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

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

3. 3 Important Terms to Remember Locusspecific region on a chromosome Allelevariant found at a locus Genotypecomposition of alleles at a locus Homozygous same allele each chromosome Heterozygous different allele each chromosome

4. 4 Genetic Differences Humans are 99% identical in DNA For 3x10 9 bases, means 3x10 7 differences On average a difference every 100 bases Types of differences Deletions Insertions Base changes MAY cause differences in people

5. 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. 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. 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. 8 Example of a Useful Marker Image from Nature Clinical Practice Cardiovascular Medicine (2007) 4, 558-569

9. 9 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

10. 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. 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. 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. 13 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

14. 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. 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. 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. 17 Simple PCR 5’3’ 5’3’ 5’3’ 5’3’ 5’3’ 5’3’ 5’3’ 5’3’ 5’ 3’5’

18. 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. 19 SNP Maps All SNPs are recorded SNP locationAll chromosomes are sequenced SNP Data

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

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

22. 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”