Application of genetics Flashcards
What was the purpose of the human genome project?
To improve knowledge and understanding of genetic disorders and consequently improve their diagnosis and treatment.
What were the aims for the human genome project?
To identify all genes in the human genome and identify which chromosome it is on.
Store information and databases.
Improve for data analysis.
Transfer related technology to the private sector to develop medical innovation.
Address the ethical, legal and social issues that might arise when undertaking the project.
How was the human genome project undertaken?
Anonymously and looked at the base sequence of individuals.
What is genomics
It’s the study of the structure, function, evolution and mapping of genomes.
What are the results of the human genome project?
It has helped determine the order of bases in the human genome as well as the identification of some jeans and sequencing and mapping this information enable scientist to scan a patient’s DNA sample for mutated sequences and also to compare the sequence of DNA bases in a patient’s gene to a normal version of the gene.
What form of sequencing did the human genome project use?
Sanger sequencing which sequences relatively small sections of DNA at a time.
What was the 100K genome project purpose?
To improve the knowledge and understanding of genetic disorders and enable healthcare to be improved by
- more accurate diagnosis
- better prediction of the effect of drugs and improved design drugs
- new and improved treatments for disease.
What did the 100 K project involve?
Do you know names from patients with cancer/rare diseases and members of the family. Before the genome was sequenced they had to give formed consent.
What sequencing was used in the 100K project?
Next generation sequencing
What are some advantages of using NGS?
It is efficient as it can sequence an entire genome in just a few hours. It enabled scientists to study variation within the human genome. And it could possibly tailor therapies to individuals for a common disease.
What moral and ethical concerns are there with sequencing your genome?
- Ownership of the information
- Misuse of the data i.e insurance
- Social discrimination against certain ancestry
- Social stigmatisation
Why is the identification of allele sequences are concern?
As DNA could be scanned for mutated sequences which could correlate with future health problems some wish not to have this knowledge but the health problem could affect relatives so must be clear whether or not relatives have the right to information.
What have been genetically screamed to detect the presence of disorders and give examples of some disorders?
Embryos have been genetically screened to detect the presence of disorders i.e CF, Huntington’s disease and thalassaemia.
What is genetic counselling?
It’s when patients meet up with a genetic counsellor on several occasions with a few weeks between each session to allow individuals to think about the consequences of finding out results.
What are the concerns for parents screening children for known diseases?
Some adults wish to screen their child for adult diseases such as Alzheimers and breast/ovarian cancer. Does the parent have the right to keep this info from the child?
What has embryonic screening led to?
Concerns of choosing alleles to ensure specific characteristics.
What do they discuss in genetic counselling?
The number of people with the condition out of the general population.
Whether parents are closely related.
Whether each parent has a history of the condition.
Inheritance of the disease.
Symptoms and treatment.
Life expectancy
How does identifying genomes help with classification?
It enables you to identify close evolutionary relationships and provide a true phylogenetic classification. It could help with conservation of species in the future.
How is malaria trying to be controlled?
Through sequencing the mosquito and the plasmodium parasite that it transmits. Trying to develop chemicals which prevent transmission of malaria.
How was the mosquito modified to prevent the spread of infection?
Genetically modified mosquitoes was produced using technology which allows genes to be written into the genome. Mosquito eggs were modified with genes which would help the synthesis of antibodies against the Plasmodium. The Plasmodium would not survive in the mosquito, meaning a mosquito couldn’t spread the infection.
How is the parasite killed?
Quinine disrupts the plasmodium’s digestion of haemoglobin in red blood cells are toxic to riveted of haemoglobin accumulate and kills the plasmodium.
How has the Plasmodium become resistant to drugs?
Through spontaneous mutations. Some drugs only require one point of mutation to gain resistance what others require more than one.
How does chloroquinine affect the plasmodium
It disrupts the digestion of haemoglobin in the Plasmodium food vacuole. Mutant Plasmodium expelled Chloroquinine from its food vacuole 50 times faster than normal Plasmodium so there’s not enough time for the drug to have an effect.
