Biology Unit 4.5 - Application of Reproduction and Genetics Flashcards
What is the human genome project?
Project designed to improve knowledge and understanding of genetic disorders and consequently improve their diagnosis and treatment
Aims:
* Identify all genes in the human genome
* Determine the sequence of the 3 million base pairs
* Improve tools for data analysis
* Transfer related technologies to the private sector
* Address the ethical, legal and social issues that may arise
Findings:
* Humans have about 20,500 genes
* More repeated segments of DNA than suspected
* Fewer than 7% of the families of proteins were specific to vertebrates
Explain Sanger sequencing…
- DNA is broken into single-stranded fragments
- Mixture is prepared containing single, short DNA strands, DNA polymerase, four deoxyribonucleotides and a single short primer
- Reaction mixture is placed in four tubes and a different deoxyribonucelotide is added to each
- DNA polymerase replicates new DNA strands, which is terminated when a ddideoxyribonucleotide is added
- DNA strand is tagged using radioactive isotopes, antigens or fluorescent markers
- Newly formed oligonucleotides are seperated by gel electrophoresis
- Shortest strands move further down
- Reading from the bottom to the top provides the correct DNA sequence
What is the 100K Genome Project?
Sequencing of 100,000 genomes from NHS patients with cancer or rare diseases and their families with the aims:
- Create an ethical, transparent programme based on consent
- Set up a genomic cervice for the NHS patients
- Enable medical and scientfic discovery
- Develop a UK genomics industry
Explain the issue of ownership of genetic information?
Ownership needs to be clarififed once a sequence of bases is known and safeguards need to be put in place if the information belongs to the individual
Explain identification of sequences correlated to health problems…
Some individuals may not want to know if their base sequence could lead to future health problems
If the mutation could affect others it needs to be made clear whether they have a right to this information
Explain carrier screening…
If there is a family history of genetic mutation, family members may be offered genetic counselling about any risks to themselves or their children
Explain screeing embryos for genetic defects…
Parents may choose not to select defective embryos for implantation
There is a legal framework which regulates the use of ‘spare’ embryos for research
Explain screeing embryos for desirable characteristics…
Technology used to screen for these characteristics could be misused and need to be debated before they are implemented
Explain screeing for sequences that pre-dispose risk…
Decision must be made about when or if parents shoudl tell the child about the test results
Society must decide whether a parent has the right to withold this information from the child e.g., who owns a child’s DNA, etc
Explain storage and security of genomic data…
Storage and security is a huge concern due to the risk of hacking computer systems and databases
Explain DNA sequencing of non-human organisms…
Species are chosen on account of their scientific or medical significanc, or their economic or cultural importance, which allos inferences to be drawn concerning evolutionary relationships
What is malaria?
Parasite transmitted by mosquito Anopheles gambiae, whose rapid evolution of insecticide resistance is responsible for over a million deaths per year
Parasite, Plasmodium sp. has also developed multi-drug resistance
Sequencing of both genomes allows for chemicals that could render the mosquito susceptible again and more effective anti-malarial drugs to be developed
What is genomics?
Study of the structure, function, evolution and mapping of genomes
Enable:
* Accurate diagnosis
* Better prediction of the effect of drugs
* Improved design of drugs
* Treatment to be tailored to the individual
What is the polymerase chain reaction?
Amplifies the quantity of DNA as it can rapidly produce billions of molecules from a single DNA molecule
Gel electropherisis is used to analyse the DNA, producing a DNA profile, where exons are regions that code for proetins, while introns contain blocks of repeated nucleotides, which rpdocue variation in individuals
What is PCR protocol?
- Sample of DNA is dissolved in buffer mixed with DNA polymerase, nucleotides and primers
- Primers act as signals which tell the DNA polymerase where to start and stop copying
- Original DNA is denatured and seperates into two single strands
- Solution is rapidly cooled allowing the primers to bind to complimentray base sequences
- Solution is heated to 70 degrees celcius, producing two identical double strands of DNA
- First three steps are repeated many times
- DNA fragments are seperated using electrophoresis gel
Explain the issue of contamination…
Any DNA that enters the system can be amplified, where most contamination comes from previous PCR reactions
Explain the issue of error rate…
DNA polymerase enzymes sometimes insert a nucelotide containing the wrong base, where taq polymerase cannot proo-read and correct their errors
Rate of error increases after each cycle
Explain the issue of DNA fragment size…
PCR is most efficient making DNA about 1000-300 base pairs long
If a lower temperature, higher pH and a proof-reading polymerase in addition to taq polymerase is used, much longer genes can be produced
Explain the issue of sensitivity to inhibitors…
Molecules may affect enzyme inhibitors
Explain the issue of limits on amplification…
At the start of PCR the number of molecules made is exponential, but after about 20 cycles it slows down:
- Reagent concentrations become limiting
- Enzyme denatures after repeated heating
- High DNA concentrations causes single-stranded molecules to base pair with each other rather than with primers
What is a genetic profile?
