4.5 Application of Reproduction and Genetics

Why was the human genome project designed?

To improve knowledge and understanding of genetic disorders and consequently, improve their diagnosis and treatment.

5 aims of the human genome project?

1. Identify all genes in the human genome and identify which chromosome each is on.

2. Determine sequence of the 3 billion base pairs in DNA to store info in databases.

3. Improve tools for data analysis

4. Transfer data related technologies to private sector, for medical innovation

5. Adress ethical, legal and social issues that may arise from the project.

3 main findings of the human genome project?

1. Humans have 20500 genes, fewer than expected

2. More repeated segments of DNA than previously suspected.

3. Fewer than 7% of families of proteins specific to vertebrates, so close relationships between all living organisms.

6 steps of Sanger sequencing

1. DNA denatured by heating.

2. A primer, complimentary to the start of the single-stranded DNA binds.

3. DNA polymerase synthesises new DNA strand.

4. DNA fragments are separated by gel electrophoresis

5. Dideoxynucleotides (ddNTPs) terminate DNA strands

6. Fluorescent tags are detected by a laser.

Main role of ddTNPs?

Terminate the reaction, so no further nucleotides can be added. There is one for each base.

Why are different length chains produced in Sanger sequencing?

ddTNPs lack 3’ hydroxyl group, so random chain termination occurs, as by CHANCE either a dTNP or ddTNP is placed.

2 types of dideoxynucleotides?

1. dTNP - C3 has OH which allows a phosphodiester bond to form.

2. ddTNP - C3 has the OH group replaced by a H, so there is no extension and so termination of the chain occurs.

How to identify fragment length?

Add a fluorescent marker to the ddTNP, after gel electrophoresis the length of the fragments can be seen. The shortest migrate the furthest, normal or altered nucleotide by CHANCE.

Next generation vs sanger sequencing

Sanger has short DNA fragments for small-scale projects whereas next generation sequencing has less reagents and lots of sequences can be done simultaneously. NGS also cheaper.

3 aims of the 100k genome project

1. Create an ethical, transparent programme based on consent

2. Enable medical and scientific discovery

3. Develop a UK genomics industry

What did the 100k genome project result in?

Improved disease diagnosis and treatment by combining sequence data with medical records for treatment to be investigated e.g. personalised cancer therapies and informed treatment decisions.

Main ethical issues of the human genome project and 100k genome project

1. Ownership of genetic information (insurance premiums, social discrimination from ancestry and company profits etc.)

2. Identification of allele sequences: mutated genes, some people may not want to know.

3. Genetic screening risk for them and their children.

4. Embryo screening for diseases like cystic fibrosis, Huntingson’s etc and issues with choosing preferred alleles.

5. Storage and security of genetic data - potential hacks.

Example to prove S and HGP are useful.

Some genetic diseases, e.g. thalassaemia, an inherited blood disease has had a reduction in cases.

What causes malaria?

The protist plasmodium causes malaria and mosquitoes are the v actors that carry the pathogen.

4 methods to cure malaria?

1. Insecticides to kill the vector

2. CRISPR-cas9 enzyme can remove and replace certain sections of DNA, so can remove the gene that causes resistance to Plasmodium in the mosquito.

3. Drugs to kill plasmodium (resistance occurs quickly however)

4. Genetically modify mosquitoes who can produce antibodies against plasmodium.

2 techniques a genetic fingerprint relies on.

1. Polymerase chain reaction (PCR) to make large numbers of DNA fragments.

2. Gel electrophoresis, to separate DNA fragments based on their size.

What parts of the DNA is used in genetic fingerprints?

Introns (non-coding) sections - either STRs or VNTRs

What are STRs and VNTRs

Short tandem repeats - unique to each person unless monozygotic twin; up to 13 base pairs with the number of repeats unique.

Variable number tandem repeats - many base pairs.

What is a polymerase chain reaction and what is the goal?

Technique to make many copies of a specific DNA region, with the goal of making enough copies to visualise and analyse.

2 main things needed for a PCR?

1. Relies on thermostable Taq polymerase that forms phosphodiester bond on growing nucleotide strand

2. Requires DNA primers designed for gene of interest (short sequence of nucleotides that provide a starting point)

4 steps of the PCR technique?

