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Identifying human disease genes
Overview Position independent methods Positional cloning Synteny Drosophila mutants that are positional candidates for human disease genes
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Identifying human disease genes
Overview of the process
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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
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Identifying human disease genes
Mouse and human synteny
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Identifying human disease genes
Drosophila and human disorders
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Genetic testing Overview Examples CF gene
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Genetic testing CF testing ARMs OLA Sequencing Heteroduplex screening
DGGE Mismatch cleavage
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Genetic testing Oligonucleotide arrays
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Genetic testing Forensics Species identification Paternity testing
DNA quantitation Human identification
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Complex Biomaterials? Presence of human DNA? No Yes Exclusively human?
If not human, What? Yes No Mixed species? Single contributor? No Yes DNA quantitation
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Non human DNA quantitation using intra-SINE PCR
Name Class Order Family Genus & species Repeat element 1 Cow Mammalia Artiodactyla Bovidae Bos taurus 1.711B bovine repeat 2 Pig Suidae Sus scrofa PRE-1 SINE 3 Chicken Aves Galliformes Phasianidae Gallus gallus CR1 SINE 4 Ruminants N/A Bov-tA2 SINE 5 Horse Perissodactyla Equidae Equus caballus Ere-1 SINE/horse 6 Dog Carnivora Canidae Canis familiaris SINEC_CF SINE/dog 7 Cat Felidae Felis catus B2_Mv SINE/carnivores 8 Rat Rodentia Muridae Rattus norvegicus L1_RN LINE/L1 9 Mouse Mus Musculus RSINE1 SINE/B4 10 Hamster Cricetulus griseus B2_Mm2 SINE/B2 11 Guinea Pig Caviidae Cavia procellus ID3 SINE/ID 12 Rabbit Lagomorpha Leporidae Lepus C_Oc SINE/rabbit 13 Birds L3b LINE/CR1 14 Waterfowl Anseriformes Anatidae
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100 bp DNA ladder Negative control Cow Horse Pig Sheep Deer Dog Cat Rat Mouse Hamster Guinea pig Rabbit Chicken Duck Dove Human 100 bp DNA ladder Negative control Cow Horse Pig Sheep Deer Dog Cat Rat Mouse Hamster Guinea pig Rabbit Chicken Duck Dove Human 100 bp DNA ladder Negative control Cow Horse Pig Sheep Deer Dog Cat Rat Mouse Hamster Guinea pig Rabbit Chicken Duck Dove Human A E I Avian Equine Mouse B F J Waterfowl Hamster Canine C G Guinea Pig Feline D H Rat Rabbit
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Our Assay Design Objectives
Human Specific a. Comparison of genomic sequences b. Testing complex biomaterials 2. Target Specific a. Comparison of target sequences b. Testing for non-specific amplification 3. Multiplex Compatible a. ABI Prism 7000 default PCR conditions b. Experimentally optimized PCR reagents FAM VIC NED
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Nuclear DNA
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Schematic of inter-Alu and intra-Alu PCR
Inter-Alu PCR 5’ Alu 3’ 3’ Alu 5’ 5’ Alu 3’ 5’ Alu 3’ tail to tail head to head tail to head Intra-Alu PCR 5’ Alu Element ’
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Nuclear DNA target design
Intra-Alu Yb8 AluY GGCCGGGCGCGGTGGCTCACGCCTGT AATCCCAGCACTTTGGGAGGC CGA 50 AluYb8 GGCGGGCGGATCACGAGGT CAG GAGATCGAGACCATCCTGGCTAACACGG 100 ......T......T A.. TGAAA CCCCGTCTCTACTAAAAATAC AAAAAATTAGCCGGGCGTGGTGGC 150 C...... GGGCGCCTGTAGTCCCAGCTACTCGG GAGGCTGAGGCAGGAGAATGGCGT 200 GAACCCGGGAGGCGGAG CTTGCAGTGAGCCGAGAT CGCGCC ACTGCA C TC 250 A T G .. A GCCT GGGC GACAGAGCGAGACTCCGTCTC AAAAAA 287 . GCAGTCCG PCR primers TaqMan-MGB probe
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Serial dilution of human nuclear DNA
100 ng 10 ng 1 ng 0.1 ng 0.01 ng 1 pg
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Nuclear DNA linear quantitation range
nDNA y = Ln(x) R 2 = 14 16 18 20 22 24 26 28 30 32 34 0.001 0.01 0.1 1 10 100 DNA (ng) Threshold PCR cycle Nuclear DNA linear quantitation range VIC