What is artimesinin?
A drug which acts at the RBC
What has caused difficulty with the eradication of malaria?
Rapid resistance to insecticide
How has sequencing genomes for malaria helped?
It has meant chemicals can be developed, resulting in mosquitoes being susceptible once again to malaria.
How has sequencing Plasmodium sp been beneficial?
it has developed multi drug resistance, so it’s allowing the development of more effective drugs.
How has genetic engineering helped?
It has allowed genes to be manipulated altered and transferred from one organism or species to another.
What are the applications of genetic engineering?
Transfer of genes into bacteria to make useful proteins i.e. insulin.
Transfer of genes into plants and animals so they acquire new characteristics i.e. resistant to diseases. Transfer of genes to humans to reduce the effect of genetic diseases.
What is the recombinant DNA
When genetic material of two species is combined. It is formed when a piece of foreign DNA is incorporated into the circular DNA (plasmid) from a bacterium.
What are transgenic organisms
Organisms which have DNA from other species introduced into their cells
What is the organism called which has been introduced to the DNA
The host
What is it called when a cell has incorporated a plasmid containing a foreign gene?
Transformed
What are the steps of genetic engineering?
- isolation of the gene fragments
- insertion of the DNA fragment into the vector
- vector carries the gene into a suitable host cell
- recipient expresses gene through protein synthesis
- identification of the host cell taking up the gene using gene markers
- cloning the transformed host cell
How can a gene be identified
Using a gene probe
What are restriction enzymes?
They cut the DNA at particular sites into fragments, these form complementary base pairing. The same enzyme should be used when cutting the plasmid/vector to ensure complementary sticky ends. Hydrolyses the sugar phosphate backbone
Why is DNA ligase used?
To anneal the sticky ends together. Binds the sugar phosphate backbone and the plasmid.
What are the human DNA fragments mixed with?
Open plasmids the gene and plasma sticky ends form complementary base pairs and DNA ligase joins together to form recombinant DNA
What are the problems with using restriction enzymes?
Difficult to locate the required gene.
The recognition sequence occurs within the gene of interest the gene will be broken into fragments with no function.
It’s recognition sequence is too far from the start of the gene DNA will contain entrance which the bacteria you’re transferring the chain cannot recognise or cut out.
What is reverse transcriptase?
It’s an enzyme which produces DNA from an RNA template. The enzymes synthesises DNA called cDNA which is complimentary to the mRNA.
What happens to the mRNA when reverse transcriptase is used?
It’s converted into a single DNA strand by reverse transcriptase. DNA polymerase then converts a single strand to a double strand of DNA (cDNA) for pulling into a plasmid.
Producing cDNA overcomes what problems?
Locating the gene.
Restriction enzymes cutting the gene into non-functional fragments.
The presence of introns.
The need for post transcriptional processing to produce functional mRNA.
Why must a vector be used?
To carry the gene into a bacterial cell i.e. a plasmid.
How are plasmids isolated?
The bacteria containing them are treated with
EDTA to destabilise the cell wall.
Detergent to dissolve the phospholipid cell membrane.
NaOH denatures the membrane proteins.
What should a good vector be?
Self replicating
small
not be broken by host cell enzymes
not stimulate an immune response
be able to be screened to confirm that the gene has actually been inserted into the plasmid
have markers to allow cells that have been successfully taken up the gene to be identified.
How can uptake of plasmid DNA be increased?
Through CaCl2 Heat shocking (chilled to 4 and then heated to 42)
What are the 3 types of bacteria which can be formed when making a recombinant DNA?
Bacteria which isn’t transformed
Bacteria which has taken up the plasmid without donor DNA (resealed with DNA ligase)
Bacteria which has taken up the recombination plasmid
What is a marker gene?
Plasma gene for antibiotic resistance. You can use these to identify the bacteria which contains the recombinant plasmid.
Plasmids contain two genes antibiotic resistance what are they?
