Application of reproduction and genetics

Asexual reproduction involves one organism, and the offspring produced are genetically identical.

Clone: A genetically identical organism formed from a single parent as a result from artificial asexual reproduction.

In Vitro fertilisation:  a technique which involves mixing gametes in a dish where fertilisation takes place.

Natural cloning occurs in bacteria and plants through suckers, bulbs and corms. Or plant cultures can grow.

Uses a few cells or part of a plant in a suitable growth medium

Meristem: growing points in a plant where cells divide rapidly by mitosis.

Totipotent (cells): cells which can mature into any cell type.

STEPS IN MICROPROPOGATION:

• Asexually produced and genetically identical
• Meristem is cut off from the plant and divided into
• Explants placed in sterile, aerated environment.
• Cells divide by mitosis to make callus.
• Callus is subdivided.
• Each piece diffentiates into plantlet.
• Plantlets are transferred into sterile soil when grown to a suitable size.

ADVANTAGES:

• Large numbers of plants produced in sterile conditions
• Less time consuming as standard fertilisation (No time taken for pollination, germination etc.)
• Good quality stock, with resistance to disease or high yield.
• Uniform crop, which is good for commercial purposes.
• Preserves genotypes.

DISADVANTAGES

• If sterile conditions are not maintained, bacterial or fungal infection may occur, and wipe out a whole crop (due to no genetic variation.)
• Genetically unstable means high mutation costs. Plantlets need to be inspected for abnormalities regularly.

PROCESS:

• Nucleus from a somatic body cell is removed, and kept.
• An egg cell is enucleated.
• The somatic diploid nucleus is inserted into the egg cell.
• The diploid egg cell is treated with a electric pulse to begin division.
• The embryo may be split before cell diffentiation, to produce many embryos.
• The embryo(s) are inserted into a surrogate mother, where they develop into a foetus.

Advantages

• Speed of production
• Production of large quantities
• identical, genetic line of organisms with advantageous genes

Disadvantages

• Animals: expensive and unreliable
• Inadvertent selection of disadvantageous genes may show up as unforseen effects, such as aging.

Cell culture is used to create

• clones of a single, identical genetic line 
• with desirable characterstics
• e.g. cancer cells for medical research.

There are a number of applications.

• Stem cells: A stem cell is an undifferentiated cell which can develop into any type of cell. They can be used to make new blood vessel, for transplant, as well as bone and cartilage repair.
• Tissue engineering also helps develop skin grafts, by growing some of the patients own ';skin' so they do not reject it.
• Stem cells can also be used to grown organs for replacemnet. 

Where do we get stem cells from?

• Surplus embryos of IVF treatment. The embryo is destroyed after removing the stem cells. Some people think this is unacceptable, as life may begin at conception. 

• Do the benefits outweigh the concerns? Stem cells could used to treat alzheimers, parkinsons, heart disease.
• Stem cells can also be obtained from bone marrow, but there are less medical applications for this, and it is a very invasive process.
• Embryonic stem cell critics say there is slippery slope to human cloning.

Determined the order of bases in the human genome, as well as the identification of some genes. Also improved tools for analysis, addressed ethical and legal problems.

Applications

• Scan patient's DNA for mutated sequences, and compare the mutated gene to a normal gene. This often uses gene probes, which are short sections of complementary DNA  to a mutated sequence.
• Carrier screening.
• Pre-implantation screneing
• Pre-symptomatic testing for disorders such as Huntington disease.
• Forensic testing.

ADVANTAGES:

• A lot of practical uses, as before mentioned.
• inc. carrier screening could reduce the number of debilitating alleles in the gene pool.

DISADVANTAGES:

• Pre-symptomatic tests can lead to discrimination and social stigmatization.
• Could also lead to symptoms appearing earlier.
• VALIDITY: The result may be incorrect due to lab error, giving a false-positive results, provoking anxiety.
• Also concerns regarding the ownership of genetic information and its misuse.
• Family testing- can parents test their children? Should people be allowed to be tested if their kids don't want to  be?

Treats a genetic disease by replacing copies of the faulty gene with copies of new gene sequences.

Somatic cell therapy replaces faulty genes in affected tissues. This is therapeutic but the changes are not inherited.

