Producing DNA fragments with reverse transcriptase
1. A cell that produces the required protein is selected (e.g. B cells of Islets of Langerhans)
2. These cells have the relavent mRNA which is extracted
3. Reverse transcriptase is used to make DNA from RNA - this DNA is complementary to the RNA and so is called cDNA
4. To make the other strand of DNA, DNA polymerase is used to build up the complementary nucleotides on the cDNA template
5. This double stranded DNA is the required gene
Identifying recombinant plasmids
1. All bacteria are grown on a medium containing ampicillin
2. Those that have taken up the plasmid will have aquired the gene for ampicillin resistance
3. These cells are able to break down the ampicillin and therefore, survive
4. Those that have not taken up the plasmid will not be resistant to ampicillin and therefore, die
However, some bacteria will have taken up the plasmid without encorporating the new gene. The next task is to identify these - this is done using gene markers.
Gene markers use genes that:
- May be resistant to an antibiotic
- May make a fluorescent protein that is easily seen
- May produce an enzyme whose action can be easily identified
Antibiotic resistant markers - replica plating
A gene (e.g. for tetracycline resistance) has been cut to encorporate the required gene - it will no longer function. In other words, the bacteria that have the required gene will no longer be resistant to TC - they can be identified by being grown on TC but will be destroyed. Replica plating is used in the following way:
1. The cells that survived the first antibiotic are known to have taken up the plasmid
2. These are cultured by spreading thinly on agar plates
3. A tiny sample of each colony is put on a 2nd (replica) plate in exactly the same position
4. This contains TC - against which the resistance gene will have been made ineffective
5. The colonies killed have taken up the required gene
6. The colonies in the same position on the original plate are the ones that have the required gene - these colonies have bacteria that have been transformed
1. A gene for GFP from a jellyfish is inserted into the plasmid
2. The required gene to be cloned is inserted into the middle of the GFP gene
3. Any bacteria that have taken up the required gene will not fluoresce
4. As the cells with the required gene are not killed, there is no need for replica plating - results can be obtained by viewing the bacteria through a microscope and keeping the ones that do not fluoresce
5. This makes the process quicker
Another gene that can be used as a marker is the gene that produces lactase - this will turn a colourless substrate blue
1. The required gene is inserted into the gene that makes lactase
2. If a plasmid with the required gene is present in a bacterial cell, the colonies grown will not produce lactase
3. Therefore, they will not be able to change the colour of the substrate
4. Where the gene has not transformed the bacteria, they will be able to change the substrate's colour
Polymerase chain reaction
It needs: The DNA fragment to be copied, DNA polymerase, primers, nucleotides and a thermocycler. It occurs in 3 stages:
1. Seperation of the DNA strand - The DNA fragments, primers & DNA polymerase are put in a vessel in the thermocycler & the temperature is raised to 95C - seperating the two DNA strands
2. Addition (annealing) of primers - The mixture is cooled to 55C, causing the primers to join (anneal) to their complementary bases on the end of the DNA fragment. The primers provide the starting point for DNA polymerase to begin copying (DNA polymerase can only attach nucleotides on to the end of an existing chain). Primers also prevent the two DNA strands from rejoining.
3. Synthesis of DNA - The temperature is increased to 72C - optimum temp for DNA polymerase to add complementary nucleotides along each of the DNA strands. It begins at the primer on both strands and adds nucleotides in sequence until it reaches the end of the chain.
Whole cycle takes 2 mins - 2 copies of original fragment - 1million copies takes 25 cycles
Advantages of in vitro & in vivo gene cloning
- Very quick - within hours, 100 million copies of a gene can be made - especially useful with a small sample (e.g. crime scene) no loss of time before forensic analysis can occur
- It does not need living cells - All it needs is base DNA sequence
- Useful for introducing gene into another organism - vectors allow the gene to be delivered into another organism (gene therapy)
- Almost no risk of contamination - a gene cut by the same RE can match the sticky ends of the open plasmid. Contaminant DNA will not be taken up.