1. Denaturation (96C) - heat to separate the hydrogen bonds to make 2 template strands.

2. Annealing (55-65C) - cool so primers can bind to template to complementary sequence.

3. Extension/amplification (72C) - raise the temperature of taq polymerase extends primers, synthesising new strands of DNA. Normal tax polymerase unsuitable as it would denature and the high temperature needed to increase rate of reaction.

4. Repeat many times (25-35)

5 limitations of PCR.

1. Contamination of DNA from previous reactions or airborne from scientist could be amplified.

2. Error rate is very high; wrong base inserted and no technology to correct it, so incorrect base sequence amplified. May create a non-functional protein.

3. DNA fragment size - most efficient at 1000-3000 bases as it cannot fix errors, but most human genes are bigger than this.

4. Sensitivity to inhibitors - PCR is very sensitive to humid acids for example in decomposition (archeology)

5. Limits on amplification - around 20 cycles it slows down as reactants limited and enzymes denatured. Also, DNA pairs with eachother, not the primers.

What is in vitro PCR?

Out of body (in lab)

What does gel electrophoresis use? (lab equipment)

Agarose gel = polysaccharide that is porous, so DNA fragments can migrate through the gel.

To move DNA, voltage applied to the cathode, so DNA moves toward anode (DNA negatively charged due to the phosphate group)

How to compare fragments on a gel electrophoresis?

Smaller fragments move further as they have less resistance, as they are lighter. There is a ladder on the far left on known DNA fragments with base pair lengths known, to compare.

What is southern blotting in a gel electrophoresis?

Nylon membrane that lifts DNA onto it and transfers the fragments. DNA probes are washed with alkaline to be single stranded again, and flourescent markers (DNA probes) added. These join to fragments and are sensitive to x-rays for visualisation.

4 pros of DNA profiles

1. Non-invasive

2. Small samples

3. Exonerate false convictions

4. Store genetic material from around the world

3 cons of DNA profiles.

1. Against privacy concerns

2. Database prone to misuse and hacking

3. Profiles have probabilities meaning answers aren’t absolutes, so possible wrongful convictions.

Define recombinant DNA.

The DNA formed when genetic material from two different species is combined (also referred to as recombinant DNA technology).

Define transgenic organisms

Organisms that have DNA from another species introduced into their cells.

Define donor DNA

The introduced DNA.

Define host

Organism into which donor DNA has been introduced.

Define transformed

When a cell has incorporated a plasmid containing a foreign gene.

Define genetic modification

Organisms that have had genes altered or deleted (these are not transgenic because they do not contain any foreign genes).

Define vector

A plasmid or virus used to carry foreign genetic material into a cell.

Define plasmid

Small circular loop of self-replicating double-stranded DNA in bacteria.

What is genetic engineering?

The process of using recombinant DNA (rDNA) technology to alter the genetic makeup of an organism.

Why is genetic engineering used?

It allows genes to be manipulated, altered and transferred from one organism or species to another, making a genetically modified (GM) organism.

3 examples of genetic engineering.

• Bacteria, so they can make useful products e.g. insulin

• Plants and animals, so they can acquire new characteristics e.g. resistance to disease

• Human, to reduce the effects of genetic diseases e.g. Duchenne muscular dystrophy.

5 steps in the basic process of genetic engineering?

1. Isolating the DNA fragments containing the desired gene

2. Inserting the DNA fragments into a vector

3. Transfer of DNA into a suitable host cell

4. Identification of host cells that have taken up the gene (using gene markers)

5. Cloning the transformed host cells.

How to identify a gene?

A gene probe (a specific segment of single stranded DNA) that is complementary to a section of a gene is then used to identify the gene.

2 methods to isolate genes

1. Restriction endonuclease – bacterial enzymes that cut DNA at specific nucleotide sequences.

2. Reverse transcriptase – enzyme which is derived from a retrovirus that catalyses the synthesis of cDNA from an RNA template.

The two ways that restriction endonuclease cuts DNA for isolation?

1. Blunt cut - straight line

2. Staggered cut - unpaired bases on both strands, known as ‘sticky ends’

Define sticky ends.

A sequence of unpaired bases on a double-stranded DNA molecule that easily base pairs with a complementary strand.

