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Analyzing DNA Differences
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  1. Analyzing DNA Differences PHAR 308 March 2009 Dr. Tim Bloom

  2. Overview • Genetic Differences • Why analyze differences? • SNP • RFLP and PCR

  3. 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

  4. 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

  5. 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

  6. 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

  7. “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

  8. Example of a Useful Marker Image from Nature Clinical Practice Cardiovascular Medicine (2007) 4, 558-569

  9. 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 Finding Genetic Variation

  10. 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

  11. 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

  12. Seeing SNPs (1) • Restriction fragment length polymorphism • Because of a change in DNA, restriction enzyme site is created or lost • DNA digestion pattern changes

  13. 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 RFLP Analysis

  14. 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

  15. 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)

  16. 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

  17. 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ Simple PCR

  18. 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

  19. SNP Maps All chromosomes are sequenced SNP location All SNPs are recorded SNP Data

  20. Individual SNP Profiles SNP profile A SNP profile F SNP profile B SNP profile E SNP profile C SNP profile D

  21. SNP Profiles and Response to Drug Therapy Breast Cancer Patients Individual SNP Profiles Are Sorted Responds to Standard Drug Treatment Does Not Respond to Standard Drug Treatment SNP profile A SNP profile B SNP profile E SNP profile C SNP profile D

  22. 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”