Gene Technology

Definition

• The manipulation of genes for our advantage.

• Methods of creating new combinations of genes is called genetic engineering.

• To create new gene combinations genetic engineers need to:

– Locate a specific gene– Isolate this gene– Transfer it into a host cell in a way that it can

be expressed

Isolating the gene

• Reverse Transcription– mRNA for a certain polypeptide is isolated– Complimentary DNA (cDNA) can be

synthesised from it by using an enzyme called reverse transcriptase

– The mRNA acts as the template– mRNA removed and DNA polymerase adds

DNA nucleotides to make the second strand of DNA.

– Result is double strand of DNA identical to the original in the cell

Inserting the gene

• The DNA sequence is inserted into a plasmid from a bacteria

• A plasmid is a small ring of DNA that replicates independently of the main bacterial DNA

• It is cut using enzymes with a specific active site.

• The active site corresponds to a sequence of nucleotide bases and will only cut at that sequence.

• Restriction Endonucleases– Cut the DNA leaving sticky ends

• DNA Ligase sticks pieces of DNA back together

Make a model

Insulin Production

• First human protein to be manufactured by gene technology

• It produces fewer side effects than insulin prepared from cow or pig extracts.– Allergic reactions– Diseases passed on– Faster reaction

• Insert gene in plasmid

• Insert plasmid into bacteria

• Reproduce bacteria

Antibiotic resistance genes

• Only bacteria that are carrying the required gene are needed.

• An antibiotic resistant gene is also attached to the plasmid

• When the particular antibiotic is then added to the bacteria colony only those with the resistant gene (and therefore the insulin gene) survive.

Cloning and harvesting

• Bacteria are grown in industrial fermenters

• Bacteria multiply rapidly in the growth phase

• A key enzyme is then added to the fermenter to ‘switch on’ the donor gene.

• The product is then made by the bacteria and then separated out.

• The fermenters have cooling jackets.

Markers for genetic engineering

• In order to identify bacteria that have taken up the plasmid, markers are used

• Antibiotic resistant genes could lead to pathogens gaining resistance

• So they use fluorescent or easily stained substances.

• Fluorescence comes from jellyfish.

Promoters

• As well as the gene to be inserted most constructs contain a promoter and terminator region as well as a selectable marker gene.

• The promoter region initiates transcription of the gene and can be used to control the location and level of gene expression, while the terminator region ends transcription. 

Project – benefits, hazards, social and ethical implications

Benefits of gene technology

• there is more variety in gene combinations.• Natural sexual reproduction limits the variety of

gene combinations. • Genetic technology can solve many of the

world's problems. • For example, researchers have tried to develop

– super-antibiotics, – designer virus or hunters that will only attack certain

diseases like cancer, – grass that will only grow 2 inches so it would never

need to be mowed, – food that can be grown in any climate

• In agriculture• improving plant breeds • and utilizing nitrogen from the atmosphere to

improve the efficiency of crop plants.• The production of new organs by

xenotransplantation may help to overcome shortages in organs for transplant surgery, so prolonging life.

• There are potentials for making human therapeutic chemicals in other animals, e.g. human serum albumin used to treat burns, could be made in huge quantities in cows milk.

Hazards of gene technology

• Issues include:• genetic manipulation can be used to create biological

warfare • organisms made through genetic engineering to clean oil

spills or toxic wastes, if released in the environment will wreak havoc and will be disastrous to human life.

• ‘super-weeds’, resistant to herbicides and spreading uncontrollably,

• genes might transfer into other closely related wild species, forming a different kind of ‘super-weed’,

• they might reduce biodiversity by genetic contamination 

Social and ethical implications• The social impact of gene technology is to do with its potential and

actual impact of human society and individuals.  

– enhance crop yields and permit crops to grow outside their usual location or season so that people have more food

 – enhance the nutritional content of crops so that people are better fed

– permit better targeted clean-up of wastes and pollutants

– lead to production of more effective and cheaper medicines and treatments

– reduce crop biodiversity by out-competing natural crops so that people are less well fed

Ethical Issues•do we really have the right to modify life?

•It is wrong to continue such research when the potential impact of the technology is unknown and many aspects of it remain to be understood.

