Analyzing DNA Differences

PHAR 308 March 2009 Dr. Tim Bloom

Overview
Genetic Differences Why analyze differences? SNP RFLP and PCR

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

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

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

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

Example of a Useful Marker

Image from Nature Clinical Practice Cardiovascular Medicine (2007) 4, 558-569

Finding Genetic Variation

Changes with effect Observation

Changes without effect? Analyze DNA itself

Alleles Genetic disease

Still must connect to a specific genetic context

Determine sequence Effect of change on DNA analytical techniques

Chromosomal stains DNA sequence

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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 5 3 3

3 5
3 5 3 5

5 3 3 5

3 5 5 3
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Using PCR
See SNP with PCR Use primer base pairing to detect
One primers end complements 1 polymorphism 5ACTGACGATCGT3 5ACTGACGATCGC3

If primer doesnt match, no DNA synthesized

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SNP Maps

All chromosomes are sequenced All SNPs are recorded SNP Data

SNP location

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Individual SNP Profiles

SNP profile A

SNP profile F

SNP profile B

SNP profile E

SNP profile C

SNP profile D
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SNP Profiles and Response to Drug Therapy

Breast Cancer Patients

Individual SNP Profiles Are Sorted Responds to Standard Drug Treatment SNP profile A Does Not Respond to Standard Drug Treatment SNP profile B

SNP profile E

SNP profile C

SNP profile D

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Highlights
DNA variations can be correlated to health problems Common variation is SNP
Detect by RFLP Detect with PCR

Another variation is VNTR Both variations used as markers

http://www.ornl.gov/sci/techresources/Human_Genome/faq/snps.shtml
Slides 19-21 from NCI tutorial Understanding SNPs and Cancer
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