Ampicillin resistance
Tetracycline resistance
Describe the identification of the recombinant DNA?
Cells are cultured in a growth medium containing the antibiotic and if the plasmid is incorporated they also contain genes antibiotic resistance. They break down the antibiotic and can grow. If they don’t contain a plasmid they don’t contain resistant genes and the antibiotics will kill them.
Which cells die?
If the bacteria hasn’t taken up the plasmid
Which cells survive?
If the bacteria has taken up the recombinant or non-recombinant plasmid.
How is it identified if the recombinant plasmid has been taken up?
The bacterial colonies are transferred onto an agar containing tetracycline. Bacteria containing recombinant DNA would die as they are resistance to tetracycline as this gene was where the insulin gene was inserted.
What happens once recombinant DNA is found?
It is cloned resulting in multiple copies of the recombinant gene. The bacteria undergoes protein synthesis to form insulin which is isolated from cells and purified.
Advantages of recombinant DNA technology?
No limit to how much protein can be synthesised. Complicated protein structures can be synthesised except by living cells.
No need to extract proteins from mammalian organs.
Disadvantages of recombinant DNA technology?
Difficulties involved in identifying the genes of value in a huge genome.
Synthesis of required protein may involve several genes each coding for a polypeptide.
Treatment of human DNA with restriction enzyme produces millions of fragments which are of no use.
Not all eukaryote genes will express themselves in prokaryotic cells.
What are the advantages of genetic engineering in bacteria?
- Medical products, large amounts of pure human products/proteins for use in medicine have been made i.e. insulin.
- Reduction in cavity formation modified strains don’t produce lactic acid which outcompete the other bacteria.
- Prevention and treatment.
- Enhancing crop growth.
- Environmental use: detecting and removing environmental hazards
What are the disadvantages of genetic engineering in bacteria?
- The use of antibiotic resistance genes and plasmids and the ready exchange of genetic material between bacteria.
- The transfer of antibiotic resistance to pathogenic bacteria.
- Newly introduced genes may disrupt the normal function of other genes.
What are the potential hazards with genetic engineering in bacteria?
Bacteria readily exchanges genetic material. Deliberate use of antibiotic resistance genes and E. coli which lives in the human gut means that these genes could be accidentally transferred to human pathogens, the possibility of transfer of DNA with linked pathogenic genes for example oncogenes increasing cancer risk.
How has disease resistance come about?
Certain species of bacteria naturally attack damage plants and stimulate the growth of a tumour scientist can replace the tumour forming genes with useful genes.
What is electroporation?
An electric field which increases cell membrane permeability enhancing gene uptake.
How are genetically modified crops produced?
Plasmid extracted from the Agrobacterium turnefaciens. Restriction enzyme is used to cut the plasmid and remove the tumour forming gene.
The DNA section responsible for the disease resistance is located and isolated using endonuclease.
The gene is inserted into the plasmid replacing the tumour forming gene.
The DNA ligase anneals these together.
Bacterial cell is introduced into the plant, which divides and the gene in inserted into the plant chromosome.
The transgenic plant cells grow in tissue culture and transformed crops are regenerated.
How has genetically modified soya bean created?
By the addition of the gene into the soya bean which allows the bean plant to be resistant to cytotoxic affects of Roundup. Farmers can spray the fields with Roundup which kills only the weeds and doesn’t inhibit growth.
What is some negatives of genetically modified soya?
Abnormal mortality is in newborns and severe reduction in fertility. Deforestation and increase flooding.
Herbicide resistant soya has led to socioeconomic and environmental problems
How has genetically modified tomatoes been created?
Tomatoes ripen once polygalactinoase is produced which breaks down pectin, a gene was introduced which was complementary to polygalactinoase. The introduced gene has a complementary mRNA strand produced by the original gene. These combine to form a double strand which prevents translocation of the original strand and production of the enzyme. Making them last longer.
What are the benefits of genetically modified crops?
High crop yield.
Superior keeping qualities.
