4.5 Application of Reproduction & Genetics Flashcards
What was the human genome project?
- project ran from 1990 to 2003
- designed to improve knowledge and understanding of genetic disorders and consequently improve their diagnosis and treatment
What were the main aims of the HGP?
- identify all genes in human genome and which chromosomes they’re on
- determine sequence of the 3 billion base pairs in human DNA
- improve tools for data analysis
- transfer technology to the private sector
- address ethical, legal and social issues associated with
Describe the ethical aspects of the HGP
- ELSI founded as an integral part of the HGP
—> worlds largest bioethics program - privacy and fairness
- integration of new genetic technology into medicine
- consent!
What were the main findings of the HGP?
- humans have about 20,500 genes
- there are more repeated segments of DNA than expected
- fewer than 7% of the families of proteins were specific to vertebrates
Define electrophoresis
- a lab technique that separates molecules on the basis of size, by their rate of migration under an applied voltage
Briefly describe how Sanger sequencing works
- DNA copied many times to make fragments of different lengths
- fluorescent nucleotide marks each fragment with four different colours
- loaded onto gel electrophoresis plate
- colours show order of bases
Describe the benefits of next generation sequencing
- small scale
- use shorter fragments
- run in parallel
- fast
Briefly describe the 100K genome project
- 2012 using NGS to sequence genome of 100,000 NHS patients who have rare genetic diseases or cancer
- aim to understand causes to improve treatment
Describe the ethical issue of ownership of genetic information
- safeguards must be in place to ensure data is not misused
—> info should not be used against people in things like health insurance claims if they have genes that predispose them to illness
—> if the genome proposes a certain ancestry then it should not be used as a pretext for social discrimination
—> no company should make financial profit from using a DNA sequence without consent
Describe the ethics of genetic screening and allele identification
- patients DNA can be scanned for mutated sequences that might correlate to future health problems
—> who should know, who has the say in consent etc
—> genetic screening of babies - genetic screening useful in genetic counselling
- embryos can be screened during IVF for alleles associated with cystic fibrosis, Huntingtons and thalassaemia
- societal issue of trying to choose genes to ensure specific characteristics ie athletic, smart, blonde etc etc
Describe the ethics of consent in genetic technology
- society must decide if parents have right to know and potentially withhold health info from children
—> ie if they show sequence for breast cancer - could choose not to tell child etc - should relatives have access to each others genome?
Why is sequencing of non-human organisms important?
- allows inferences to be drawn about evolutionary relationships
- understanding primate sequences contribute to understanding the origin of mankind
Describe attempts to control malaria by killing the vector
- insecticide sprays
—> mosquitos become resistant and then resistant to all insecticides in that particular class - DNA sequencing of the mosquito done in 2002
- develop genetically modified mosquito in 2015 using CRISPR-Cas9
—> eggs modified with addition of a gene that allows them to synthesise a Plasmodium antibody
—> prevents Plasmodium spreading from human to mosquito to human
—> not released into wild but provides model for future research
Describe attempts at controlling malaria by killing the parasite
- extracts from Cinchona bark were first used as drugs
—> contain quinine which disrupts Plasmodium digestion of Hb in RBC
—> toxic derivative builds up, killing the Plasmodium - spontaneous mutations have led to quinine resistance
Describe 3 chemicals used to kill Plasmodium
- chloroquine
—> disrupts Hb digestion
—> mutant Plasmodium expels chloroquine 50x quicker than normal - not enough time to have an impact so drug is considered resistant - atovaquone
—> kills Plasmodium by acting on ETC on mitochondria
—> resistance develops rapidly due to a mutation on the cytochrome b gene - artemisinin
—> acts on Plasmodium in RBC
—> resistance has been detected
What 2 techniques does genetic fingerprinting rely on?
- polymerase chain reaction making large numbers of DNA fragments
- gel electrophoresis to separate fragments based on size
Differentiate between exons and introns
Exons: code for proteins
Introns: do not code for proteins
What is meant by a short tandem repeat?