What is EcoR1?

A restriction endonuclease from E.coli, leaving sticky ends. Form a palindrome as four base pairs at each end are in reverse order.

2 main problems of restriction endonuclease?

1. If recognition sequence is within the gene, non-functional fragments will be made.

2. Eukaryotic genes have introns that are transcribed and removed from RNA, bacteria have no enzymes to remove them, so non-functional proteins will be made.

What does reverse transcriptase do?

Reverse transcriptase is an enzyme that produces DNA from an RNA template. The synthesised DNA is called copy DNA or cDNA and is complementary to the RNA.

3 steps of using reverse transcriptase for gene isolation

1. mRNA extracted from cell.

2. Reverse transcriptase produces single-stranded DNA from the mRNA template.

3. DNA polymerase then synthesises the complementary strand of DNA to produce a double-stranded DNA molecule.

Reverse transcriptase example

Only two copies of insulin gene, but many mRNA transcribed from it. Especially in B-cells in pancreas which synthesises and secretes insulin.

4 advantages of reverse transcriptase

1. This method can produce many copies of cDNA that are complementary to the RNA.

2. There are no issues with extra amino acids being produced due to introns already being removed from the RNA.

3. Overcomes the problem of locating the gene as RNA can be extracted.

4. No chance of cutting into the gene making it non-functional.

5. No need to further process the RNA to make it functional.

How to isolate a plasmid to make a recombinant plasmid?

1. Bacteria treated as EDTA destabilises the cell walls

2. Detergent that dissolves the phospholipid bi-layer.

3. Sodium hydroxide makes alkaline environment to denature membrane proteins.
   
   Now the plasmids can be separated from cell debris

How to cut open a plasmid to make a recombinant plasmid?

Using the same restriction endonuclease used to isolate the gene, so it has the same nucleotide sequence on its sticky ends.

How to join the gene and plasmid to make a recombinant plasmid?

1. Mix vector and gene, so complimentary base pairs (due to sticky ends) occurs.

2. Enzyme DNA ligase permanently binds the sugar-phosphate backbones together.

3. Gene now spliced and so is recombinant DNA

6 features of a good vector structure

1. Not broken down by host cell enzymes

2. Small

3. Able to be screened to confirm insertion of gene to plasmid

4. Not stimulate immune response in recipient

5. Self-replication

6. Markers to identify uptake in host cells.

What increases the uptake of plasmids into host cell?

Calcium chloride as positive charge of calcium ions bind to negatively charged backbone of plasmid and LPS.

How to use genetic markers?

Marker gene added to same region as human insulin gene e.g. antibiotioic resistance. Then grow on an agar plate with antibiotics. If the plasmid is incorporated, then resistance will occur, proving insulin gene must be present.

What are genetically modified crops?

Transferring a piece of DNA from one organism to another different organism, through targeted removal of the desired genes from the DNA of one organism and adding them to the other organism.

Why do we need GM crops? [4]

1. Rapid increase in population, so need to provide enough food to feed the growing population.

2. Increased affluence links to an increased consumption of meat which is an inefficient use of land, water and food.

3. More effective use of land is using organisms at lower trophic levels.

4. Unfortunately, 70% crops lost between harvesting an supplying homes

5 reasons GM crops help feed the population?

1. Increasing nutritional value (e.g. Golden rice with vitamin K for eyesight)

2. Increasing the size, and so yield

3. Making crops disease resistant

4. Making crops drought resistant

5. Increasing shelf life

4 ways to introduce novel genes into plant cells.

1. Using a ‘gene-gun’ firing small spheres of gold or tungsten coated with a preparation of the gene at plant cells. Some will penetrate the cell wall and are taken up through the cell membrane.

2. Electroporation - electric field increases permeability of cell membranes, enhancing gene uptake.

3. Microinjection where the membrane is pierced with an ultra-fine needed and gene is injected into the cytoplasm or nucleus, so better for use in animal cells.

4. Using a bacterial vector - Agrobacterium tumefaciens is the most common method for making transgenic plant cells.

6 steps of transforming plants with Agrobacterium tumefaciens.

1. Plasmid extracted from the A. tumefaciens

2. Restriction enzyme is used to cut the plasmid and remove the tumour-forming gene

3. A section of DNA containing a gene for disease resistance is located and isolated using the same endorestriction nuclease.