Pesticide reduction: genes for pathogen resistance and insect attack
Improved food
Herbicide resistance, decreases crop loss
Pharming - modified to make proteins/antibodies, hormones and blood products
Addition of N fixing bacteria - reduce fertiliser use
What are the concerns of genetically modified crops?
Dispersal of pollen from crops engineered for herbicide resistance to wild relatives.
Unknown effects of eating new protein produced in the crop.
Reduction in biodiversity: decreases the range of potentially useful genes.
Organic farm produce may be compromised
What are exons?
Regions of DNA which codes for proteins
What are introns?
Regions of non-coding DNA which contain blocks of repeated nucleotides called short tandem repeats
What does the number of STR indicate?
The variation between individuals and the number is used to build up a unique fingerprint. They enable us to see differences between individuals as they don’t code for proteins so have no observable differences. The coding regions are generally identical between all individuals.
What is the function of PCR?
It is used to amplify small sections of DNA rapidly. A large number of copies of specific fragments of DNA are produced using semi conservative replication. This enables test to be carried out on very small samples accurately and more rapidly regardless of the age of the sample. Modern development of the technique have increased routine use
Give an example of a STR?
D7S280, is where the GATA bases repeat on chromosome 7, different alleles have from 6-15 tandem repeats, the more repeats the larger the fragment.
What is used to amplify STR?
PCR is used to amplify the ST eyes by using a primer (single-stranded DNA) which is complimentary to the start of the sequence. These are needed in the sequencing reaction and polymerise reaction to bind to a section of DNA because DNA polymerase enzymes can only combine free nucleotides to an existing DNA backbone
Give the steps of PCR?
- Heating to 95 to separate the strands and break the H bonds
- Cooled to 55 degrees to allow primers to anneal to the strand
- Heat to 70 which allows taq polymerase to add complementary nucleotides by forming phosphodiester bonds in the backbone.
- Cycle repeated, producing millions of copies.
How is PCR different from DNA replication?
Can only replicate small DNA sequences not entire DNA.
Requires primers.
A cycle of heating and cooling to separate them by the strands: DNA helicase is used in DNA replication.
What are the uses of PCR?
Classification of organisms mutation detection sequencing cancer research DNA fingerprinting genetic engineering prenatal diagnosis pathogen detection gene matching drug discovery
What are limitations of PCR?
- Even the smallest contamination with DNA could affect amplification.
- Error rate – DNA polymerase sometimes insert a nucleotide containing the wrong base.
- Taq polymerase can’t proof read.
- DNA fragment size – more efficient for DNA fragments of 1000 to 3000 bases long.
- Sensitivity to inhibitors
- After 20 cycles the process slows down because reagent concentrations become limiting enzymes denature after repeated heating.
- DNA in high concentrations causes single-stranded molecules to base pair with each other rather than primers.
What is gel electrophoresis?
A method of separating DNA fragments according to size. The gel is made from agarose which contains pores in its matrix, allowing small molecules to move through.
Where does the DNA travel to?
As a voltage is applied across the gel the negatively charged fragments (PO4-) migrate towards the positive electrode. Smaller fragments move more easily through the pores so migrate through the gel faster than larger fragments smaller fragments travel further.
How can fragment size be estimated?
By running a DNA ladder (which contains fragments of a known size) alongside samples in the gel.
Why are radioactive DNA used?
They used to produce banded patterns which are effectively unique to an individual. They attach to specific single-stranded sequences and any unbound probes are washed off.
What do the dark bands indicate?
The dark bands indicate the position of the radioactive probe
Why is NAO H used in gel electrophoresis?
To separate the DNA strand into single-stranded fragments, the gel is then covered with a nylon membrane which touches and picks up DNA fragments – southern blotting.
What do the patterns of bands and electrophoresis indicate?
The genetic fingerprint
What are used to indicate and confirm the identity of an individual?
The position and thickness of the band of DNA.
Where can a sample of DNA be obtained from?