- sequences of nucleotides where up to 13 nucleotides repeat several hundred times
- number of repeats is inherited so can be used to trace family lines
What is meant by PCR?
- polymerase chain reaction
- semi-conservative replication of DNA in a test tube
What is the DNA sample dissolved in in a PCR?
- buffer
- Taq polymerase (polymerase from bacterium living in hydrothermal vents)
—> optimum temp of 80°C - nucleotides containing the four bases
- short, single stranded pieces of DNA called primers
What is meant by a primer?
A strand of DNA about 10 nucleotides long that base pairs with the end of another longer strand making a double stranded section to which DNA polymerase may attach
Describe the main stages of a PCR
- reaction mixture containing DNA sample, primers, free nucleotides and DNA polymerase is set up
- original ‘target’ DNA is heated to 95°C, separating it into two single strands by breaking hydrogen bonds
- solution cooled to 55°C which is cool enough for primers to anneal to the complimentary base sequences on the single strands
- solution heated to 70°C and Taq polymerase catalyses synthesis of a complimentary strand by causing phosphodiester bond formation in the elongation stage
—> for each fragment of DNA, two identical double strands are produced - sequence is repeated many times
Explain the limitations of PCR
- contamination: any DNA that enters by accident can be amplified, often some left from previous PCR in the apparatus
- error rate: all DNA polymerase sometimes insert a nucleotide with the wrong base, Taq polymerase cannot be proofread. Error rate of 1 per 300
- DNA fragment size: most efficient for making DNA about 1000-3000 base pairs long - human is significantly longer
- sensitivity of inhibitors: sensitive to phenolics, humic acids, haem breakdown products and the blue dye used on denim
- limits on amplification: at the start it increases exponentially but it soon slows down and becomes linear
—> reagent conc is limiting, enzyme denatures, DNA in high conc causes single stranded molecules to base pair with others not the primer
What is meant by genetic modification?
- organisms that have had genes altered or deleted
—> not transgenic as do not contain foreign genes
Define recombinant DNA
DNA produced by combining DNA from 2 different species
Define a transgenic organism
An organism that has been genetically modified by the addition of a gene or genes from another species
What is meant by donor DNA and a host?
- introduced DNA
- organism into which DNA is introduced
What is meant by saying a cell is ‘transformed’?
When a cell has incorporated a plasmid containing foreign gene
—> organisms that have had genes altered or deleted are not transgenic as they don’t have foreign genes
What are the steps in producing a genetically engineered protein?
- identifying location of the gene
- isolation of DNA fragments
- insertion of the DNA fragments into vector
- transfer of DNA into host cell
- identification of host cell using gene markers
- cloning the transformed host cells
Describe the process of isolating a gene with restriction endonucleases
- bacterial enzymes that cut DNA at specific nucleotides
- cuts sample into smaller fragments
- some cut across the double helix making a blunt cut, some make a staggered cut which leaves unpaired bases on both sides, called sticky ends
- unpaired bases in reverse order bind to the ends, forming a palindrome
What is meant by sticky ends?
A sequence of unpaired bases on a double stranded DNA molecule that readily base pair with a complimentary strand
What are 2 drawbacks of restriction enzymes?
- if the recognition sequence occurs within the gene of interest, the gene will be broken into fragments that have no function
- eukaryotic genes contain introns which get incorporated into plasmids. Bacteria cannot process RNA to remove introns so proteins will be non-functional
Describe how reverse transcriptase is used to isolate a gene
- reverse transcriptase is an enzyme derived from a retrovirus that catalyses the synthesis of cDNA from an RNA template
- many copies of cDNA that is complimentary to the insulin mRNA can be made
- does not have an intron problem as the RNA in the nucleus transcribed from DNA has already been processed
- reverse transcriptase produces single stranded DNA from mRNA template
- DNA polymerase catalyses the synthesis of DNA that is complimentary to cDNA making a double stranded molecule containing the insulin gene
Define a vector and a plasmid
- virus or plasmid used as a vehicle for carrying foreign genetic info into a cell
- small circular loop of self replicating DNA in bacteria
How are bacteria treated to isolate plasmids?
