Flashcards
Genetic variation and treating genetic diseases
Flashcards do conjunto (35)
- 1Approximately what % of the human genome is made up of genes?1.5%
- 2What is the rest of the genome made up of?DNA which encodes non-coding RNA (such as miRNA) and evolutionary conserved non-coding regions, enhancer functions, transposons and microsatellites.
- 3How many base-pairs did the human genome project sequence?3.2*10^9
- 4what % of DNA is identical between individuals?99%
- 5How many variants are there in protein coding regions?22,000
- 6How many of those mutations in protein coding regions are heterozygous? Homozygous?13,000
9,000 - 7What is a polygenic disorder?Due to the combined effects of many genes, or in combination with environmental factors (such as diabetes)
- 8What are somatic disorders?Disorders of somatic cells (not germline), such as uncontrolled cell growth, or cancer
- 9What may cause nevis?Mosaic somatic mutations
- 10What is a splice-site mutation?The insertion or deletion of a number of nucleotides in the specific site at which the splicing of an intron takes place
- 11Describe how PCR works (4)1) There are 30-40 cycles of 3 steps: 18-25 nucleotide long oligomers are used to target specific sequences in DNA.
2) S1 - denaturation occurs by heating the DNA for one minute at 94C
2) S2 - annealing (forward and reverse primers) by heating the sample at 45 s at 54C
3) S3 - Extension using dNTPs and Taq DNA polymerase by heating the sample for 2 minutes at 72C - 12How does the amount of DNA increase throughout PCR? Roughly how many copies do you end up with?1) Exponential increased (1st cycle = 2^2, 2nd cycle = 2^3, 3rd cycle = 2^4 etc.)
2) 2^36 - 68 billion - 13What year was Maxim and Gilbert sequencing, and Sanger/Dideoxynucleotide sequencing invented?1977
- 14How does dideoxynucleotide sequencing work?The length of each ddNTP terminated DNA chain is the basis of sequence detection - four reactions are run on polyamide gels; have to physically read the sequence of DNA and is only appropriate for examining a small amount of genes.
- 15When was Sanger-cycle sequencing invented?1986
- 16How does cycle sequencing work? How many base pairs can be sequenced at a time?1) Use a Taq thermostable polymerase. ddNTPs are each labelled with their own fluorophore - this can then be read using a laser and the data can be collected electronically. Uses capillary electrophoresis.
2) 800bp - 17What do ddNTPs lead to?Chain termination - incorporated by the primer, stopping the sequence so that gene is sequenced in fragments.
- 18How many base-pairs are in the entire human genome?3.2 billion
- 19How much electronic storage do the protein coding genes take up?35.9mb
- 20What is next generation sequencing called?Massive parallel sequencing
- 21Describe the steps involved in the generation of an exome profile.1) Fragmentation - ultrasonic shearing breaks genomic DNA into fragments of DNA ~150 bps
2) Library preparation - end repair and 'A' overhang and adaptor ligation.
3) Exome capture - sequence specific biotinylated RNA oligos are used to select the desired coding region
4) Sequencing - low cycle PCR amplifcation
5) Sequence read alignment against a reference genome
6) Target coverage reporting
7) Variant calling
8) Variant annotation - 22What % of the population have a rare inherited disease? How many people is that in the UK?5% - 3 million in the UK
- 23How many rare disorders are there?7,000
- 24What % of rare disorders have a single gene defect?85%
- 25What are the stages of the 100,000 genome project?1) Stage 1 - 100,000 volunteers spit in a tube, health records
2) Recall all of 1,000 volunteers (based on genetic results and health records) a year - Whitechapel centre - volunteer can agree or decline each further stage 2 study - 26What are the long-term aims of the 100,000 genome project?1) Resource many research programs to improve diabetes, cholesterol, heart disease, new ailments
2) Increase health awareness
3) Tackle under-representation of south asians in genetic studies and health research in general - 27At what age do individuals with epidermolysis bullosa usually die and why?Early 20s due to squamous cell carcinomas
- 28What do you need to know to genetically target embryos (3)1) The genetic variant/mutation you want to select to exclude
2) Need to legally be able to select for it
3) In vitro fertilisation followed by preimplantation genetic diagnosis - 29Explain how pre-implantation genetic diagnosis by chromosome or mutation analysis works?1) Egg and sperm are combined using IVF techniques.
2) One or two cells are then removed from the blastomere for testing
3) Healthy embryos are implanted while unhealthy are discarded - 30What is mutated in Harlequin Ichthyosis?A to T
- 31Which mutation causes multiple complicated ulcers of the small intestine?Loss of function mutations in cPLA2a (Phospholipase A2 group, PLA2G4A)
- 32Which cascade does the loss of cPLA2a affect? Describe the cascade. What does this result in a deficit of?1) Eicosanoid biosynthesis (20-carbon essential fatty acids)
2) Phospholipids - arachidonic acid (Omega-6) - a) prostaglandins via actions of COX1/2 or b) leukotrienes by 5-lipoxygenases
c) Deficit of prostaglandins (causes stomach ulcers) and leukotrienes - 33Having discovered that a non-functional cPLA2a group is to cause for the complex ulcers of the small intestine, how is the condition treated?Prostaglandin agonists (such as misoprostol) and diet supplements are used to bypass the defective protein and increase prostaglandin activity.
- 34Why don't drugs work in all patients?Largely dependent on genetic variation in various cellular pathways
1) Variation in absorption
2) Distribution
3) Metabolism
4) Elimination
5) Variability of target receptors
6) "same" disease can be due to independent and diverse pathological and genetic problems - 35Given the card before, what is a key benefit of genetic based medicine?Drug-response testing, can determine which patients need a 'standard' dose, lower dose or a high dose.