1. Identifying human disease genes Overview Position independent methods Positional cloning Synteny Drosophila mutants that are positional candidates for human disease genes

2. Identifying human disease genes Overview of the process

4. Identifying human disease genes Position independent methods –Complementation

5. 14_13.jpg

6. Identifying human disease genes Position dependent methods –CF gene chromosome jumping and walking

11. Identifying human disease genes Mouse and human synteny

13. Identifying human disease genes Drosophila and human disorders

15. Genetic testing Overview Examples CF gene

17. Genetic testing CF testing –ARMs –OLA –Sequencing –Heteroduplex screening –DGGE –Mismatch cleavage

24. Genetic testing Oligonucleotide arrays

26. 18_09_3.jpg

27. Genetic testing Forensics –Species identification –Paternity testing –DNA quantitation –Human identification

28. Presence of human DNA? Complex Biomaterials? Yes No Exclusively human?If not human, What? Yes Single contributor? No Mixed species? DNA quantitation Yes No

29. NameClassOrderFamilyGenus & speciesRepeat element 1 CowMammaliaArtiodactylaBovidaeBos taurus1.711B bovine repeat 2 PigMammaliaArtiodactylaSuidaeSus scrofaPRE-1 SINE 3 ChickenAvesGalliformesPhasianidaeGallus gallusCR1 SINE 4 RuminantsMammaliaArtiodactylaN/A Bov-tA2 SINE 5 HorseMammaliaPerissodactylaEquidaeEquus caballusEre-1 SINE/horse 6 DogMammaliaCarnivoraCanidaeCanis familiarisSINEC_CF SINE/dog 7 CatMammaliaCarnivoraFelidaeFelis catusB2_Mv SINE/carnivores 8 RatMammaliaRodentiaMuridaeRattus norvegicusL1_RN LINE/L1 9 MouseMammaliaRodentiaMuridaeMus MusculusRSINE1 SINE/B4 10 HamsterMammaliaRodentiaMuridaeCricetulus griseusB2_Mm2 SINE/B2 11 Guinea PigMammaliaRodentiaCaviidaeCavia procellusID3 SINE/ID 12 RabbitMammaliaLagomorphaLeporidaeLepusC_Oc SINE/rabbit 13 BirdsAvesN/A L3b LINE/CR1 14 WaterfowlAvesAnseriformesAnatidaeN/AL3b LINE/CR1 Non human DNA quantitation using intra-SINE PCR

30. 100 bp DNA ladder Negative control Cow Horse Pig Sheep Deer Dog Cat Rat Mouse Hamster Guinea pig Rabbit Chicken Duck Dove Human A B C D E F G H I J Equine Canine Feline Rat Mouse Hamster Guinea Pig Rabbit Avian Waterfowl

32. 1.Human Specific Our Assay Design Objectives 2. Target Specific 3. Multiplex Compatible a. Comparison of genomic sequences b. Testing complex biomaterials a. Comparison of target sequences b. Testing for non-specific amplification a. ABI Prism 7000 default PCR conditions b. Experimentally optimized PCR reagents FAMVICNED

33. Nuclear DNA

34. Schematic of inter-Alu and intra-Alu PCR 5’ Alu 3’3’ Alu 5’5’ Alu 3’ Inter-Alu PCR tail to tailhead to headtail to head Intra-Alu PCR 5’ Alu Element 3’

35. Nuclear DNA target design Intra-Alu Yb8 AluY GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGA 50 AluYb8..................................................50 AluY GGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACGG 100 AluYb8......T......T.................................A..100 AluY TGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGC150 AluYb8...........................................C......150 AluY GGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGT200 AluYb8..................................................200 AluY GAACCCGGGAGGCGGAG CTTGCAGTGAGCCGAGAT CGCGCCACTGCACTC 250 AluYb8..........A........................ T...........G..250 AluY CA-------GCCTGGGC GACAGAGCGAGACTCCGTCTC AAAAAA287 AluYb8.GCAGTCCG................................... 294 PCR primersTaqMan-MGB probe

36. Serial dilution of human nuclear DNA 100 ng10 ng1 ng 0.1 ng0.01 ng1 pg

37. Nuclear DNA linear quantitation range nDNA y = -1.4761Ln(x) + 21.236 R 2 = 0.9998 14 16 18 20 22 24 26 28 30 32 34 0.0010.010.1110100 DNA (ng) Threshold PCR cycle VIC

38. Mitochondrial DNA

39. Mitochondrial DNA assay Incorporates specific diagnostic bases at the 3’ ends of each primer PCR primersTaqMan-MGB probe

40. Mitochondrial DNA linear quantitation range mtDNA y = -1.4769Ln(x) + 22.547 R 2 = 0.9999 14 16 18 20 22 24 26 28 30 32 34 0.0010.010.1110100 DNA (ng) Threshold PCR cycle FAM

41. Y chromosome DNA

42. Human Y-chromosome DNA assay design Human X-chromosome 90 bp deletion in the X-Y homologous region PCR primersTaqMan-MGB probe

43. Human Y chromosome locus fixation PopulationMalesFemalesTotal African-American150141 291 European-American4960 109 Hispanic-American97 16 North-American7554 129 South-American712 19 Asian1514 29 Total305288593

44. Male Y DNA linear quantitation range NED Male Y y = -1.4935Ln(x) + 34.74 R 2 = 0.9994 27 29 31 33 35 37 39 0.1110100 DNA (ng) Threshold PCR cycle

45. Multiplex Analysis

46. 1.Amplicon compatibility Multiplex Analysis 2. Human specificity 3. Background amplification a. Test assays and modify amplicons a. Analyze mixed DNA samples a. Analyze DNA from nearest neighbors

47. “DNA mixtures” to test human specificity

48. Linear quantitative range of nDNA / mtDNA duplex The open symbols along each standard curve represent detection of human DNA within a mixed sample. nDNA mtDNA nDNA y = -1.4761Ln(x) + 21.236 R 2 = 0.9998 mtDNA y = -1.4769Ln(x) + 22.547 R 2 = 0.9999 14 16 18 20 22 24 26 28 30 32 34 0.0010.010.1110100 DNA (ng) Threshold PCR cycle VIC FAM

49. Duplex background amplification

50. Linear quantitative range of a nDNA / mtDNA / male triplex PCR assay The open symbols along each standard curve represent accurate detection of human DNA within a mixed sample. VIC FAM NED

51. Triplex background amplification

52. Linear quantitative ranges Y Male Y DNA template 100 ng 10 ng 1 ng 100 pg 10 pg 1 pg nDNA mtDNA Each assay individually Duplex Triplex

53. Duplex PCR for Human Sex Typing X PCR primersTaqMan-MGB probesY PCR primers

54. Duplex PCR for Human Sex Typing

55. Mobile elements serve as “ genomic fossils ” of human ancestral lineages.

56. 1. Since the dispersal from Africa, mobile elements have continued to expand in the human genome. 2. Many elements are polymorphic and occur at high/low frequencies in human populations. 3. These elements can be exploited to examine human population history. 4. Display-based PCR methods can be used to “extract” recent, population-indicative elements. Human Population Biology and Investigative Forensics

57. Inferring Geographic Affiliation 1)Series of genetic markers (100 Alu loci) 2)Database of human variation (currently 715 individuals of known ancestry) 3)Genotype unknown sample 4)Analytical approach (Structure analysis) Forensic Sci. Intl. (In press)

58. Identifying 18 Unknown DNAs Forensic Sci. Intl. (In press)