- EDTA to destabilise cell walls
- detergent to dissolve phospholipid cell membrane
- sodium hydroxide to make alkaline environment which denatures proteins
- plasmids separated from cell debris and the circular DNA is cut open using restriction endonuclease
- vector and gene are mixed, and complimentary base pair to the sticky ends
- gene bound to plasmid by DNA ligaments
- plasmid is now recombinant DNA
Define DNA ligase
An enzyme that joins together portions of DNA by catalysing the formation of phosphodiester bonds between the sugar-phosphate backbone
What makes a good vector?
- self replicating
- small
- not be broken down by host cell enzymes
- not stimulate an immune response
- be able to be screened to confirm the gene is inserted in the vector
- have markers to allow host cells that have taken up the vector to be identified
Describe the transfer of DNA into a host
- number of cells that are taken up by a plasmid can be increased by CaCl2
- positive charge on the Ca ion binds to the negatively charged DNA backbone of plasmids and the LPS membrane
- plasmid DNA passes into cells with a heat shock
What is meant by a clone?
A population of genetically identical cells or organisms formed from a single cell or parent
Describe the method used to test if bacteria have taken up a gene
- antibiotic resistance marker
- blue-white screening used to distinguish which transformed bacterial cells have taken up an empty plasmid - grown on X-gal
- white means plasmid with gene was taken up, blue means empty plasmid
Explain the gel electrophoresis process
- DNA extracted from biological material and cut into thousands of fragments using restriction endonucleases
- DNA fragments separated by length with genetic engineering on an agarose gel
- DNA samples loaded into wells on one end and voltage is applied across the gel
- phosphate groups have a negative charge and so are attracted to the anode, and fragments of known lengths are separated to make a DNA ladder
- separated into strands by flooding gel with alkali and the trough is covered in a nylon membrane which picks up DNA fragments (also known as southern blotting)
- radioactive or luminescent probes that contain sequences complimentary to the STRs attach to fragments by base pairing
- a film that is sensitive to the wavelengths emitted by the probe is placed over the southern blot overnight
- film is exposed and the audio graph reveals a banding pattern in which dark bands show the position of the probe
—> genetic fingerprint
Describe the pros of genetic engineering in bacteria
- medical products (ie mass insulin production from bacteria)
—> safer than older products which were purified from cadavers and transmitted disease - tooth decay
—> oral bacteria strep makes lactic acid and is a major contributor to tooth decay, modified strains that don’t make the acid are available and they outcompete the lactic producing strep, decreasing cavity formation - prevention and treatment of disease
—> bacteria used to produce vaccines and treat disease - enhancing crop growth
- environmental uses
Cons of genetic engineering in bacteria
- plasmids are easily transferred
—> may exchange genes with other bacteria which could lead to potential for antibiotic resistant marker genes to be transferred - fragments of human DNA used to make gene samples and cytoplasmic mRNA used to make cDNA may contain oncogenes or gene switches that activate proto-oncogenes in recipient cells
- a microorganism with a new gene may become a threat if released into a new environment
- a newly introduced gene may disrupt the normal function of other genes in ways not yet understood
Advantages of using reverse transcriptase
- produce many copies of cDNA that is complimentary to the RNA
- no issues with introns and non functional proteins as already removed
- overcome locating problems because mRNA can be extracted
- no chance of cutting into gene and making it non-functional
- no need to further process the RNA to make functional
Explain why introns are more useful for genetic fingerprinting than exons
- introns are more unique to individuals due to STRs
- introns are non-coding pieces of DNA
Briefly describe CF
- genetic disorder cased by the mutation of a single gene which codes for the CTFR protein
—> channel protein which transports chloride ions out and into the mucus, making it watery as water moves in by osmosis - sticky and thick mucus causes problems in gas exchange, reproduction, and digestion
Describe how CF impacts the respiratory system
- build up of mucus in lungs traps bacteria, increasing the risk of infection
- build up of mucus in airways decreases number of alveoli exposed to fresh air, therefore reducing surface area for gas exchange
Describe how CF impacts the reproductive system
- cervical mucus prevents the sperm from reaching the egg
- blocks sperm duct so sperm cannot leave testes
Describe how CF impacts the digestive system
- the pancreatic duct becomes blocked so digestive enzymes don’t reach the small intestine, therefore fewer nutrients are absorbed
- mucus lining the duodenum is very thick which reduces absorption of nutrients
- mucus can cause cysts to form in pancreas and damage insulin secreting cells, leading to diabetes
Differentiate between somatic gene therapy and germ line gene therapy
- somatic: allele introduced to target cells only
- germ line: allele introduced to embryonic cells, so every cell contains the allele
—> somatic is short term and needs repeating, whereas germ line is permanent and will be passed down to offspring
Define gene therapy
The insertion of a normal allele into target cells to replace a faulty allele
How can CF be treated using gene therapy?