Combination of different sequences of different length non-functional DNA, which are passed on to offspring and show the difference between individuals
What is electrophoresis?
- DNA is extracted and cut into small fragments using restriction endonuclease
- Fragments are pipetted into pits within the electrophoresis gel
- Electric current is applied to the gel; DNA fragments are pulled through the gel
- Small fragments move through the gel
- Banding pattern is formed which can then be compared to other known samples
What is paternity testing?
White blodd cells are taken from the mother and the possible father, where bands in the mother and child’s profile are compared and those which match are removed
Remaining bands will only match wil the biological father
Explain application of genetic engineering in bacteria…
Bacteria produced useful procuts such as insulin and enzymes
Explain application of genetic engineering in plants and animals…
Plant or animals aquire a new characteristic
Explain application of genetic engineering in humans…
Gene therapy
Explain isolating the gene from a donor DNA molecule…
- Gene is located using a gene probe and then isolated
- Restriction endonuclease cut the DNA into small pieces
- Enzyme cuts between specific base sequences
- Most split the two strands in a staggered sequence
- Unpaired bases form sticky ends
Explain inserting the gene into a vector?
- Vector carries the gene into the new host cell
- Plasmid is a small circular piece of DNA found in bacterial cells
- Plasmid is obtained by dissolving bacterial cell walls and then seperating the plasmid from the rest of the debris
- DNA molecule is cut open using restriction endonuclease
- Enzyme makes staggered cuts, allowing the donot DNA to be spliced into the vector DNA
- Splicing occurs when the donor and vector DNA are mixed together
- Sticky ends are complimentary and pair up
- DNA ligases are used to join the donor DNA to the vector DNA
- DNA molecule created is called recombinant DNA
Explain the manufacture of insulin…
Recombinant DNA is taken up by bacterial cells, which reproduce asexually to produce clones, making many copies of the human insulin gene
Genetically modified bacteria are cultured on a large scale using a fermenter and produce insulin which is extracted and purified
What are marker genes?
Needed to show which bacteria have taken up the recombinant DNA
Those which confer ampicillin resistance, are spliced into the bacterial plasmid along with useful donor gene
Bacteria which have taken up the plasmids will have aquired the gene for ampicillin resistance and are able to metabolise the antibiotc and survive
How are reverse transcriptase used to copy a useful gene?
- mRNA is extracted from the cytoplasm
- Reverse transcriptase is added and makes a copy called copy DNA or cDNA
- Many copies of cDNA are made
- DNA polymerase adds complimentary free nucleotides to form a double strand for insertion into a plasmid
- No useful sticky ends are formed
Explain the use of reverse transcriptase…
- Extract the mRNA molecule which codes for a particualr protein
- Reverse transcriptase synthesis a single strand of cDNA, using the mRNA as a template
- cDNA is seperated from the mRNA template
- DNA polymerase catalyses the addition of free complimentary nucelotides to form a double strand of DNA
- Sticky ends are added to each end
- DNA is amplified by PCR
What are transgenic plants?
Plants that have altered genotypes and express a new useful characteristic in their phenotypes
Describe the gene gun…
Fires small spheres, coated with a preparation of the gene at plant cells, where some penetrate the cell wall and are taken up by the cell membrane
Describe electroporation…
Electrical field increses the cell membrane’s permeability, enhancing gene uptake
Describe microinjection…
Cell membrane is pierced with a needle and the gene is injected into the cytoplasm or nucleus
Describe bacterial vector…
Agrobacterium tumefaciens is the most common method for making transgenic plant cells, where they infect the plant cells, and carry useful genes into the target cells
What are the benefits of transgenic plants?
- Higher yield
- Superior keeping qualities
- Substantial reduction in pesticide use
What are the concerns of transgenic plants?
- Dispersal of pollen from crops engineered for herbicide resistance to wild relatives
- Unkown effects of earing new protein produced
- Reduction in biodiversity
Describe the genetiv engineering of plants…
- Find and isolate the chromosome/useful gene, which codes for a useful protein
- Cut out the gene using a suitable restriction endonuclease, leaving sticky ends which makes splicing easier
- Isolate a suitable chromosome from the soya, and cut it using restriction endonuclease leaving complimentary stikcy ends
- New gene is spliced into the chromosome, where hydrogen bonds between complimentary bases allow for splicing, which must be secured by DNA ligase
- cDNA is inserted into a callus, which can be sub-divided many times and cloned by mitosis, where the callus is allowed to differentiate into a plant
What are the advantages of genetically engineered plants?
- Less chemicals are used on the crops
- Less damage to the crops by insects
- Better yield
What are the disadvantages of genetically engineered plants?
- Gene could spread to closel related plants
- Plants could be toxic to humans
- Biodiversity could decrease
- Pollinators could decrease in numbers
What is genetic counselling?