Germ line therapy introduced the correction into the germ-line cells, and the change is inherited.

Usually uses a vector, such as a virus.

CYSTIC FIBROSIS

• Caused by an autosomal recessive allele. The parents must be carriers of the recessive gene. A blood test can be used for diagnosis.
• CFTR gene transports chloride ions out of the cell and into mucus. Sodium ions follow, and water moves out the cell by osmosis, creating watery mucus. The removal of one amino acid results in the faulty protein, and the chloride ions are not transported.

It causes thick, sticky mucus  to be produced by epithelial cells.

• This blocks the pancreatic duct, stopping pancreatic enzymes reaching the duodenum and stops food digestion.
• Bronchioles and alveoli become clogged, creating congestion, breathing problems and infections.

Daily Treatment:

• Daily chest physiotherapy massage is needed to keep airways open.
• Impaired digestion and impaired food absorption leads to larger appetites and low weight. 

Somatic cell therapy

Liposomes: 

Normal genes are inserted into liposomes which can be inhaled through aerosol, and absorbed into the epithelial cells in the lungs. The gene is then expressed as the protein is synthesised.

• The virus is rendered harmless by removing harmful genes.
• Cultured in epthelial cells along with plasmids with the normal CFTR gene. The gene becomes incoorporated into the virus.
• The virus is isolated and introduced into a patient by inhaler. 
• The virus injects the plasmid DNA into the epithelial cells of the lungs.

Effective??

Limited success, as the effect is short lived and needs to be repeated. There could also be an immune response . However, the benefits outweigh the negatives, as the sufferer could lead a more normal life.

Advantages:

• Helps people make an informed decision about their pregnancy.
• Reduces harmful alleles in the gene pool
• Reduces suffering
• Less cost for the NHS

Disadvantages:

• Ethics:
• Privacy invasion
• Increase number of abortions
• Insurance companies may target those with genetic defects.
• Fears of eugenics, and choosing characteristics for a child is also widespread.

FOR GERMLINE THERAPY:

-tampering with genes (which we don't know the true function of) may result in unforeseen consequences for future generation

There are means of testing if a child has a faulty/defected allele before it is born. 

• A blood test- this can detect cystic fibrosis
• Amniocentesis- This withdraws some of the amniotic fluid at 15-20 weeks, and removes cells from the surface of the embryo, for microscopic analysis.
• Chlorionic villus sampling- tiny samples of foetal tissue are cultured and examined.

These, however, can lead to an increased chance of miscarriage.

If a family has a history of a genetic defect, members can seek advice of their chances of having an affected child. Advice can include:

• Whether there is a history of the disorder in the family
• Whether the parents are closely related
• Whether the fault gene is frequent in the population

It helps people make important decisions about their life and their potential offspring.

USES PROBES.

These probes contain the complementary sequence for the beginning of the gene. The probe will match and combine with the gene of interest.

ISOLATION via Restriction Enzymes

• The plasmid is a vector, and it carries recombinant DNA (foreign DNA.)
• A restriction enzyme cuts the foreign DNA at particular sites into fragments. The unpaired bases form sticky ends.
• The enzyme produces the same sticky ends in the plasmid DNA.
• Enzymes such as DNA ligase, joins (anneals) these two sticky ends.
• Human DNA fragments are mixed with broken plasmids and the ligase recombines them with their complementary bases. 

REVERSE TRANSCRIPTASE

Reverse transcriptase-- an enzyme that makes DNA out of mRNA in specific cells.

• mRNA is extracted from suitable cells and reverse transcriptase enzyme is used to form a single stranded of the desired gene.
• DNA polymerase converts it into a double strand to incooporate it into a plasmid.
• The plasmids are added to a growing culture of bacteria but actual uptake is in a tiny proprtion of cells.
• Antibiotic resistant sequences are used to identify those bacteria with the recombinant DNA.
• Bacteria are cloned results in many copies of the new gene.

Intron- a portion of DNA within a DNA that does not code for polypeptide.

• This method is good as it misses out introns, which bacterial cells cannot deal with.
• mRNA is only for one polypeptide, so if we know a particular cell secretes a lot of this mRNA, we can isolate the gene more easily.