- somatic cell therapy
- use of liposomes which serve as means of delivering the gene for a normal CTFR gene into epithelial cells of lungs
- liposomes inhaled and fuse with phospholipid bilayer of the lung cells, DNA enters and is transcribed, the gene remains functional, relieving symptoms
- as soon as epithelial cells that took up the gene are replaced, the treatment needs repeating
Why do we need GM crops?
- rapid increase in population creates challenges to provide enough food for the growing population
—> GM characteristics can aid this
What are the different types of ways crops can be modified?
- increasing nutritional value
- increasing size/yield
- making crops disease resistant
- making crops drought resistant
- increasing shelf life
Describe how gene guns can introduce new genes into plants
- fire small spheres coated with the gene at the plant cells
- some penetrate cell wall and are taken up through the membrane
Describe how electroporation is used to introduce new genes to plants
- electric field increases permeability of cell membranes, enhancing gene uptake
Describe how microinjection can be used to introduce new genes to a plant
- membrane pierced with an ultra-fine needle and gene injected into cytoplasm or nucleus
Describe the bacterial vector Agrobacterium
- Agrobacterium is a pathogenic bacterium that causes tumours
- contains the Ti plasmid which integrates into host plant chromosomes
- contains vir genes which control infection and transfer of T-DNA to the chromosome
—> T-DNA contains the tumour inducing genes that express opines, used by the bacterium as a carbon source
Describe how the bacterial vector Agrobacterium is used to introduce new genes to plant cells
- tumour inducing genes are removed to make a disarmed plasmid and the gene of interest is placed in between left and right borders of the T-DNA
- the Agrobacterium is co-cultivated with target cells to enable modified T-DNA to integrate into the plants chromosomal DNA
- transformed transgenic cells are grown into plants
What enzyme joins DNA strands together?
DNA ligase
Describe how ‘round up ready’ soya beans become herbicide resistant
- round up herbicides contain glyphosate which inhibits the production of amino acids needed for protein synthesis by binding to an essential enzyme, killing the plant
- round up soya beans produce a modified enzyme that does not bind with the glyphosate which then breaks down in the soil to harmless compounds
Describe Bacillus thuringiensis
- contains plasmid with a gene that encodes for a protein that is toxic to immature insects and acts as an insecticide
- known as the cry protein as it crystallises
Applications of the cry protein in tomatoes
- scientist able to locate the cry gene and incorporated it into the cells of Bt tomatoes
- only expressed in the leaves of the plants which are eaten by insects, not the tomato which is eaten by humans
- when insects feed on the leaves, the cry protein destroys their gut wall, enters the blood stream and kills them by food poisoning
What is the benefit of Bt tomatoes making their own insecticide?
