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Analyzing DNA Differences

PHAR 308 March 2009 Dr. Tim Bloom

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 3 .

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 4 .

brown  Blood type A.Genetic Differences Alleles represent genetic differences Blue eye vs. B. AB and O  Genetic diseases Muscular dystrophy  Cystic fibrosis  Sickle cell anemia  “Non-genetic” diseases or therapeutic targets Increased susceptibility  Decreased sensitivity  5 .

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 6 .

“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  7 .

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

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 9 .

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 10 .

position 46.Single Nucleotide Polymorphism Variability in one nucleotide  Example Rs17822931 Chromosome 16.815.699  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  11 .

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

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 13 .

edu/Courses/Molbio/MolStudents/spring2003/Williford/assignment1_home.htm 14 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.

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)  15 .

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 16 .

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

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 18 .

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

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

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 21 .

ornl.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.shtml Slides 19-21 from NCI tutorial “Understanding SNPs and Cancer” 22 .gov/sci/techresources/Human_Genome/faq/snps.