A group of viruses best known for HIV. The genetic information of retroviruses is in the form of RNA.

Polymerase chain reaction.

A method of copying fragments of DNA. It is automated, so efficient and rapid.

Severe combined immunodeficiency (SCID).

A rare inherited disorder, do not show any cell mediated immune response and they are not able to produce antibodies. Individuals inherit a defect in the gene that codes for the enzyme ADA, this enzyme destroys the toxins that would otherwise kill white blood cells. Survival depended upon patients being raised in a strictly sterile environment/ bone marrow transplants/ injections of ADA.

DNA probes.

A short, single stranded section of DNA that has some sort of label attached to make it easily identifiable. Used to find where a particular gene is located.

Genetic modification.

Altering the genetic make up of organisms by transferring genes between individuals of the same or different species.

PCR stage two.

Annealing of the primers. Mixture cooled to 55 degrees, causing the primers to join to their complementary bases at the end of the DNA fragments. Primers provide the starting sequence for DNA polymerase to begin DNA copying. DNA polymerase only attaches to nucleotides at the end of an existing chain. Primers prevent the two separate strands from rejoining.

Probability of someone else's DNA matching.

Based on the assumption that the DNA which produces the banding pattern is randomly distributed within a community. This may not always be the case, for example it may not apply where religious or ethnic groups tend to have partners from within their small community.

DNA polymerase.

Builds up complementary nucleotide bases on a single strand of DNA.

Genetic screening and cancer.

Can help detect: 1) Oncogene mutations, deterring the type of cancer and the most effective drugs to use. 2) Gene changes that predicts which patients are more likely to benefit from certain treatments and have the best chances of survival. 3) A single cancer cell amongst millions of normal cells, to help identify patients at risk of relapse of certain forms of leukaemia.

Genetic screening is valuable in the detection of oncogenes.

Cancers may develop as a result of mutations of tumour suppressor genes that inhibit cell division. Mutations of both alleles must be present, to inhibit and initiate development of a tumour. People who inherit one mutated gene are at risk. Can be detected by genetic screening, then individuals can be informed decisions about lifestyle and future treatment. Avoid mutagens. Check more regularly, leading to early diagnosis and better chance of successful treatment. Could also undergo gene therapy.

Reverse transcriptase.

Catalyses the production of DNA from RNA.

Plasmid.

Circular lengths of DNA founds in bacteria which is separate to the main bacterial DNA. Almost always contain the gene for antibiotic resistance.

Sticky ends.

Cut in a staggered fashion, leaves an uneven cut, in which each strand of the DNA has exposed, unpaired bases. Palindrome.

Automation of DNA sequencing and restriction mapping.

DNA sequencing and restriction mapping can be carried out by automatic machines, and computers analyse the data that they produce. Instead of radioactively labelling the DNA, the four terminators are labelled with fluorescent dye. Each base has a different colour. DNA synthesis takes place in a single test tube and is speeded up using PCR cycles. Electrophoresis is carried out in a single narrow capillary gel. Results are scanned by lasers and interpreted by computer software. DNA sequence can be obtained rapidly.

Restriction endonucleases.

Enzymes that cut up DNA. Many types, each one cuts the DNA double strand at a specific sequence of bases (recognition sequence).

Core sequences.

For every individual, the number and length of core sequences has a unique pattern. They are different in all individuals, except identical twins. The probability of two individuals having identical sequences of these repetitive non coding bases is extremely small. The more closely related two individuals are, the more similar their core sequences.

Huntingtons disease.

Genetic disorder of the nervous system, AGC repeated over and over at one end of the gene on a chromosome. People with fewer repeats are unlikely to get the disease, more than 38 are almost certain to get it. over 50, the onset will occur earlier than average. A sample of DNA with the allele for huntingtons can be cut out with restriction endonuclease. DNA fingerprint can be prepared. Can be matched with fingerprints from people with various forms of the disease and though without, probability of developing the symptoms can be established.

Genetic counselling.

Genetic screening goes hand in hand with genetic counselling. Helps individuals to understand results and implications of screening to make appropriate decisions. Advice and information given to enable people to make informed choices about themselves and their offspring. Research family history of inherited diseases. Inform about the effects, and emotional, psychological, medical, social and economic consequences. Choose whether or not they would like to have children. Aware of any further medical tests to give a more accurate prediction of whether the child will have the condition.

Genetic screening.

Genetic techniques can determine whether an unborn child might be affected by a genetic disorder.

Gene markers.

Identify whether a gene has been taken up by bacterial cells. Uses a second, separate gene on the plasmid which is easily identifiable because it may be resistant to an antibiotic, make a fluorescent protein or produce an enzyme whose action can be identified.

Dominant and recessive alleles.

If the mutation results in a dominant allele, all individuals will have the genetic disorder. If the allele is a recessive allele, then it will only be apparent in homozygous recessive individuals. Heterozygous are carries of the allele. Capacity to pass disease onto offspring.

PCR stage one.

Separation of the DNA strand. DNA fragments, primers and DNA polymerase are placed in a vessel in the thermocycler. Temperature increased to 95 degrees, the stands of DNA fragments separate.

Restriction mapping.