- saves money for farmer
- protects non-target species
- prevents eutrophication
Describe how GM has been used to alter the ripening of tomatoes
- tomatoes ripen when they produce the enzyme polygalacturonase which breaks down the pectin in cell walls
- Agrobacterium introduces a second copy of the polygalacturonase gene that has a base sequence complimentary to the normal gene, making it an antisense gene
- mRNA transcribed from the antisense gene is complimentary to the mRNA of the original gene and so they base pair and prevent the original gene being transcribed, blocking the production of polygalacturonase
—> not transgenic!
Describe how GM is being used to reduce the amount of fertiliser used
- nif genes from nitrogen fixing bacteria are being transferred into plants
- allow plants to make their own fertiliser, reducing the amount added artifiically
Arguments for GM of crops
- higher crop yield
—> enables plants to grow in disease, droughts and floods - pesticide reduction
- improved food
—> higher nutritional value, better flavours, longer shelf life - decreased plant loss
—> maintain biodiversity
—> herbicide resistant genes - pharming
—> production of pharmaceutical molecules in GM plants ie antibiotics, hormones etc
Arguments against GM crops
- transfer of herbicide resistance to wild plants generating super weeds
- pest resistance
- marker genes may transfer to bacteria inside the intestines of customers
- organic farming may be compromised by pollen from GM crops
- monopoly of the market
—> few commercial companies will use GM so farmers are very limited
—> could lead to decrease in biodiversity - new proteins
—> adverse health effects from eating crops that express new genes and make new proteins - economic concerns
—> intellectual property law & expenses on the farmers
Describe some of the uses of genetic testing
- carrier screening
—> identify someone who carries recessive allele associated with a genetic disease & can lead to antenatal testing too - pre-implantation genetic diagnosis (screening embryos in IVF)
- pre-natal diagnostic testing
- newborn baby screening
- pre-symptomatic testing for adult onset disorders
- confirmation of suspected disease
- forensic and identity testing
Describe somatic gene therapy
- targets body cells in the affected tissues
- does not appear in future generations as somatic = non reproductive
- needs repeated treatment
What are some of the problems with somatic cell therapy?
- acquiring the human genes for the patients cells
- insertion of gene into right cells
- making sure gene is activated
Describe DMD
- DMD caused by mutation in dystrophin gene which stops individuals from producing dystrophin, a protein that protects muscles
- almost always affects boys - sex-linked disease
- caused by one or more deletions in the gene
—> gene has 79 exons and deletion of any alter the reading frame of dystrophin mRNA
Describe the use of germ-line therapy for DMD
- using the antisense oligonucletoide drisaopersen which binds to the mRNA over the exon with the deletion
- that portion of RNA becomes double stranded and so ribosomes do not translate it
- this restores the reading frame so that a shorter, partially functional dystrophin can be synthesised
—> exon skipping
How is drisapersen delivered?
Subcutaneous injections
What may impact the effectiveness of gene therapy?
- only a small proportion of the introduced genes are expressed
- there may be an immune response in the patient
How can genomics lead to improvements in healthcare?
- more accurate diagnosis
- better prediction of the effect of drugs
—> individual differences affect metabolic pathways so genetic info can inform decisions about doses - new and improved treatments
- NGS technology sequences genomes very quickly
—> individual/personalised therapies
What is the purpose of tissue engineering?
- regenerate injured tissue and produce bio-artificial organs
- hoped to treat diabetes, traumatic spinal injury, DMD, and vision and hearing loss
How are telomeres used in tissue engineering?
- technique to elongate telomeres developed
- cells can be separated from tissues with enzymes and following elongation of their telomeres can be used to grow tissue replacements
Explain autologous cells
- from the same individual
- fewest problems with rejection and pathogen transmission
—> not always available (if ill, old, burnt etc)
Explain allogenic cells
Cells from a donor of the same species
Explain xenogenic cells
- another species
—> pig cells used to develop cardiovascular implants - dangers from viral sequences in pig DNA but research has been able to remove them
Explain syngenic/isogenic cells
Cells from genetically identical organisms
What is meant by scaffolds?
