analyzing genetic variation.ppt
DESCRIPTION
Analysis of genetic variants.TRANSCRIPT
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Analyzing DNA Differences
PHAR 308
March 2009
Dr. Tim Bloom
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Overview
Genetic Differences
Why analyze differences?
SNP
RFLP and PCR
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Important Terms to Remember
Locus specific region on a chromosome
Allele variant found at a locus
Genotype composition of alleles at a locus
Homozygous same allele each chromosome
Heterozygous different allele each chromosome
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Genetic Differences
Humans are 99% identical in DNA For 3x109 bases, means 3x107 differences On average a difference every 100 bases
Types of differences Deletions Insertions Base changes
MAY cause differences in people
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Genetic Differences
Alleles represent genetic differences Blue eye vs. brown Blood type A, B, AB and O
Genetic diseases Muscular dystrophy Cystic fibrosis Sickle cell anemia
“Non-genetic” diseases or therapeutic targets Increased susceptibility Decreased sensitivity
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Genetic Differences
Majority of differences show no effect Differences in inactive DNA “Silent” differences in protein
Neutral change in amino acid Silent codon change
Requirement for environmental effects CCR5 receptor on T-cells
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“Useful” Genetic Differences
Used to identify specific populations
“Polymorphisms” Found in sizeable fraction of population Can be used as “markers” Can be made into family tree Can be correlated with other traits
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Example of a Useful Marker
Image from Nature Clinical Practice Cardiovascular Medicine (2007) 4, 558-569
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Finding Genetic Variation
Changes with effect
Observation Alleles Genetic disease
Still must connect to a specific genetic context Chromosomal stains DNA sequence
Changes without effect?
Analyze DNA itself Determine sequence Effect of change on DNA
analytical techniques
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Genetic Markers
Detectable difference
Can be associated with a condition Down syndrome Schizophrenia Sensitivity to chemotherapy
Associated means those afflicted or at risk have or are more likely to have the marker
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Single Nucleotide Polymorphism
Variability in one nucleotide Example Rs17822931
Chromosome 16, position 46,815,699 Either C or T Homozygous T = dry ear wax Heterozygous or homozygous C = wet ear wax
Can be used forensically to ID race T > 90% in Asian C > 95% European or African
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Seeing SNPs (1)
Restriction fragment length polymorphism Because of a change in DNA, restriction
enzyme site is created or lost DNA digestion pattern changes
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RFLP Analysis
RE's cut DNA based on sequence If recognition sequence
present, cuts If recognition sequence
absent, no cut
Compare bands created by digestion with one enzyme
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Using RFLP
Link an RFLP to a disease as marker
Link an RFLP to drug resistance
Link multiple RFLPs to make a forensic identification
Image from http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Williford/assignment1_home.htm
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Finding RFLPs
Empirical Must have DNA samples from separate sources Must find polymorphism (Many are found in genome projects) Polymorphism must affect restriction enzyme Must be able to show relation to something
(for usefulness as a marker)
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Seeing SNPs (2)
Polymerase chain reaction
Technique for targeted DNA replication Uses DNA polymerase Uses synthetic DNA primers Primers direct polymerase action
Repeated cycles of replication of DNA target
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Simple PCR
5’3’5’ 3’
5’3’
5’ 3’5’3’
5’ 3’
5’3’
5’ 3’
3’5’
3’ 5’
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Using PCR
See SNP with PCR
Use primer base pairing to detect One primer’s end complements 1 polymorphism 5’ACTGACGATCGT3’ 5’ACTGACGATCGC3’
If primer doesn’t match, no DNA synthesized
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SNP Maps
All SNPs are recorded
SNP locationAll chromosomes are sequenced
SNP Data
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SNP profile A
SNP profile B
SNP profile F
SNP profile E
SNP profile DSNP profile C
Individual SNP Profiles
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SNP Profiles and Response toDrug Therapy
Does Not Respond to Standard Drug Treatment
Breast Cancer Patients
Individual SNP Profiles Are Sorted
SNP profile A SNP profile B
SNP profile D
SNP profile E SNP profile C
Responds to Standard Drug Treatment
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Highlights
DNA variations can be correlated to health problemsCommon variation is SNP Detect by RFLP Detect with PCR
Another variation is VNTRBoth variations used as “markers”http://www.ornl.gov/sci/techresources/Human_Genome/faq/snps.shtmlSlides 19-21 from NCI tutorial “Understanding SNPs and Cancer”