•It is wrong to use the results of such research when this involves release of gene technology into the environment as once it is released it cannot be taken back – the genes are self-perpetuating, and the risks that they might cause in future are unknown

Electrophoresis

• Electrophoresis is – a method of separating substances – and analyzing molecular structure – based on the rate of movement of each component – in a liquid medium – while under the influence of an electric field.

• In genetic fingerprinting and DNA sequencing, the components being separated are fragments of DNA.

•  In this case, the type of electrophoresis used is– gel electrophoresis 

• the gel appears solid but is actually a colloid in which there are spaces between the molecules through which other molecules can move.

• Electrodes are placed at either end of the gel, as a result of which the DNA molecules move under the influence of an electric current. 

• DNA is cut into fragments using a restriction enzyme.

• The sites are randomly distributed so the fragments are of varied lengths.

• Fragments of DNA are negatively charged and so move towards the positive electrode (anode). 

• The distance moved in a given time will depend on the mass of the fragment. The smaller fragments move further in a given time.

• The DNA is transparent and invisible, so the fragments must be treated to make them visible. 

• There are two key ways of doing this:1. staining all of the DNA fragments,2. creating a gene probe that is complementary:

– either to a commonly repeated bit of DNA that will therefore be present on many of the fragments,

– or to a base sequence that is specific to a particular gene or allele of a gene which will therefore be present on no more than one of the fragments.

 

The gene probe is a single stranded piece of DNA with a base sequence complementary to the DNA that you wish to identify. In order to make it possible to locate which fragment the gene probe has attached itself to, the gene probe must be labelled.

The most common forms of labelling are:

• With radioactivity, using X-ray photographs

• With fluorescence stain that will fluoresce with bright visible light when placed in ultraviolet light,

• In humans the base sequence of every chromosome 1 in every human is similar, but not identical due to the existence of mutations and therefore of different alleles of genes. 

• What this means is that when the DNA is fragmented with a restriction enzyme, the fragments are similar but not exactly the same in DNA from different people.

DNA Sequencing

• DNA sequencing determines the order of the nucleotide bases in a molecule of DNA.

Process• A mixture is made containing:

– A DNA template– A primer to start the reaction– DNA polymerase, an enzyme that drives the

synthesis of DNA– Four deoxynucleotides (G, A, C, and T)– One dideoxynucleotide, either ddG, ddA, ddC,

or ddT• dideoxynucleotides are missing a special group of

molecules, called a 3'-hydroxyl group, needed to form a connection with the next nucleotide

http://biochem.co/wp-content/uploads/2008/08/deoxynucleotide-dideoxynucleotide.pn

• DNA polymerase moves along the template and adds base after base.

• Until a dideoxynucleotide is added, blocking further elongation.

• Only a small amount of a dideoxynucleotide is added to each reaction, allowing different reactions to proceed for various lengths of time until by chance, DNA polymerase inserts a dideoxynucleotide, terminating the reaction.

• Therefore, the result is a set of new chains, all of different lengths. 

• The molecules generated in the presence of ddATP are loaded into one lane of the gel,

• the other three families, generated with ddCTP, ddGTP, and ddTTP, are loaded into three adjacent lanes.

• After electrophoresis, the DNA sequence can be read directly from the positions of the bands in the gel. 

http://biochem.co/wp-content/uploads/2008/08/electrophoresis-dna-sequencing1.pn

• Variations of this method have been developed for automated sequencing machines.

• In cycle sequencing, the dideoxynucleotides are tagged with different coloured fluorescent dyes; thus, all four reactions occur in the same tube and are separated in the same lane on the gel.

• As each labelled DNA fragment passes a detector at the bottom of the gel, the colour is recorded, and the sequence is reconstructed from the pattern of colours representing each nucleotide in the sequence. 

Cystic Fibrosis

• Recessive genetic condition

• Thick mucus produced by lungs and other parts of the body.

• Prone to bacterial infection

• Need daily therapy

• Pancreatic duct may become blocked.

Causes

• Caused by a recessive allele which codes for a transporter protein called CFTR.

• CFTR allows chloride ions to pass out of cells.• Cells lining airways pump chloride ions out to allow a

high concentration of chloride ions outside of the cell.• This lowers water potential so water moves out by

osmosis.• It mixes with the mucus making it thin enough for the

cilia to move it.• The recessive allele codes for a faulty version of this

protein.

Treatment

• The traditional treatments of CF depend on the severity of the disease and the organs involved.