Process which provides advice on the risk of bearing a child afffected with a genetic defect, based on:
* Whether there is a history of the disorder in the family
* Whether the parents are closely related
* The frequence of the faulty gene in the general population
What is genetic screening?
Tests to determine whether the child is affected:
* Chorionic villus testing - Tiny sample of the foetal tissue is withdrawn from the uterus, and cells are cultured and analysed
* Amniocentesis - Amnietic fluid is withdrawn at 15-20 weeks, which contains cells from the surface of the embryo, which can be analysed
* Blood test - There is a simple blood test for cystic fibrosis
What are the advantages of genetic screening?
- Parents can educate themselves about a genetic disorder and prepare fully
- Provides choice
- Redcue the frequency of a defective allele in the population
What are the disadvantages of genetic screening?
- Considered to be an invasion of privacy
- Increases the number of abortions
- Individuals may be placed in high risk group for insurance, future health care may become more expensive
- Misuse of the technology to modify or choose the phenotype
Explain gene therapy…
- Attempts to insert a normal, functioning gene into a person’s cells
- Main problem is developing a gene delivery system and ensuring the new gene functions correctly once it’s there
- Inserting a functional gene into a cell does not remove the defective gene; cells will produce both functional and non-functional proteins
- Majority of procedures use viruses as vectors, as virsuses insert their own genetic material into cells and are highly adapted to carry out this function
- Some use liposomes which fuse with the plasma membrane of the cell and release the new gene into the cell by endocytosis
What is somatic cell therapy?
Targets cells in the affected tissues, and is a therapeutic method but cannot be inherited
What is germ-line therapy?
Involves introduction of corrective genes into germ-line cells; gene is replaced in the egg and will enable genetic corrections to be inherited
What is cystic fibrosis?
Genetic defect due to a defective autosomal recessive allele, where sufferers produce thick, sticky mucus from the epithelial cells lining parts of the digestive system and lungs
These secretions cause:
* Pancreatic duct becomes blocked, preventing enzymes from reaching the duodenum, leading to incomplete digestion of food
* Bronchioles and alveloi of the lungs become clogged, causing congestion and difficulty breathing
What is the traditional treatment for CF?
- Frequent daily chest physiotherapy massage, which keeps the airways open
- Digestive enzymes can be taken orally before eating, while children with the condition have huge appetites to trey to compensate
How is cystic fibrosis inherited?
- Both parents must be carriers (heterozygous)
- Identified using a blood test
- Normal gene codes for a protein found in the cell membrane, called cystic fibrosis transmembrane regulator, which transports chloride ions out of cells into mucus, sodium ions follow
- Lowers the water potential of the mucus, water leaves, viscosity of mucus is reduced, making it watery and runny
- Protein of cystic fibrosis sufferers lack just one amino acid, so the protein produced cannot perform its junction
Explain gene therapy in cycstic fibrosis sufferers…
Gene which codes for the CFTR protein is inserted into liposomes, by wrapping the gene in lipid molecules that can fuse with the cell membranes of lung epithelial cells
These liposomes are delivered by an aerosol inhaler
Liposomes fuse with the phospholipid bilayer, where DNA enters the cell and the cells start to express the inserted gene by making the CFTR protein
What is Duchenne muscular dystrophy?
Recessive, sex-linked form of muscular dystrophy which affects more than on in 3500 live births, caused by one or more deletions in the dystrophin gene, causing the ribosome to reach the stop codon too soon, so dystrophin is not produced
How is DMD treated?
Diasperen introduces a molecular patch over the exon with the mutation, making the gene readable again, producing a shorter form of dystrophin
This is known as exon skipping, where teh drug is delivered to the patient in subcutaneous injections
Trials have not yet revealed the best age for treatment of the duration of treatment
What are the advantages of gene therapy?
- Correct proteins are expressed in some cells
- Milder symptoms
- Children with genetic diseases have a chance of braing healthier
What are the disadvantages of gene therapy?
- Small proportion of the introduced genes are expressed
- May can an immune response in the patient
- Therapy must be reapplied
- Commercial companies may abuse technologies to modify characteristics of health children
What is tissue culture?
Technique of growing cells in a laboratory, where the medium on which the cells are grown need to be carefully controlled and monitored
What is tissue engineering?
Inducing living cells to grow on a framework of synthetic material to produce a tissue such as skin, which has applications for treatment of extensive, deep skin burns
What are stem cells?
Undifferentiated cells that can form specialised cells, and that can be used from embryos and children/adults
What are the benefits of using stem cells?
- Using a patient’s stem cells means there is no risk of rejection and there’s no need to find a donor, and no ethical concerns
- Embryonic stem cells can differentiate to form any specialised cell
What are the concerns of using stem cells?
- Stem cells from the patient are limited and can only form specialised cells from the original organ
- To use embryonic stem cells an embryo is destroyed, which is controversial and is an ethical dilemma