An artificial structure that can support a 3D tissue
What are essential features of scaffolds?
- allow cells to attach and move
- deliver and retain cells and biological molecules
- be porous to allow diffusion of nutrients and waste products
- be biodegradable and absorbed by surrounding tissues - rate of degradation should match rate of tissue formation so that it leaves ‘neotissue’
Define a stem cell
An undifferentiated cell capable of dividing to give rise to daughter cells, which can develop into different types of specialised cells or remain undifferentiated
Describe embryonic stem cells
- found in 3-5 day old embryos
- blastocyst has ESCs which can form every type of cell in the body and the cells that support embryonic development
- described as totipotent
Describe pluripotent stem cells
- derived from ESCs
- form every type of cell in the body (ie a zygote)
Describe adult stem cells
- resident in the ‘stem cell niche’
- replace cells that are lost through normal wear and tear, injury or disease, but cannot form all cell types
- multipotent cells
—> have since been genetically reprogrammed to become induced pluripotent stem cells which can differentiate into any cell type
Describe the potential of stem cells
- tissue engineering to regenerate tissues and organs
—> regenerating bone, developing insulin producing cells, and repairing cardiac muscle - cell-based therapies to treat disease
- screen new drugs
—> stem cells allow drugs to be tested in numerous bodily cell types - develop model systems to study normal growth and identify causes of birth defects
- investigate the events that occur during human development, and how gene switches turn undifferentiated stem cells into differentiated cells, and form tissues and organs
What are some disadvantages of using stem cells?
- techniques for extracting, culturing, and manipulating are still under development and the behaviour of cell cultures is not always predictable
- results of stem cells allow technology are exceedingly rare and expensive
- uses of stem cells are very new and long term studies have not been conducted
—> concerns to premature aging of cells and unpredictable events
Describe the ethical problems surrounding the use of embryonic stem cells
- the source of embryonic stem cells
—> only recently allowed to create embryos purely for stem cell research - prev had to be spare from IVF as considered unethical
—> cannot be legally transferred to a uterus - moral status of the embryo
—> do they have rights? are they considered humans?
—> religious beliefs - potential rights of an embryo vs the large benefits people will gain from research into the treatment
- fear that it could lead to humans being cloned which is illegal
Why is the use of embryonic stem cells in research important?
- clarify fundamental biological mechanisms
- indicate which types of stem cells into will be most useful in cell-based treatments
What is meant by apoptosis?
- programmed cell death
- where telomeres degenerate
How is the shortening of telomeres countered in tissue engineering?
Enzyme telomerase protects the telomere and prevents its degradation
What is tissue culture?
Growth of tissues or cells in an artificial medium separate from the organism
Differentiate between therapeutic and reproductive cloning
Therapeutic: cloning cells to generate tissues
Reproductive: cloning a whole organism (illegal)
Conditions needed by chondrocytes
Low oxygen to mimic their development in skeletal tissue
Conditions needed for endothelial cells
Need shear stress to mimic blood flow in blood vessels
Conditions needed for cardiovascular tissue
Mechanical stimuli such as pressure pulses to stimulate their development
What is the difference between pluripotent and totipotent?
Totipotent - all body cells and embryonic tissue (ie placenta)
Pluripotent - not reprogrammed to make embryonic tissue, only all body cells
What two features of DNA profiles help identification?
Position and thickness
What is the function of DNA polymerase in interphase?
Replication of DNA
Why is it important to use primers which are specific to a certain gene on each chromosome?
Enables specific gene to be amplified
Why is the quantity of genes expressed as a ratio?
- ratio will be the same no matter the number of copies made
Explain the advantage to farmers of having crops resistant to roundup
- round up won’t kill crop but will kill weeds
- reduces competition in field
- therefore increased yield
Explain why environmentalists might have objections to using GM crops resistant to roundup
- increased use of herbicide
- decreased biodiversity may lead to super weeds
