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

Identifying human disease genes Overview of the process

Identifying human disease genes Position independent methods –Complementation

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

Identifying human disease genes Mouse and human synteny

Identifying human disease genes Drosophila and human disorders

Genetic testing Overview Examples CF gene

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

Genetic testing Oligonucleotide arrays

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

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

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

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

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

Nuclear DNA

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’

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

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

Nuclear DNA linear quantitation range nDNA y = Ln(x) R 2 = DNA (ng) Threshold PCR cycle VIC

Mitochondrial DNA

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

Mitochondrial DNA linear quantitation range mtDNA y = Ln(x) R 2 = DNA (ng) Threshold PCR cycle FAM

Y chromosome DNA

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

Human Y chromosome locus fixation PopulationMalesFemalesTotal African-American European-American Hispanic-American97 16 North-American South-American Asian Total

Male Y DNA linear quantitation range NED Male Y y = Ln(x) R 2 = DNA (ng) Threshold PCR cycle

Multiplex Analysis

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

“DNA mixtures” to test human specificity

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 = Ln(x) R 2 = mtDNA y = Ln(x) R 2 = DNA (ng) Threshold PCR cycle VIC FAM

Duplex background amplification

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

Triplex background amplification

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

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

Duplex PCR for Human Sex Typing

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

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

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)

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