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Mitochondrial DNA
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Mitochondrial DNA assay
PCR primers TaqMan-MGB probe Incorporates specific diagnostic bases at the 3’ ends of each primer
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Mitochondrial DNA linear quantitation range
mtDNA y = Ln(x) R 2 = 14 16 18 20 22 24 26 28 30 32 34 0.001 0.01 0.1 1 10 100 DNA (ng) Threshold PCR cycle FAM
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Y chromosome DNA
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Human Y-chromosome DNA assay design
Human X-chromosome 90 bp deletion in the X-Y homologous region PCR primers TaqMan-MGB probe
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Human Y chromosome locus fixation
Population Males Females Total African-American 150 141 291 European-American 49 60 109 Hispanic-American 9 7 16 North-American 75 54 129 South-American 12 19 Asian 15 14 29 305 288 593
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Male Y DNA linear quantitation range
y = Ln(x) R 2 = 27 29 31 33 35 37 39 0.1 1 10 100 DNA (ng) Threshold PCR cycle NED
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Multiplex Analysis
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Multiplex Analysis Amplicon compatibility 2. Human specificity
a. Test assays and modify amplicons 2. Human specificity a. Analyze mixed DNA samples 3. Background amplification a. Analyze DNA from nearest neighbors
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“DNA mixtures” to test human specificity
Contents Human male Dog Cat Total template Mix DNA (ng) % 1 50 25 100 2 5 2.5 10 3 0.5 4.5 45 4 0.05 4.95 49.5 0.005 4.995 49.95
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Linear quantitative range of nDNA / mtDNA duplex
y = Ln(x) R 2 = mtDNA y = Ln(x) = 14 16 18 20 22 24 26 28 30 32 34 0.001 0.01 0.1 1 10 100 DNA (ng) Threshold PCR cycle The open symbols along each standard curve represent detection of human DNA within a mixed sample. nDNA VIC mtDNA FAM
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Duplex background amplification
18 20 22 24 26 28 30 32 34 Human genomic Human nuclear Bonobo Common chimp Gorilla dog cat rat mouse rabbit cow horse sheep pig deer chicken NTC DNA Source Threshold PCR cycle nDNA mtDNA
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Linear quantitative range of a nDNA / mtDNA / male triplex PCR assay
y = Ln(x) R 2 = nDNA y = Ln(x) = 1 Male Y y = Ln(x) = 18 20 22 24 26 28 30 32 34 36 38 40 0.01 0.1 1 10 100 DNA (ng) Threshold PCR cycle The open symbols along each standard curve represent accurate detection of human DNA within a mixed sample. VIC FAM NED
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Triplex background amplification
24 26 28 30 32 34 36 38 40 Human Male Human Female Bonobo Common chimp Gorilla dog cat rat mouse rabbit cow horse sheep pig deer chicken NTC DNA source Threshold PCR cycle nDNA mtDNA Male Y
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Linear quantitative ranges
DNA template 100 ng 10 ng 1 ng 100 pg 10 pg 1 pg nDNA Each assay individually mtDNA Male Y Duplex Triplex
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Duplex PCR for Human Sex Typing
X PCR primers Y PCR primers TaqMan-MGB probes
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Duplex PCR for Human Sex Typing
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Mobile elements serve as “genomic fossils” of human ancestral lineages.
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Human Population Biology and Investigative Forensics
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.
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Inferring Geographic Affiliation
Series of genetic markers (100 Alu loci) Database of human variation (currently 715 individuals of known ancestry) Genotype unknown sample Analytical approach (Structure analysis) Forensic Sci. Intl. (In press) Forensic Sci. Intl. (In press)
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Identifying 18 Unknown DNAs
Forensic Sci. Intl. (In press)
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