4. The gene is inserted into the plasmid, replacing the tumour-forming gene. DNA ligase is used to join the donor and vector DNA together.

5. The bacterial cell is introduced into plant cell. The bacterial cell divides and gene is inserted into plant chromosome.

6. Transgenic plant cells are grown in tissue culture and transformed plants are regenerated.

How and why are soya beans genetically modified?

Routinely treated with herbicides, but this can damage the plant, so they are modified to be ‘Round-up Ready’ and herbicide resistant, to increase yield and not inhibit growth.

Bt tomatoes

1. How are they insecticide resistant?

2. How is this not a problem for human consumption?

3. Benefits

1. A bacterium that has an insecticide protein and is incorporated into the cells of the Bt tomatoes.

2. Only expressed in the leaves, which insects eat, and then kills them through blood poisoning.

3. Less of an ecological footprint than tomato farming normally is, and increases farm income.

Antisense tomatoes:

1. Why are they needed?

2. What does the GM do?

3. Method to make this work?

1. Transporting over long distances from supplier may over-reopen them and not be suitable to sell.

2. Flavr Save tomato was developed through the use of antisense RNA to regulate the expression of the naturally producing enzyme polygalacturonase in ripening tomato fruit.

3. Second copy of the gene added, so two identical mRNA sequences are made that base pair with each other to block the production of the enzyme.

Genetic difference between FlavrSavr tomatoes and Bt tomatoes?

Bt tomatoes are genetically modified and transgenic. FlavrSavr tomatoes are genetically modified but they are not transgenic as they do not contain a gene from another species.

4 arguments in favour of GM crops?

1. Higher crop yield

2. Pesticide reduction

3. Improved food e.g. Golden rice

4. ‘Pharming’ is the production of pharmaceutical molecules in GM crop plants.

7 arguments against GM crops?

1. Gene transfer e.g. pollen from GM crops to wild plants, creating ‘superweeds’.

2. Pest resistance, leading to a population all resistant.

3. Marker genes: antibiotic resistance may be transferred to bacteria in the intestine of the consumer.

4. Biodiversity decrease: decreases amount of useful genes.

5. New proteins: possible adverse health affects

6. Organic farming: pollen from GM crops could compromise this

7. Economic concerns: intellectual property law with the expense borne by the farmer e.g. monopoly of the market.

4 potentials of genetic screening

1. Confirm a diagnosis

2. Indicate an appropriate treatment

3. Allow families to avoid having children with devastating diseases (pre-natal diagnosis)

4. Identify people at high risk for conditions that may be preventable.

5 concerns about widespread genetic screening?

1. Invasion of privacy

2. Defective alleles in prenatal tests may increase abortion rate, which has seen to be disproportionately of female foetus’

3. Defects may lead people to be in a high-risk group for insurance purposes making it expensive and difficult to obtain

4. Commercialised gene tests are not tightly restricted as they’re not regulated by health bodies, so there is an increased error rate.

5. May increase anxiety and social stigma, perhaps even discrimination.

7 main uses of genetic testing

1. Carrier screening to identify a recessive allele for a genetic disease, so may not have children.

2. Pre-implantation genetic diagnosis to screen embryos generated from IVF

3. Pre-natal diagnostic testing

4. Newborn baby screening

5. Pre-symptomatic testing for predicting adult-onset disorders like Huntington’s, cancers and Alzheimer’s.

6. Confirmation that an individual has a suspected disease.

7. Forensic and identity testing.

What is gene therapy and what are the 3 methods?

Replacing defective allele with a healthy one:

• Virus as a vector

• Plasmid as a vector

• Injection of naked plasmid DNA

2 main approaches to gene therapy?

1. Somatic cell: therapy targets body cells in the affected tissues, but not inherited in daughter cells of treated cells and do not appear in future generations. Therapeutic, but do not rectify DNA.

2. Germline therapy: introduces corrective genes into herm-line cells (oocyte or sperm or zygote). The genetic correction will be in the offspring and the offspring’s gametes.

3 problems of somatic cell therapy

1. Aquiring human gene for patients cell

2. Insertion of gene into the right cells

3. Making sure the gene is activated

Why is gremline therapy controversial?