Blood, cheek epithelial cells from the mouth lining or cells clinging to the root of hair.
What are the uses of DNA profiling?
Paternity – DNA is taken from the white blood cells to construct a DNA profile. The bands in the child’s profile are compared with the mothers any shared bands are inherited from her the remaining bands in the child’s profile must be inherited by the dad.
Twins - monozygotic have identical banding profiles where is dizygotic twins don’t
Siblings
Immigration – some Visa applications depend on proof of relatedness
Forensic use – to identify and rule out suspects
Phylogenetic studies – profiles of members of different taxa can be compared to determine if they’ve been classified correctly and determined genetic relatedness.
What are the pros of DNA profiling?
Non-invasive methods to obtain biological samples.
Can be used on samples too small for blood testing. Reverse wrongful convictions when used with other forensic tools.
Can rule out non-matches of DNA samples to exonerate people who have been falsely accused.
What are the cons of DNA profiling?
Requesting DNA samples is a violation of an individuals right to privacy.
DNA profiles are held on a computer database vulnerable to misuse and hacking.
They offer possibilities not absolutes.
Access to and use of genetic data must be carefully regulated insurance companies could deny coverage or claims. Employers could involve eight applying individuals who have certain genetic traits or risks of illnesses.
Data profiling may produce wrongful convictions.
How can genetic diseases be treated?
By replacing genes or replicating the function of genes with drugs.
What is gene therapy?
To treat genetic diseases by inserting functionalists; DNA sequences into cells to counteract the effect of the effective gene ( new DNA sequence replaces the faulty gene)
What does gene therapy use
A virus as a vector
Plasmid as a vector
infection of naked plasmid DNA
What are the 2 methods for GT
SOMATIC CELL - targets body cells of affected tissues, new genes aren’t inherited, so can’t prevent it from being passed on.
GERM LINE GENE THERAPY- gene inserted into embryo or gamete, all new cells contain the new gene, the correction is inherited. Ethical issues have meant it hasn’t been performed yet.
What is Duchenne Muscular Dystrophy
It is a recessive sex linked form of muscular dystrophy affecting up to one in 3500 live male births caused by mutation in the dystrophin gene.
What is DMD caused by
It’s caused by deletion which introduces the stop codon too soon so protein dystrophin is not produced. Dystrophin is a structural protein in in muscle so people with DMD have muscle loss and come wheelchair-bound by the time they are teenagers. It causes progressive muscle weakness due to lack of protein muscle fibre breakdown. This is replaced by fibrous and fatty tissue causing gradual muscle weakness. The life expectancy is 27 and the usual cause of death is respiratory complications.
How is DMD treated?
Excellent skipping aim to treat DMD by introducing a molecular patch. By using a drug called drisapersen the patch over the exon that contains the mutation makes the gene readable again. A shorter form of dystrophin is produced but one thought to be more functional than the untreated version. The molecular patch binds to the pre-mRNA over the Exxon with the deletion. They are a neighbour comes double-stranded again and x150 one is spliced out removing the stop codon. A shorter form is produced.
Describe the effectiveness of gene therapy?
Only a small proportion of the introduce genes are expressed. It initiates an immune response.
What are the advantages of somatic gene therapy?
Relief of symptoms. No need for medication. Prevent the development of cancer. Doesn't permanently damage genome. No negative effects would be inherited.
Disadvantages of somatic?
More than one treatment required.
Difficult to get the gene to integrate into the chromosome to function correctly.
Genetic disorder can still past offspring.
Long-term affects unknown.
Immune response to use of viral methods
Advantages of germline?
Children can be born free of genetic diseases
What are the disadvantages of germline?
Maybe used to modify the characteristics of a child not used just to treat does genetic disorders.
Permanent modification raises difficult moral ethical and social issues.
Could create new diseases
Advantages of gene therapy?
More accurate diagnosis can be made.
Better prediction of the effect of drugs.
New improved treatments for disease.
NGS technology sequences genomes quickly and allows individuals to have therapy is based on the DNA sequence.
