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”