popgenspring2011 - BSC 2010L BSC Population Genetics I In...

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Unformatted text preview: BSC 2010L BSC Population Genetics I In this lab, we will: In A. Set up PCR samples for Human Set Genetics exercise. Genetics B. Discuss Hardy-Weinberg Equilibria C. Review single-gene human genetic traits Review PCR • 2 ways to get many copies of DNA: – Cloning (plasmids) – Amplification (PCR) • Components of PCR: – – – – – – DNA polymerase Oligonucleotide primers Template DNA (cheek cells) Dioxynucleotides (dNTP’s) Buffer Mg2+ PCR involves repeated cycles of a 3 step process: step 1. Denature--PCR mixture is heated to around 95°C to separate the complementary DNA strands from each other. 2. Anneal--temperature is reduced to 50-60°C to allow the primers to bind to the template DNA. 3. Elongate--temperature is raised slightly to about 70°C to allow the polymerase to replicate the template DNA starting at the primers. See http://www.sumanasinc.com/webcontent/anisamples/molecularbiolog See http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/ Procedures Procedures 1. We will use your cheek cell DNA to We amplify a short DNA sequence called an Alu element. Alu 2. Use PCR to get lots of copies of your 2. Use DNA DNA 3. Use electrophoresis to determine each Use person’s genotype with respect to the Alu insert First Week Procedure First • • • Remind the students to use sterile technique - wear gloves. Remind wear Remind students how to use the pipettes DNA Extraction a. a. b. I have aliquoted 25 µ L iif master mix into a sterile 0.5 mL f have microcentrifuge tube for each of you. microcentrifuge Student will scrape inside of cheek with a toothpick and insert Student swab into master mix and mix vigorously. and Label tube and place in PCR machine. What is in the Master Mix What 1. GoTaq Hot Start Green master Mix (12.5µL) 2. Upstream primer (1µL) 3. Downstream primer (1µL) 4. Nuclease free water (10.5µL) Thermocycler Thermocycler a. Mark your tubes b. Place them in the thermocycler. Program of the thermocycler Program a. 94oC, 2 min (break cells and denature DNA) C, b. 94oC, 30 sec b. C, c. 54oC, 30 sec c. C, d. 72oC, 30 sec - loop step b-d 39 times d. C, e. 72oC, 5 min (allows DNA polymerase to e. C, finish elongation) f. 4oC, pause f. C, Duration: 3 Hours and 10 minutes • We will continue the lab next week – You will run their amplified samples with gel electrophoresis to determine your genotype Electrophoresis: separates DNA of different sizes Ladder Ladder How to read the final gels: How Population genetics Population • Evolution = the change in allele frequencies through time – a change from generation to generation – for groups called populations • Population = a group of individuals of the same species – living in the same area so they have the potential to interbreed • Allele frequencies = the proportions of different alleles of a gene in a population Allele Frequencies of Populations (assume these are haploid moths) Population A Population B Pop. Size: 1,000 Proportion red allele: 500/1000 = 0.50 Pop. Size: 100 Proportion red allele: 60/100 = 0.60 Hardy-Weinberg Law Hardy-Weinberg • If all other factors remain constant, the frequency of particular alleles will be constant from generation to generation = genetic equilibrium. Hardy-Weinberg Law Hardy-Weinberg • If all other factors remain constant, the frequency of particular alleles will be constant from generation to generation = genetic equilibrium. • Evolution = the change in allele frequencies through time – Evolution: occurs when genetic equilibrium is upset • Gene frequencies of a particular generation depend upon the gene frequencies of the previous generation and not upon the genotype frequencies. Factors that upset genetic equilibrium a. Nonrandom mating b. Migration = gene flow c. Selection d. Mutation e. Small population size = genetic drift Assumptions of Hardy-Weinberg Equilibrium Equilibrium Conditions for genetic equilibrium: a. random mating b. very large population = no genetic drift c. no selection: all genotypes are equally viable d. no mutation e. no migration = no gene flow Gene flow vs genetic drift Red morph Orange morph Gene flow = Migration Population A Population B proportion of red allele: proportion of orange allele: proportion of red allele: proportion of orange allele: Gene flow = Migration Population A Population B proportion of red allele: proportion of orange allele: proportion of red allele: proportion of orange allele: Gene flow = Migration Population A Population B proportion of red allele: proportion of orange allele: proportion of red allele: proportion of orange allele: Gene flow = Migration Changes allele frequency because individuals move, with their alleles, b/w pop’s Population A Population B proportion of red allele: proportion of orange allele: proportion of red allele: proportion of orange allele: Genetic Drift and Infinite Population Size Population A Population B Pop. Size: 1,000 Proportion red allele: 500/1000 = 50% Pop. Size: 10 Proportion red allele: 5/10 = 50% Population A (1,000 moths) 5 red moths randomly killed by bus Population B (10 moths) 5 red moths randomly killed by bus Moth populations after bus passes by: Population A Population B Pop. Size: 995 Proportion red allele: Pop. Size: 5 Proportion red allele: Moth populations after bus passes by: Population A Population B Pop. Size: 995 Proportion red allele: 495/995 = 49.7% Pop. Size: 5 Proportion red allele: 0/5 = 0% Genetic Drift and Infinite Population Size • Genetic drift is random change in allele frequencies • Magnitude of effect of random changes on population is dependent on population size • 2 common ways of getting small populations that are more prone to the effects of genetic drift • Population bottleneck • Founder effects – In the process of becoming small, allele frequencies usually change and some alleles can be lost = more genetic drift Genetic Drift: Bottleneck effect Genetic Drift: Bottleneck effect Source: brooklyn.cuny.edu Genetic Drift: Founder effect Source: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=glossary&rendertype=box&id=further_illus-63 Natural selection Mutation Random Mating Assumptions of Hardy-Weinberg Equilibrium Equilibrium Conditions for genetic equilibrium: a. random mating b. very large population = no genetic drift c. no selection: all genotypes are equally viable d. no mutation e. no migration = no gene flow H/W Equation • H/W equation predicts the number of individuals of each genotype if the population is in equilibrium – Need to know the allele frequencies in the population – Plug those allele frequencies into the equation to get the expected genotype frequencies for a population in equilibrium – Compare those predicted genotype frequencies to the observed genotype frequencies – Remember: gene frequencies of a particular generation depend upon the gene frequencies of the previous generation and not upon the genotype frequencies How do we get allele frequencies? • If you know the genotype of all the individuals in the population, then you can calculate the allele frequencies • For example: – – – – – 25 individuals are AA 17 are AB 30 are BB What is the allele frequency of A? What is the allele frequency of B? Calculating Allele Frequencies Genotype AA AB BB Totals: # of individuals 25 17 30 ___ #A __ __ __ ∑ ___ #B __ __ __ ∑ ___ A allele frequency = p = ∑A / (# of individuals x 2) B allele frequency = q = ∑B / (# of individuals x 2) To relate allele frequencies to genotypic frequencies for 2 alleles (A & B): 1. p = proportion of the A allele q = proportion of the B allele 2. since there are only two alleles: p + q = 1 – and so: p = (1-q); and q = (1-p) (1-p) 3. Hardy Weinberg Equation: p2 + 2pq + q2 = 1 4. p2 = probability (proportion) of homozygous ‘AA’ 2pq = probability heterozygous ‘AB’ q2 = probability homozygous ‘BB’ To test if a population is in HardyTo Weinberg equilibrium • Compare observed genotypes with expected genotypes 1. To get the expected number of each genotype: – p2 x # individuals in sample = expected number of AA individuals. individuals. – 2pq x # individuals = expected number of AB individuals. – q2 x # individuals = expected number of BB individuals. 2. Compare the expected genotypic numbers obtained Compare above with those observed from the gel electrophoresis. electrophoresis. – Use a chi-square test • Note that in a chi square test you must compare numbers of Note individuals, not frequencies. individuals, – Degrees of freedom are equal to 1. (If p + q =1, once you (If know either p or q, you know the other.) know Hardy-Weinberg Equilibrium • If evolution did NOT occur then within 1 generation alleles in population will be distributed according to equation: p2 + 2pq + q2 = 1 • All we need to know are the frequencies of each allele in the population – then see if the observed genotype frequencies match those we predicted from HW equation Is our population in Hardy-Weinberg Equilibrium for the Alu insert? Equilibrium Alu Insert primer Alu insert (300bp) primer 400 bp Alu insert missing primer primer 100 bp Is our population in HW Equilibrium for the Alu insert? How will we test this? Lab Overview 1. Get DNA samples 2. Amplify DNA – PCR 3. Genotype samples – Gel electrophoresis 4. Use genotype data to calculate H/W statistics 5. Compare observed genotypes with expected H/W genotypes Observable human traits Observable Single locus, two distinct alleles 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. PTC tasting positive - autosomal dominant PTC Sodium benzoate tasting positive - autosomal dominant Sodium Widow's peak pointed - autosomal dominant * Widow's pointed Dimpled chin dimple - autosomal dominant * Dimpled dimple Convex nose convex - autosomal recessive * Convex convex Tongue rolling roll - autosomal dominant Earlobe attachment attached - autosomal recessive Interlacing fingers left over right - autosomal dominant Hitchhiker's thumb bent - autosomal recessive Relative finger length second finger shorter – sex-influenced Relative Bent little finger bent - autosomal dominant, sex-influenced * bent Mid-digital hair hair - autosomal dominant Mid-digital Pigmented irises blue, gray - autosomal recessive Pigmented Red/green color blindness positive - X-linked recessive Red/green ABO blood types codominance ABO Human genetics worksheet (5 pts) (5 A. Complete the table. (May need help of a partner A. to score some traits.) to How individual is an individual? How Diseases due to single-gene traits: traits: A. Cystic fibrosus A. Cystic B. Phenylketonuria (PKU) B. Phenylketonuria C. Galactosemia C. Galactosemia D. Huntington's disorder D. Huntington's E. Sickle cell anemia E. Sickle F. Hemophilia A F. Hemophilia Complete the Population Genetics prelab Assignment before the next lab. before ...
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This note was uploaded on 04/24/2011 for the course BSC 2010L taught by Professor Herrerabaerbolker during the Spring '08 term at University of Florida.

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