What methods are used in tissue engineering?
Biochemistry, self biology, engineering and materials science to repair, improve or replace biological functions. It has allowed the replacement of many tissues and organs.
What are autologous cells?
From the same individual, serious problems with rejections and pathogen transmission
What are allogenic cells?
From a donor of the same species
What are xenogeneic cells?
From another species
What are syngeneic or isogenic cells?
From genetically identical organisms
What are scaffolds and what must they do?
These are cells which are seeded onto a scaffold
It must:
- Allow cells to attach and move
- Deliver and retain cells and biological molecules
- Allow diffusion of nutrients and waste products
- Biodegradable and absorb surrounding tissues. Degrades proportional to formation
What is a stem cell?
A stem cell is an undifferentiated cell capable of dividing to give rise to cells which can develop into different types of specialised cells
What type of stem cells are there?
Embryonic stem cells are found in 3-5 day old embryos, the blastocyst has ESC which forms every cell type in the body.
Adult tissues e.g bone marrow, muscle and brain contain SC, residing in a specialised area - stem cell niche. Replacing cells which are lost through normal wear, injury and disease. CANT form all cell types.
What can stem cells be used for?
- Tissue engineering to regenerate tissues and organs.
- Cell based therapies in which SC different into specific cell types, replacing cells and tissues to treat diseases. e.g heart disease
- To screen new drugs
- To develop model systems to study normal growth and identify causes of birth defects.
To investigate human development and how gene switches undifferentiated stem cells into differentiated ones.
Advantages of SC
Embryonic stem cells can become any cell type, adult stem cells for more limited.
A blastocyst contains about 100 ESC.
ESC grows easily in culture and large quantities can be readily produced.
Speed of production and large-scale production.
SC be used to resolve organs shortage problems.
A patient with ESC less likely to need a new suppressive drugs.
A A patient with ESC less likely to need a new suppressive drugs. A patient and patient’s own adult stem cells are less likely to be rejected after transplantation. Production of genetically identical/organisms.
A patient’s own adult stem cells are less likely to be rejected after transplantation.
Production of genetically identical cells/organisms
Disadvantages of SC
In mammals the technique is expensive and unreliable.
In plant disease, entry of pathogens may cause problems.
In adverting selection of disadvantageous alleles.
Long term/unforseen effects ie premature ageing.
What are the requirements for SC?
- Any SC must be able to be used in different projects
- Deposited in SC bank to be used by other research group.
- No financial reward
- Donors must give specific consent for ESC to be used.
What are the ethical issues of SC?
- ESC used contravenes with the principle that human life shouldn’t be created as a means to end.
- That the embryo has human rights as new life starts at contraception, the foetus rights increase as it develops.
- The use of ESC is unjustified as adult stem cells can be used.
- Cloning- research knowledge could be used to clone humans
Why are ESC important?
Clarify biological mechanisms
Indicate the type of SC most beneficial in treatment
A pre 14 day embryo has no possibility of independent existence
Why do people undergo genetic screening?
To determine the nature and inheritance of a genetic condition
Why is genetic screening useful?
Confirm diagnosis
Indicate appropriate treatment
Allow families to avoid having children with disease
Identify high risk people for illnesses which are preventable
What are the concerns for genetic screening?
Invasion of privacy
Defective alleles identified in prenatal tests could increase abortion rate
Individuals could be placed in high risk group for insurance
Uses for GS?
- Carrier screening to identify if the unaffected person carries the the recessive allele
- Preimplantation genetic diagnosis to screen embryos generated in IVF
- Pre natal diagnosis testing
- New born screening
- Pre symptomatic testing for predicting adult onset diseases
- Confirmation of a specific disease
- Forensic and identity testing
What are the limitations of gene tests?
- Not regulated, independently verified
- Only a small number of genes in the human genome
- Difficult to interpret a positive result, some carry the mutation never develop the disease.
- Lab errors
- Anxiety
- Discrimination and social stigmatisation who has taken the test.