- Very accurate - Few if any errors, at one time, the PCR cloned about 20% of DNA wrong
- Cuts out specific genes - Very precise procedure as the culturing of transformed bacteria produces copies of just the specific gene, not the whole DNA sample
- Produces transformed bacteria that produce gene products - Transformed bacteria produce proteins for commercial or medical use (e.g. hormones like insulin)
Examples of GM plants
- GM tomatoes - developed with insertion of a gene that prevents them from softening - they develop flavour without the problems of them being soft (storage, transporation ect.)
- Herbicide resistant crops - They have a gene introduced that gives resistance to a specific herbicide. The weeds competing for resources are killed but the crop plants aren't
- Disease resistant crops - Have genes introduced that give resistant to specific diseases (e.g. GM rice can withstand infection by a particular virus)
- Pest resistant crops - E.g. Maize, can have a gene added that gives the plant the ability to produce a toxin. This toxin kills insects that eat the maize but is harmless to other animals, including humans
- Plants that produce plastics - Are currently being explored. It's hoped that we can genetically engineer plants that have the metabolic pathways necessary to make the raw materials needed for plastic production
Examples of GM microorganisms
There are three main groups of substances produced by GM bacteria:
- Antibiotics - are produced naturally by bacteria. Genetic engineering has not vastly improved the quality of antibiotics but it has produced bacteria that increase the amount of antibiotics produced & the rate at which they are made.
- Hormones - insulin is needed daily by diabetics. Previously, insulin from cows or goats was used but this gave some side effects. With genetic engineering, bacteria have the human insulin gene. The insulin produced is identical to human insulin so produces no side effects. It also avoids killing animals and negates the need to modify the insulin before it is injected into humans. Other hormones produced in this way include human growth hormone and the sex hormones, oestrogen and testosterone
- Enzymes - Many enzymes used in the food industry are made by GM bacteria. These include amylases used to break down starch during cheese production, lipases, used to improve the flavour of cheese and proteases used to tenderise meat.
Examples of GM animals
- An example of GM in animals is the transfer of genes from an animal that has resistance to a totally different animal - this second animal is now resistant to that disease. In this way domestic animals can be more economic to rear.
- More examples - fast-growing food animals such as sheep & fish that have a growth hormone gene added, so (in the case of salmon) they can grow 30x larger at 10x the normal rate
- Another example is the production of rare & expensive proteins for use in medicine. Domestic milk-producing animals can be used. The gene for the required protein is inserted alongside the gene that codes for proteins in goat's milk - the protein is then produced in the milk. The gene can also be inserted into a fertilised goat's egg so all female offspring will produce the protein in the milk. Anti-thrombin is an example of a protein produced in this way
- Some people have an inherited disorder that affects an allele that codes for Anti-thrombin - they therefore produce less AT & are at risk of blood clots - they are currently treated with drugs that thin the blood or given AT extracted from donated blood
- Small amounts of AT can be extracted from human blood, but far more can be produced in the milk of genetically transformed goats.
Benefits of DNA technology
- Microorganisms can be engineered to produce a range of substances
- Microorganisms can be used to control pollution (e.g. they can break down and digest oil slicks or destroy harmful gases released from factories)
- GM plants can be transformed to produce a specific substance in a particular organ of the plant. The organs can then be harvested & the desired substance extracted. If a drug is involved, this is called plant pharming. Plants can be made to manufacture antibodies to pathogens & the toxins they produce, or they can produce antigens, which stimulate antibody production when injected into humans
- GM crops can be modified to have economical & environmental advantages. These include producing plants that are more tolerant to environmental extremes (e.g. temperature extremes, drought, flood, salt or polluted soils etc.) This allows plants to be grown comercially in areas where they currently can't grow. Growing GM plants more suited to the extremes allows land to be productive again.
- GM crops can help prevent disease. Rice can have a gene for Vitamin A added
- GM animals can produce expensive drugs, hormones, proteins & enzymes relatively cheaply
- Gene therapy can be used to cure genetic disorders such as CF
- Genetic fingerprinting can be used in forensic science
Risks of recombinant DNA technology
- Can't predict ecological impact of a GM organism
- A recombinant gene may pass from the organism it was placed in to a different one
- Manipulation of DNA leads to consequences for metabolic pathways
- GM bacteria often have antibiotic resistance - they can spread resistance to other bacteria
- All genes mutate - what could the consequences be of a transformed gene mutating?
