1. IDENTIFICATION OF POLYMORPHIC ALLELES 14.04.09

2. Quiz If you are to prepare a %3 agarose gel, what should be the amount of agarose in the 75 ml 1xTAE buffer? Show your calculations. If you are to prepare a %3 agarose gel, what should be the amount of agarose in the 75 ml 1xTAE buffer? Show your calculations. If you cross a heterozygous wildtype with an ebony mutant what will be the phenotypic ratio of your F1 generation? If you cross a heterozygous wildtype with an ebony mutant what will be the phenotypic ratio of your F1 generation?

3. Quiz If you are to prepare a %3 agarose gel, what should be the amount of agarose in the 75 ml 1xTAE buffer? Show your calculations. If you are to prepare a %3 agarose gel, what should be the amount of agarose in the 75 ml 1xTAE buffer? Show your calculations. In 75 ml for %1 percent: 0,75 gr %3 percent: 2,25 gr

4. e+ eee+e eee+e 1:1 ebony: wildtype

5. Answer: e+ eee+e ++e++ F1 generation++e+e+e e+e+e F2 generation Phenotype ratio  3:1, wild-type:ebony Genotype ratio  1:2:1, homozygot WT, heterozygot WT, homozygot ebony

6. Today’s experiments: You will work as 4 groups First prepare 2% agarose gel 1. Weigh 0,8 gr agarose into a flask 2. Put 40ml 1X TBE buffer 3. Dissolve the agarose completely by heating in the microwave 4. When boils, remove the flask from the microwave 5. Waits until it cools to 55C 6. Add 2.5µl EtBr and mix 7. Pour the melted agarose into gel apparatus 8. Let it to harden 9. Mix 5 µl of loading dye+ 5 µl of DNA sample, load on agarose gels 10. Run agarose gels at 150 V for 15 min. 11. Observe under UV light

7. Electropherosis: A method to seperate, identify and purify DNA fragments 2 types of gels: 1. Agarose gels 2. Polyacrylamide gels

8. Agarose Gels: From sea weed Cheap, non—toxic Low resolving power, but a higher range (200bp-50kb) Run in horizantal configuration In order to observe, EtBr is used EtBr intercalates DNA and it flouresces under UV light, so we can detect the location of the DNA fragments on the gel

9. Agarose Gels: Agarose gel electrophoresis unit

10. Agarose Gels:

11. Polyacrylamide gels: Highly toxic, synthetic chemicals Prepared with acrylamide and bisacrylamide. In the presence of free radicals, it polymerizes into long chains

12. Polyacrylamide gels: By changing acrylamide and bisacrylamide ratio, you can change the size of the pores

13. Polyacrylamide gels: High resolution power, but a shorter range (5bp-500bp) Vertical configuration

14. Polyacrylamide gels:

15. In order to visualize DNA, silver staining method can be used Ag + ions bind to (-)ly charged DNA, Reduced to Ag which has a brown color

16. Polymorphisms: Common variation in DNA sequence It is a kind of variation related to biodiversity, genetic variation, and adaptation Presence of more than one genetically distinct type in a single population Useful tools in genetic studies for linkage analysis, prenatal diagnosis, criminal cases and paternity tests 1. RFLP (restriction fragment length polymorhism) 2. VNTR (variable number of tandem repeats)

17. RFLP: Restriction enzymes can recognize and cut specific DNA sequences Ex: Msp I enzyme can recognize CCGG Cfo I enzyme can recognize GCGC EcoR I can recognize GAATTC

18. RFLP: -/- +/- +/+

19. RFLP:

21. VNTR: (variable number of tandem repeats) Can be found on many chromosomes, and often show variations in length between individuals Each variant acts as an inherited allele, can be used for personal or parental identification Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting.

23. Today’s experiments: You will work as 4 groups First prepare 2% agarose gel 1. Weigh 0,8 gr agarose into a flask 2. Put 40ml 1X TBE buffer 3. Dissolve the agarose completely by heating in the microwave 4. When boils, remove the flask from the microwave 5. Waits until it cools to 55C 6. Add 2.5µl EtBr and mix 7. Pour the melted agarose into gel apparatus 8. Let it to harden 9. Mix 5 µl of loading dye+ 5 µl of DNA sample, load on agarose gels 10. Run agarose gels at 150 V for 15 min. 11. Observe under UV light

24. Today’s experiments: You will make a paternity test 1) On each bench, you have 5 DNA samples: mother, child and 3 father candidates 2) We will identify the father by checking 2 polymorhisms on different chromosomes 1. group  RFLP (on chromosome 2) 2. group  VNTR (on chromosome 5)

25. Today’s experiments: 1. group  RFLP (on chromosome 2) DNA fragment includes a Single nucleotide polymorhism (G or T) Msp I enzyme  CCGG 600bp CCGGCCTG Digest with Msp I 400bp200bp 600bp

26. Today’s experiments: On agarose gel: load 3 µl 100bp marker bp mother child father3 father1 father2 marker -/- +/- +/+ +/- -/- 100 200 300 400 500 600

27. Today’s experiments: 2. group  VNTR (on chromosome 5) DNA fragment includes different number of (CA) repeats You dont need to load marker

28. Expected results:

29. RFLP: 1. group: 616bp 400bp 200bp mother child father3 father1 father2 marker -/- +/- -/- +/+ +/- 100 200 300 400 500 600 700 800

30. VNTR: 2. group: mother child father3 father1 father2 marker 2/4 1/2 2/6 1/2 1/3 100 200 300 400 500 600 43214321 800 700

31. Monohybrid cross results Ebony vs. wildtype

32. Hypothesis: Body color is an autosomal trait and ebony type is recessive to the wild type” Body color is an autosomal trait and ebony type is recessive to the wild type” To test the accuracy of our hypothesis, we need to calculated to which extent our observed results are departed from the expected results To test the accuracy of our hypothesis, we need to calculated to which extent our observed results are departed from the expected results

33. e+ eee+e ++e++ F1 generation++e+e+e e+e+e F2 generation Phenotype ratio  3:1, wild-type:ebony Genotype ratio  1:2:1, homozygote WT, heterozygote WT, homozygote ebony Count Ebony vs. Wildtype Drosophila