ADVANTAGES

• A large amount of a complex protein can be made quickly.
• Proteins dont have to be extracted from animals anymore, or corpses, which was a dangerous practice.
• Health benefits for people with diseases

Disadvantages

• Complicated process
• Expensive on a industrial scale
• Huge genome= difficulty in finding appropriate gene
• One protein may require many genes, and many polypeptide synthesis for different enzymes involved.
• Produces millions of fragments which are of no use.
• Not all eukaryote genes will be expressed in bacteriums.

HAZARDS:

• Bacteria readily exchange material, so a bad gene could be produced on a huge scale and then infect thousands recieving medical treatment.
• Deliberate use of antibiotic resistant genes for identification means that bacteria could become very resistant (e.g. E.Coli.)
• Human pathogenic genes, such as oncogenes, could also be passed on by accident.

Marker gene-

A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species e.g. antibiotic genes in bacteria that have uptaken plasmids.

Often uses a species of bacteria which attacks plants to cause a tumour. The bad gene can be replaced with useful genes.

Examples of GM crops are soya and tomatoes.

• Herbicide resistant soya beans allow specific herbicides to kill weeds without harming the crop. The weed killer breaks down into harmless components.
• A special tomato called FlavrSavr. It has an additional gene, which is complementary to the gene for the enzyme that breaks down pectin in tomatoes. This means the two mRNA strands join to form a double helix, and the enzyme isnt made. The tomatoes have a longer shelf life and a better taste.

• Superior keeping qualities
• Higher yield
• A reduction in pesticide use in agriculture, as plants are resistant.
• Reduces food shortages- crops that can grow with little water= useful in droughts.

• Geneticly engineered pollen could reach wild plants, affecting variation between species. This will lead to a greater number of restistant insects and bacteria.
• Unknown effects of eating new protein in crops
• ^^ including, antibiotic resistant genes transferred to bacteria in gut of consumer
• a reduction in biodiveristy
• Farmers may find they have less variation of crops to choose from providers. This could lead to highly susceptible crops, and monocultures.

An individuals genetic fingerprint or DNA profile is completely unique.

Exons are regions of DNA that codes for proteins, they are inbetween regions called introns, which don't code for proteins. Introns are usually made up of repeating blocks of nucleotides. The number of times these blocks are repeated is what changes our DNA fingerprint.

ORIGINAL TECHNIQUE

• DNA is extracted and cut into fragments using restriction enzymes.
• The fragments are then separated by Electrophoresis. The DNA fragments are negatively charged, so when exposed to an electric current, they move to the positive terminal. 
• Smaller pieces move further than large pieces. Bands form according to size of fragments.
• DNA fragmnets are transferred to a nylon membrane, and exposed to radioactive probes that attach to DNA fragments.
• Unbounded fragments are washed off. Bands left are completely original for that individual.
• The Nylon membrane is exposed to xray film, which is exposed by the radioactive probes.
• This creates an autoradiograph==== genetic fingerprint.

Human paternity testing.

• (Child contains 50/50 of parents DNA. Autoradiograph has mothers bands removed, and the remaining bands must match the fathers.)

Forensic science

Polymerase Chain Reaction produces a large number of specific fragments of DNA. This allows us to do tests on very small samples of DNA regardless of the age of the sample.

Mix DNA with DNA polymerase, nucleotides and primers (short pieces of DNA which trigger PCR)

• Heat DNA to 95°C, separating the strands.
• Cool to 55 °C activating primers to join complementary sequences. This then triggers DNA replication.
• Solution is heated to 70°C, allowing DNA polymerase to catalyse the synthesis of complmentary strands to the DNA strands.
• This doubles the amount of DNA.

95- seperates helix

55- activates primers

70- dna polymerase begins work

• The mass amount of data recieved from genetic fingerprinting must be stored appropriately, and given sufficent privacy. 
• Insurance companies may use the data to target those with larger risk of genetic disease.

HOWEVER:

• Efforts are being made to store the genomes of as many ethnicities, races and tribes of the world before they are intermixed and lost.
• Privacy must be maintained and the information shouldnt be misused.