- Long term consequences of new gene combinations? Can't be certain of effects on evolution
- Economic consequences of developing plants and animals to grow in new regions? Developing bananas to grow in the UK could effect the Carribean countries that rely on bananas for income
- How far can we take gene therapy? Designer babies?
- Will this lead to eugenics?
- Consequences of the ability to manipulate genes falling into wrong hands?
- Is the cost justified? Should the money spent fighting hunger and poverty?
- Genetic fingerprinting - how easy could swapping DNA be? (leading to incorrect convictions)
- Is it moral to tamper with genes at all?
- Human genome project - is it right that people can patent, & effectively own genes?
Treating cystic fibrosis (CF) using gene therapy
Two ways gene therapy can be used:
- Gene replacement - the faulty gene is replaced with one that functions properly
- Gene supplementation - one or more copies of functional gene added alongside faulty gene. As the added genes have dominant alleles, the effects of the recessive alleles of the defective gene are masked
Two different techniques of gene therapy:
- Germ line gene therapy - replaces or supplements gene in the fertilised egg. All cells of the organism will develop normally, as will all cells of any offspring. Much more permanent solution. Moral and ethical?
- Somatic- cell gene therapy - targets just the affected tissues (e.g. lungs). Additional gene not present in sex cells - not passed on to offspring. Because the cells in the lungs are constantly dying and being replaced, this needs to be done repeatedly, as often as every few days. Currently, this treatment has limited success. The long term aim is to target undifferentiated stem cells that give rise to mature tissues. The treatment would then be effective for the individual's whole life.
Delivering CFTR genes using adenoviruses
Viruses, called adenoviruses, cause colds and other respiratory diseases by injecting their DNA into the epithelial cells of the lungs. They therefore make useful vectors for the transfer of the normal CFTR gene into the host cells.
The process works as follows:
- The adenoviruses are made harmless by interfering with a gene involved in their replication
- These adenoviruses are then grown in epithelial cells in the laboratory, along with plasmids that have had the normal CFTR gene inserted
- The CFTR gene becomes incorporated into the DNA of the adenoviruses.
- These adenoviruses are isolated from the epithelial cells and are purified
- The adenoviruses with the CFTR gene are introduced into the nostrils of the patients
- The adenoviruses inject their DNA, which will include the normal CFTR gene into the epithelial cells of the lungs
Treating CF by wrapping the CFTR gene in lipids
Genes are wrapped in lipid molecules because they can relatively easily pass through phospholipid bilayers of cell-surface membranes. The process of delivering the CFTR genes to their target cells using lipid molecules is as follows:
- CFTR genes are isolated from healthy human tissue and inserted into bacterial plasmids
- The plasmids are reintroduced to the bacteria hosts. Gene markers are used to detect which bacteria have taken up the plasmid with the CFTR gene.
- These bacteria are cloned to make copies of the plasmid with CFTR
- The plasmids are extracted from the bacteria and wrapped in lipid molecules to form a liposome
- The liposomes containing the CFTR gene are sprayed into the nostrils of the patient as an aerosol where they are drawn down into the lungs during inhalation
- The liposomes pass across the phospholipid bilayer of the cell surface membrane of the lung epithelial cells
Treating SCID using gene therapy
An introduction for this is in the book. The process works as follows:
- A normal ADA gene is isolated from healthy human tissue using RE enzymes
- The ADA gene is inserted into a retrovirus
- The retroviruses are grown with host cells in the lab to increase their numbers, and therefore the number of copies of the ADA gene
- The retroviruses are mixed with the patients T cells (white blood cells)
- The retroviruses inject a copy of the normal ADA gene into the T cell
- The T cells are reintroduced into the patients blood to provide the genetic code needed to make ADA
Effectiveness of this is limited as T cells only live for 6 months to a year and so the treatment has to be repeated at regular intervals.
More recent treatment involves transforming bone marrow stem cells rather than T cells. Bone marrow stem cells divide to produce T cells, so there is a constant supply of the ADA gene and hence the enzyme that it codes for. Although not totally effective (increased risk of leukaemia) early results look good.