• Lungs - vigorous percussion on the back using cupped hands to dislodge the thick mucus from the lungs

• Varying the positions of the patient and where the therapy is given drains the mucus from various parts of the lung.

• Antibiotics help with respiratory infections • Digestive system - supplements replace the

enzymes needed for digestion. 

Gene Therapy for CF

• Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, such as an hereditary disease in which a deleterious mutant allele is replaced with a functional one. 

How?

• basic concept is to identify the defective gene and to correct the defect with a normal gene.

• There are two forms of gene therapy1. germ line gene therapy.

– This helps the individual and his or her children. It would change the genetic pool

2. somatic gene therapy. – Somatic means of, relating to or

affecting the body. – This therapy involves changing the

defective gene in the individual but the change won't be inherited by the next generation.

– Somatic gene therapy would be the therapy of choice since it doesn't have the ethical considerations that germ line therapy creates.

How?

• There are two vectors that could be used to transport the CFTR gene.

• A harmless virus which will deposit the gene into cells as viruses naturally do. – A new gene is injected into a virus vector, which is

used to introduce the modified DNA into a human cell.• Or a liposome  which would be inhaled and

hopefully would pass the gene through the cells.

• If the treatment is successful, the new gene will make a functional protein.

Liposome

• Subsequent studies have tested other methods of gene delivery, such as: fat capsules, synthetic vectors, nose drops, or drizzling cells down a flexible tube to CFTR cells lining the airways of lungs.

• Researchers are now testing aerosol delivery using nebulizers.

Issues with Gene Therapy• con - germ line therapy changes the gene expression

forever, what will this do to the gene pool that exists afterwards.

• - researchers aren't sure how long the results of gene therapy will work or how often it would have to be repeated to get results.

• - ethics involved in changing a human beings make-up. Is this the right thing to do, even though it may help a person afflicted with CF?

• - it isn't permanent as epithelial cells have a short life and are constantly being renewed, so the treatment would have to be taken regularly and this would be expensive.

• - CF affects the entire body; and we can’t treat everything and whilst the lungs may be temporarily cured, the pancreas still will not function properly and the sufferer will still need to take enzyme supplements.

Genetic Screening

• “systematic searches for persons with a specific genotype” or “tests to identify persons who have an inherited predisposition to a certain phenotype or who are at risk of producing offspring with inherited diseases or disorders”.  

• a sample of a patient’s DNA is tested for mutated sequences by comparing a sequence of DNA of a mutated patient to that of a normal version of the gene.

Process

DNA extracted from a body cell (often skin or wbc). Mixed with restriction enzyme to cut the strands into particular sequences of bases.

DNA fragments separated by electrophoresis, and the correct fragment for the gene concerned detected according to how far it moves on the gel.

Correct fragment extracted and mixed with a DNA probe (known sequence of bases) which has a fluorescent molecule added which will activate if the bases pair up, indicating the presence of the defective gene.

• There are many uses of genetic screenings, which can take place to help against – infertility, – miscarriage, – stillbirths, – neonatal deaths, – multiple malformations, – retardation in growth and development, – mental illness, and mental retardation.

• Prenatal screening is done on a mother’s fetus to see if there are any risks, or problems with the baby.

• Carrier screening tests are done to determine whether an individual is a carrier of a certain disease. 

• Susceptibility testing is used in many workplaces to see if their employees are susceptible to different toxins in the work place and could have devastating effects later on in life.

Discussion

• Many religious groups feel that genetic screening is a very bad idea because they feel that you should not alter the course of your life, everything happens for a reason.

• The importance of the debate about what constitutes a disease is underscored by the two extensive questions that underlay the current debate,– who decides whether testing is done; – and what happens to that information?

• Genetic discrimination. Some people may feel that people with genetic flaws, which may not show up as dysfunctions, may be denied life insurance.

Genetic Counselling

• The genetic counsellor can help a person or family understand their risk for genetic conditions (such as cystic fibrosis, cancer, or Down syndrome), educate the person or family about that disease, and assess the risk of passing those diseases on to children.

• A genetic counselor will often work with families to identify members who are at risk.

• If it is appropriate, they will discuss genetic testing, coordinate any testing, interpret test results, and review all additional testing, surveillance, surgical, or research options that are available to members of the family.