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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

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analyzing dna differences

Analyzing DNA Differences

PHAR 308

March 2009

Dr. Tim Bloom

overview
Overview
  • Genetic Differences
  • Why analyze differences?
  • SNP
  • RFLP and PCR
important terms to remember
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
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
genetic differences1
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 differences2
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
“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
Example of a Useful Marker

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

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

Finding Genetic Variation
genetic markers
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
single nucleotide polymorphism
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
seeing snps 1
Seeing SNPs (1)
  • Restriction fragment length polymorphism
    • Because of a change in DNA, restriction enzyme site is created or lost
    • DNA digestion pattern changes
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

RFLP Analysis
using rflp
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

finding rflps
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)
seeing snps 2
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
simple pcr

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Simple PCR
using pcr
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
slide19

SNP Maps

All chromosomes are sequenced

SNP location

All SNPs are recorded

SNP Data

slide20

Individual SNP Profiles

SNP profile A

SNP profile F

SNP profile B

SNP profile E

SNP profile C

SNP profile D

slide21

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

highlights
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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