Negatives of Somatic cell therapy
- Effect is short lived - The somatic cells, which have the cloned gene added, are not passed on to offspring, repeat treatments are needed for the therapy to have any effect
- It can cause an immune response - Both the gene and the structure used to deliver it (vector or liposome) can cause an immune response - this means it's often rejected. Made worse by antibodies responding to a future infection (secondary response)
- Using viral vectors to deliver genes presents problems - Viruses are the usual way of getting genes to their target cells. But they can lead to toxic, inflammatory and immune responses in the recipient. There is also the posibility that the disabled virus could regain the ability to cause disease once inside the patient
- The genes are not always expressed - Even if successfully delivered to their target cells only a small proportion of the introduced genes are usually expressed.
- It is not effective in treating conditions that arise in more than one gene - Gene therapy works best in disorders that are the result of a single mutation. However, many commonly occuring disorders, such as arthritis, heart disease, diabetes and Alzheimer's are the result of variations in a number of genes.
Problems relating to somatic cell gene therapy don't apply to germ line gene therapy. However, germ - line gene therapy isn't allowed in humans.
- Short, single-stranded section of DNA that is labelled (radioactively with 32P or flourescently)
DNA probes are used to identify particular genes in the following way:
- A DNA probe is made that has bases complementary to the portion of the DNA sequence that makes up part of the gene whose position is being determined
- The DNA being tested is treated to seperate its two strands
- The seperated DNA strands are mixed with the probe, which binds to the complementary bases on one of the strands. This is known as DNA hybridisation.
- The site at which the probe binds can be identified by the radioactivity or fluorescence that the probe emits.
But, before we can make the specific probes, we need to know the sequence of nucleotides in the particular gene that we are trying to locate.
- Uses modified nucleotides that can't attach to the next base in the sequence when they are being joined together.
- These nucleotides act as terminators, ending synthesis of a DNA strand
The first stage is to set up four test tubes, each containing:
- Many single stranded fragments of the DNA to be sequenced. This acts as a template for the synthesis of its complementary strand.
- A mixture of nucleotides with the bases A, T, C & G
- A small quantity of one of the four terminator nucleotides - each one in a seperate test tube
- A primer to begin the DNA synthesis
- DNA polymerase to catalyse DNA synthesis
The binding of nucleotides to the template is random - the addition of a normal or terminator nucleotide is equally likely. DNA sequencing may be stopped after only a few nucleotides have been attached, or after a long sequence of DNA has been synthesised.
Gel electrophoresis is then used to seperate these different length fragments of DNA.
1. The order of nucleotides on the mutated gene is determined by DNA sequencing. Genetic libraries store the sequences of many genes responsible for genetic diseases
2. A fragment of DNA with complementary bases to the gene is produced
3. A DNA probe is formed by radioactively labelling the DNA fragment
4. PCR techniques used to produce lots of copies of the probe
5. Probe is added to single stranded DNA from the person being screened
6. If they have the mutated gene, some DNA fragments will have a nucleotide sequence complementary to the probe and the probe will bind to donor DNA
7. These DNA fragments will be radioactive & can be distinguished using X-Ray film
8. If complementary fragments are present, the probe will expose the film
9. If complementary fragments aren't present, the probe won't bind & won't expose the film
- Extraction - DNA taken from sample (e.g. hair or blood)
- Digestion - DNA cut into fragments using RE enzymes
- Seperation - Gel electrophoresis and southern blotting
- A nylon membrane placed over the gel
- The membrane is covered with several sheets of absorbant paper which draws up the liquid containing the DNA by capillary action
- This transfers the DNA fragments to the nylon membrane in exactly the same relative positions that they occupied on the gel.
- The DNA fragments are fixed to the membrane using UV light.
- Hybridisation - Labelled probes bind with DNA fragments
- Development - Xray film put over membrane and is exposed by the probes. A series of bars is revealed. The pattern of bands is unique to everyone, except identical twins.
- Samples are checked visually, if there appears to be a match, the samples are passed through an automatic scanner, which calculates the length of the DNA fragments (it does this using data obtained by measuring the distance travelled in gel electrophoresis). Finally the odds are calculated of someone else having an identical fingerprint. The closer the match between the two patterns, the greater the probability that the two sets of DNA are from the same person