Identifying human disease genes

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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
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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 3
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Nuclear DNA target design Intra-Alu Yb8
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
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
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
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Male Y DNA linear quantitation range
NED
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Multiplex Analysis

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Multiplex Analysis

• Amplicon compatibility

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
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Linear quantitative range of nDNA / mtDNA duplex
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
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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
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Triplex background amplification
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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
TaqMan-MGB probes
Y PCR primers
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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)

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