DNA Paternity Test RFLP analysis (Restriction Fragment Length Polymorphism) -analyze the size of DNA fragments that result when a segment of DNA from the genome is cut with special enzymes -Restriction Enzymes cut DNA at specific sequences -each enzyme recognizes and cuts DNA at a different base sequence e.g. BamHI XXXXXXXXGGATCCXXXXXXXXXX XXXXXXXXCCTAGGXXXXXXXXXX -due to spontaneous mutations over time, different people have slightly different base sequences in their DNA -if mutation creates or deletes a restriction site in the DNA, the new DNA will generate more or less fragments/different sized fragments when cut with a particular enzyme

...XXGGATCCXXXXXXGGATCCXXXX... ...XXCCTAGGXXXXXXCCTAGGXXXX… cut with BamHI generates three fragments ...XXGGATCCXXXXXXAGATCCXXXX... ...XXCCTAGGXXXXXXTCTAGGXXXX… single base mutation, BamHI will not cut 2nd site, generates only two fragments -RFLP analysis targets DNA segments known to have variability between individuals and cuts with enzymes that show those differences -cut DNA fragments are then analyzed on agarose gel electrophoresis 1 2 3 1 2

DNA Gel Electrophoresis -use to separate DNA by size to visualize it -Agarose gel = matrix with pores -place in running chamber with electrolyte buffer -electrical current runs through buffer between electrodes on opposite sides of gel -DNA samples loaded into wells near negative electrode -DNA has negative charge due to phosphate backbone -DNA moves through gel away from negative toward positive electrode -gel matrix separates moving DNA by size: -smaller molecules “squeeze” through gel easier thus moving faster -smaller molecules end up further away from the wells -DNA will need to be stained to see it after running the gel

_ Bigger + Smaller

(one gene from mom, one gene from dad) -each person has two copies of every gene: one from their mother, one from their father -a resulting child’s DNA will have characteristics of both parents’ DNA Mom 200bp 500bp 800bp 1000bp 200bp 500bp 800bp 1000bp Dad 200bp 1300bp 1000bp 200bp 1300bp 1000bp Child 200bp 500bp 800bp 1000bp 200bp 1300bp 1000bp (one gene from mom, one gene from dad)

_ + Mom Child Dad 1300bp 1000bp 800bp 500bp 200bp *do practice problem on worksheet

8. Given the following DNA agarose gel electrophoresis RFLP analysis, who is the father of the child in question? Mother Child Dad 1 Dad 2 Dad 3

8. Given the following DNA agarose gel electrophoresis RFLP analysis, who is the father of the child in question? Mother Child Dad 1 Dad 2 Dad 3 First circle and ignore all the fragments that could have been inherited from the mom...

8. Given the following DNA agarose gel electrophoresis RFLP analysis, who is the father of the child in question? Mother Child Dad 1 Dad 2 Dad 3 Then find the only man who could have contributed all the remaining fragments to the child

Agarose gel electrophoresis = “submarine gel” -submerged in running buffer -DNA must be suspended in loading buffer: contains: 1. glycerol to make sample dense to sink through running buffer into well 2. tracking dye to follow movement through gel (DNA is colorless) -bromophenol blue: co-migrates with ~300bp (small DNA) -xylene cyanol: co-migrates with ~4000bp (big DNA) -After gel runs, DNA must be stained with methylene blue to visualize it *practice use of pipettors *practice gel loading *set up and run pre-cut paternity test DNA samples

Genetics and Inheritance Genetic diversity comes from: 1. Meiotic recombination 2. Random fertilization Play GeneticVariation.swf

Human Genetics and Inheritance Humans: 46 chromosomes 23 homologus pairs (one of each from mom, one of each from dad) -2 copies of each gene, each is 1 allele -allele = a version of a gene -the 2 alleles could be the same or they could be different: same = homozygous (GG or gg) different = heterozygous (Gg) genotype = ones genetic makeup: all the alleles a person has phenotype = appearance of a person due to the genes/alleles -many traits or phenotypes will depend on the contribution of many alleles -some traits are determined by a single pair of alleles -in this case, one allele type will be dominant over another and the dominant allele will be the phenotype

e.g. alleles G and g G = green spots (dominant, capital letter) g = no spots (recessive, lower case letter) Genotype GG or Gg = Phenotype green spots Genotype gg = Phenotype no spots In order to have the recessive phenotype, both alleles have to be recessive A single dominant allele will control the phenotype *do genetics problems in book *do genetics problems on worksheet Note for genetics problems: Phenotype = what it looks like e.g. freckles Genotype = the genes: write as letters, but say: - “homozygous dominant” (FF) - “homozygous recessive” (ff) - “heterozygous” (Ff)