Involves cutting DNA with a series of different restriction endonucleases. The fragments produced are then separated by gel electrophoresis. - The distance between recognition sites can be determined by the patterns of fragments that are produced. - If a plasmid is circular, then one restriction endonuclease can be used to produce a single piece of DNA. - Restriction endonucleases can be used in pairs, to get two fragments of DNA from a circular plasmid. The length of each fragments depends on the restriction endonucleases used.

Screening.

It is important to screen individuals who may carry the mutant allele. Such individuals often have a family history of the disease. Screening can determine the probabilities of a couple having offspring with a genetic disorder.

DNA ligase.

Joins the phosphate sugar framework of the two sections of DNA and unites them as one.

Enzyme markers.

Lactase will turn a colourless substrate blue. the required gene is transplanted into the gene that produces lactase. Colonies that grow from it will not produce lactase. Will be unable to turn the substrate blue.

The importance of sticky ends.

Leaves unpaired nucleotide on a single strand that is complementary to the bases on the opposite side. If the same restriction endonuclease is used then all the fragments will have ends complementary to one another. Can combine the DNA of one organism with another.

cDNA.

Made up of the nucleotides complementary to the mRNA.

Calcium ions and temperature.

Makes the bacteria permeable, allowing the plasmids to pass through the cell membrane into the cytoplasm.

Cannot prove if someone did the crime as...

May have been left on another occasion, DNA may be of a close relative or the DNA sample may have been contaminated after the crime, by the suspects DNA or by chemicals that affect the action of the restriction endonucleases used in preparing the fingerprint.

Symptoms of CF:

Mucus congestion in the lungs so higher risk of infection as the mucus trapping disease causing organisms cannot be removed. Breathing difficulties and less efficient gas exchange. Accumulation of thick mucus in the pancreatic ducts, preventing pancreatic enzymes from reaching the duodenum leading to the formation of fibrous cysts. Accumulation of thick mucus in sperm ducts which could lead to infertility.

Why do only some of the bacterial cells possess DNA fragments?

Only a few bacterial cells (1%) take up the plasmids when the two are mixed together and some plasmids would have closed up again without incorporating the DNA fragment.

Gene therapy.

Replacing defective genes with genes cloned from healthy individuals.

Primers.

Short sequences of nucleotides that have a set of bases complementary to those at one end of each of the two DNA fragments.

Sickle cell anaemia.

Still common in parts of the world, as it is the result of a gene that has two co dominant alleles. The malaria parasite cannot exist in sickled red blood cells. 1) Homozygous for Haemoglobin S. Suffer from sickle cell anaemia. Considerably disadvantaged. Rarely live long enough to pass on genes to the next generation. Outweighs positives of being resistant to malaria. 2) Homozygous for haemoglobin A. Normal healthy lives. Susceptible to malaria in areas of the world where it is an endemic. Selected against only in these regions. 3) Heterozygous for haemoglobin. Sickle cell traits, not badly affected except when oxygen conc in their blood is low, for example in exercising muscles. May become tired easily. Have a resistance to malaria. Advantage weighs out disadvantage of begin tired. Selected against where malaria is not present, but for where it is.

PCR stage three.

Synthesis of DNA. Temperature increased to 72 degrees. Optimum temperature for DNA polymerase to add complementary nucleotides along the separated DNA strands. Begins at the primer on both strands and adds nucleotides until it reaches the end of the chain.

Required for the PCR:

The DNA fragment to be copied, DNA polymerase obtained from bacteria in hot springs to tolerate heat (thermostable) and will not denature in high temperatures of the process, primers, nucleotides (ATGC) and a thermocycler.

Recombinant DNA.

The DNA of two organisms that has been combined.

Transformation.

The plasmids must be reintroduced into the bacterial cells. Involves the plasmids and bacterial cells being mixed in a medium containing calcium ions.

Genetically modified organism.

The resulting organism that is formed from recombinant DNA.

Fluorescent markers.

Transferring a gene from a jellyfish into the plasmid, produces a green fluorescent protein (GFP). Any bacterial cel that has taken up the plasmid will not produce GFP and will not fluoresce. No need for replica plating. View under a microscope, more rapid.

In vivo gene cloning.

Transferring fragments to a host cell using a vector. Genes are cloned within living organisms.

DNA sequencing.

Used to sequence the exact order of nucleotides in the section of DNA.

Delivering cloned CFTR genes in somatic gene therapy. Using a harmless virus.

Using a harmless virus. Adenoviruses cause colds and other respiratory diseases by injecting their DNA into epithelial cells of the lungs. 1) Adenoviruses are made harmless by interfering with a gene involved in their replication. 2) Adenoviruses are grown in epithelial cells in the laboratory along with plasmids with the normal CFTR gene inserted. 3) The CFTR gene becomes incorporated in the DNA of adenoviruses. 4) These adenoviruses are isolated from epithelial cells are purified. 5) Adenoviruses with the CFTR gene are introduced into the nostrils of patients. 6) Adenoviruses inject their DNA, including the normal CFTR gene into epithelial cells of the lungs.

In vitro gene cloning.

Using the polymerase chain reaction.

Genetic diseases.

Usually caused by a missing gene or a gene that does not express itself properly.

Blunt ends.

When restriction endonucleases cut between two opposite base pairs, leaving two straight edges.