Seen as ‘playing God’ and could switch on other genes e.g. protooncogenes with unpredictable effects in future generations.

What is Duchenne Muscular Dystrophy (DMD) and how is it obtained?

Genetic disease that causes muscle weakness and wasting. It is a recessive X-lined disease, so more common in males as they only have one X chromosome (nothing to counteract defect). The mother is normally the carrier.

Genetic change in DMD?

Faulty gene cannot produce Dystrophin protein, that protects muscles in contraction. As a result, muscles are worn down, so one or more deletitions in the gene leads to the disease.

What is the treatment for DMD?

The drug drisapersen is injected into subcutaneous tissue. It is an antisense oligonucleotide which is complimentary to the mutated sequence to block the sequence in transcription, so is known as an ‘exon patch’, so it is partially functional as corrective protein is synthesised.

What is cystic fibrosis?

An inherited condition where sticky mucus is in lungs (clogging alveoli) and digestive system (pancreatic duct blocked, so no enzymes reach leading to malnourishment), and reproductive system (males infertile as the vas defferens is blocked). Symptoms seen in early childhood, so symptoms get worse.

How is cystic fibrosis obtained?

Homozygous for autosomal recessive allele.

Both parents heterozygous and are carriers, so 25% change of cystic fibrosis (nn).

What is the genetic issue with cystic fibrosis?

The cystic fibrosis transmembrane regulator (CFTR) is a transmembrane protein that transports chloride ions out of the cell. Mutant version is no regulator.

The protein uses co-trasnport, so water potential decreases, so water leaves cell into the mucus to make it watery. No protein or faulty with cystic fibrosis, so mucus remains thick and sticky.

1. Chest physiotherapy needed and tablets needed to bring digestive enzymes.

2. Initially virus used to deliver gene, but was unsuccessful. Liposomes used, so inhaled with aerosol and fuses with phospholipid bi-layer to provide relief, targeting somatic cells.

3. Modern treatment is ivacaftor drug that corrects the protein folding.

What does tissue engineering do?

Creates bioartificial tissues e.g. skin graft

4 types of cells for tissue engineering.

1. Autologous cells are from the same individual. Fewest problems with rejection and pathogen transmission, but not always available if there is a genetic disease or severe burns, or is very ill or old.

2. Allogeneic cells come from a donor of the same species.

3. Xenogeneic cells are from another species e.g. pig cells for cardiovascular tissue, but danger in viral sequences in pigs DNA.

4. Syngenetic or isogeneic cells are from genetically identical organisms.

What is a scaffold, and what are the 4 properties needed for an effective one?

Cells are ‘seeded’ on to a scaffold:

1. Allow cells to attach and move

2. Deliver and retain cells and biological molecules

3. Be porous to allow diffusion of nutrients and waste products.

4. Be biodegradable and be absorbed by the surrounding tissues. The rate it degrades should match the rate of tissue formation, so eventually it will break down leaving the ‘neotissue’.

Uses for cell cultures?

Been used for ages for research and medical purposes. Now its used for cell replacement therapy and tissue engineering.

Define stem cells?

An undifferentiated cell capable of dividing to give rise to daughter cells, which can develop into different types of specialised cell or remain undifferentiated.

Define the 4 types of stem cells?

1. Embryonic = totipotent: make all cell types and cells supporting embryonic development. Blastocyst has ESCs that can form every cell type.

2. Pluripotent: make all cell types.

3. Adult = multipotent: makes several cell types, in stem cell niches. Replace cells lost by normal wear and tear, injuries or disease but cannot form all cell types. Found in meristem and in bone marrow e.g. femur.

4. Induced pluripotent: genetically reprogrammed adult stem cells can make several cell types, behave like ESCs.

4 examples of uses of stem cells.

1. Treat type 1 diabetes by developing pancreatic beta-cells.

2. Damaged heart muscles with cardiac muscle cells with tissue engineering.

3. Macular degeneration - vision dicreases with age, so stem-based therapies can rectify.

4. Can be used to screen new drugs on tissue cultures.

Problem with a patient receiving tissues derived from ESCs?

Need immunosuppressant drugs, which may cause side effects. Own adult stem cells like likely to provoke an immune response as they have the persons own antigens.