Half of Gene Technology Flashcards
How and when was the first ‘designer baby’ created?
Officially known as pre-implnation genetic diagnosis (PGD), first performed in 1989. Father’s sperm is mixed with mother’s oocytes (standard IVF). At eight-cell stage, one cell is removed, and DNA analysed to predict whether embryo would have the disease. Embryo not carrying disease causing allele was implanted, and the rest discarded.
Describe aminocentesis
Ultrasound scanning to visualise foetus, and position of placenta, foetus and umbilical cord. Suitable point of insertion is chosen and marked on skin. Ultrasound probe placed so inserted needle will show up as a bright spot. Skin is swabbed with disinfectant at insertion point. 20ml of amniotic fluid is wihdrawn using sterile hypodermic synringe with narrow needle. Insertion site protected frmo ifnection by dressing.
Used for karotyping to look for chromosomal mutations, and checking health of foetus. 15-16 weeks.
Slight increased chance of miscarriage.
Describe chorionic villus sampling
Small sample part of placenta (chorion) removed by narrow needle, procedure monitored by ultrasound scanning.
Slgiht increase in miscarriage rate. Allows earlier warning of genetic abnormalities than amniocentesis. 10-13 weeks.
What is ELECTROPHORESIS for?
Separates DNA fragments of different sizes for genetic profiling and sequencing.
How is electrophoresis carried out? (5)
1) Region of genetic variance chosen. Contains variable numbers of repeated DNA sequences or VNTRs (VARIABLE NUMBER TANDEM REPEATS).
2) Quantity of DNA increased using PCR (POLYMERASE CHAIN REACTION)
3) DNA chopped into pieces using restriction enzymes known to cleave it close to VNTR regions.
4) DNA put into wells in AGAROSE gel. A direct current is passed continuously through the gel. DNA fragments attracted to the anode.
5) Shorter strands of DNA move further through gel, DNA fragments separate according to length.
How is the DNA in agarose made visible for genetic profiling? (5)
1) DNA transferred onto absorbent paper placed on top of gel
2) Paper heated to make two strands of DNA separate
3) Short sequences of single stranded DNA (PROBES) added with complementary base to VNTR regions.
4) Probes contain radioactive phosphorous isotope, and pair up with separated DNA strands on paper.
5) Paper placed on X-ray film, radiation makes film go dark.
How is electrophoresis used to sequence a gene? (eg plant) and how is PCR performed? (7)
1) Leaf cells crushed, DNA precipitated, washed, resuspended in soln
2) Primer added to DNA (short section of DNA with complementary code to start of the gene)
3) Free nucleotides and enzymes added to DNA and primer. many copies of teh gene are made, starting at the primer.
4) Modified free nucleotides are added, they have a dye that will fluroesce when illuminated, joined onto them, and are also doubly deoxidised (stops reaction when it happens to be joined to the growing DNA).
5) Results in may copies of DNA of different lengths, each with a coloured ‘tag’ identifying it. There are as many different lengths as the number of bases in the gene.
6) Mixture of DNA pulled along capillary tube by electrophoresis, shortes lengths move fastest. Near end, a laser shines on tube and the ‘tag’ colour for each band of DNA is recorded automatically.
7) Computer reads sequence of colours that pass the ‘window’ (short lengths arrive first) so it is in order.
What is cystic fibrosis and how does it affect a patient?
It is a genetic disease in which abnormally thick mucus is produced in the lungs (and other parts of body). An affected person is prone to bacterial infection (as mucus removal is difficult, allowing bacteria breeding), and needs daily therapy to help cough up mucus. Also blocks pancreatic duct (patients take pancreatic enzymes by mouth), and 90% men are sterile )thick secretion blocks ducts in reproductive system).
What causes cystic fibrosis?
RECESSIVE allele of gene which codes for a faulty CFTR (transporter protein). Mucus stays thick and sticky. Mutations in the CFTR gene (chrom 7) produces several different defect alleles.
Most common is deletion of 3 bases. CFTR protein made is missing one amino acid. Cell recognises its not the right protein, and protein isnt placed in cell membrane.
What is the function of the CFTR protein?
Sits on cell surface membranes of alveoli cells (and elsewhere), allowing chloride ions to pass out of cells. Normally, cells lining airways/lungs pump Cl- ions through channel in cell surface memb formed by CFTR, resulting in high concn of Cl- outside cell. WP out side cells are reduced, and water moves out of cell by osmosis, mixing with mucus and making it thin enough for easy removal by sweeping movements of cilia.
Describe the attempts at gene therapy trials for cystic fibrosis (3)
1) UK - Normal alleles inserted into liposomes (tiny balls of lipid), and sprayed as aerosol into noses of 9 volunteers. Succeeded in introducing gene, but effect only lasted for a week as the cells had a short life span.
2) US - Gene introduced into normally harmless viruses, and used to carry gene into gas exchange system. Gene did enter some cells but patients experienced unpleasant side effects (infection by virus).
3) Recently drug used to treat one type of cystic fibrosis where a stop codon is wrongly placed in the middle of the gene. Drug PTC124 allows translation to go across stop codon so entire protein is made (though with a missing or wrong AA).Hopeful signs. Easier than ‘classic’ gene therapy as it only involves one pill a day.
What does gene therapy need to achieve to be used a treatment?
Gene needs to get to many cells throughout respiratory system, including ones that divide to form new surface cells. So far not achieved.
When would a couple be referred to a genetic counsellor? (5)
1) Either has a genetic disease.
2) Belongs to high risk group
3) History of miscarriages
4) Woman is over 38 (high risk of Down’s syndrome)
5) Part of management of high-risk pregnancy
What is the role of a genetic counsellor?
Provide couple with information and help them understand risks of having their child. Will encourage them to make own decision based on information.
What is genetic screening?
The analysis of a person’s DNA to check for the presence of a particular allele. Can be done in adults, foetus or embryo (uterus or newly formed in IVF).
