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biological markers

biological markers

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biological markers biological markers Presentation Transcript

  • Genetic and Biochemical Markers
    • Biological markers can be anything that distinguishes one individual or population from another
    • Can be phenotypic
      • color: yellow colony vs white colony
      • texture: smooth colony vs rough colony
      • shape: round colony vs irregular colony
    • Can be a biochemical or genetic difference
  • Marker Uses
    • Phenotyping and Genotyping
    • Evolutionary relatedness
    • Contamination detection
    • Disease diagnosis
    • Forensic evidence
    • Marker assisted breeding
  • Biochemical Markers
    • Carbohydrates
    • Blood typing
      • ABO blood group
      • LM blood group
      • Bombay phenotype
    • Food adulteration
      • Sweetened juices
    View slide
  • Biochemical Markers
    • Proteins
    • Isozyme analysis
      • heart attack
        • creatine kinase & alanine aminotransferase
    • Gel electrophoresis
      • S-hemoglobin
    View slide
  • Biochemical Markers
    • ELISA - enzyme linked immunosorbant assay
    • Uses engineered antibodies to detect a specific protein
    • Sensitive - Can detect ng quantities
    • Quick - 1 - 2 hours
    • Can be easily automated
  • Lipids
    • Not widely used as markers
    • Can be used for variety identification
    • Several diseases associated with altered lipids
    • Can be used a health predictors
      • cholesterol
      • HDL/LDL
  • Cytological Markers
    • Mostly used in genetic counseling
    • Abnormal chromosome number or morphology can be easily detected
      • Down Syndrome
      • Turner’s Syndrome
    • Cytological markers are used in plant breeding
  • Down and Turner’s Syndrome
  • Other Biochemical Markers
    • Involves detection of specific metabolite
    • Usually involves addition of 2nd chemical or enzyme to catalyze reaction
    • Detection usually based on color change
    • Detection of cyanogenic glycosides
      • purple color when reactants added
  • DNA Markers
    • Most widely used markers
    • Can be hybridization or PCR-based
    • Time range - 2 hours to 1 week
    • Can detect single nucleotide difference
      • SNP - single nucleotide polymorphorism
    • DNA markers need not be the DNA responsible for the difference
  • Types of DNA Markers
    • RFLP - restriction fragment length polymorphism (pm)
    • AFLP - amplified fragment length pm
    • RAPD - random amplification of polymorphic DNA
    • VNTR - variable number tandem repeat
    • SSR - simple sequence repeat
  • RFLP Markers
    • Oldest DNA marker technology
    • Relies on the difference in DNA sequences between two samples that creates or removes a restriction site
    • Restriction sites are palindromic sequences of 4 or more bases which are recognized and cut by specific endonucleases (restriction enzymes)
  • RFLP Markers
    • DNA samples from contrasting individuals or populations are digested with an RE
    • DNA is separated by gel electrophoresis
    • The DNA is then subjected to Southern blotting
    • The DNA probe used must span the restriction site
  • RFLP Analysis
  • RFLP Analysis
  • RFLP Markers
    • Reliable - very reproducible
    • Hard to develop unless the DNA sequence of interest is known
    • Labor intensive - little automation
    • Take time - minimum 2 days, normal time frame 3 - 7 days
  • VNTR Markers
    • A variation of RFLP analysis
    • Main difference is RE does not cut within the probe but on flanking regions
    • Technique relies on short sequences (10 - 100 bp) repeated within the restricted fragment
    • Differences in the number of repeats can be detected by difference in length
  • VNTR Markers
    • VNTRs exist in most eukaryotes but largely exploited in humans
    • VNTRs have no known genetic function
    • The presence of some VNTRs has been associated with some diseases
    • VNTRs exist in multiple allelic forms
    • May be repeated from 2 - 100 times
  • VNTR Markers
    • Used in forensics and paternity cases
    • Referred to as DNA fingerprinting
    • Dozens of different VNTR loci are present in the human
    • Variability in the alleles associated with each VNTR locus determines its utility
  • VNTR Analysis
  • VNTR Analysis
  • VNTR Markers
    • How many for a conclusive match?
    • The probably for a random match in a population will depend on the number of alleles a VNTR has
    • 2 alleles 1:3 probability
    • 4 alleles 1:10 probability
    • 10 alleles 1:65 probability
  • VNTR Markers
    • Most widely used VNTR loci have 10 or more alleles
    • Most samples are analyzed for a minimum of 4 loci
    • When probabilities are combined they are multiplied
    • ie. 1/65 x 1/65 x 1/65 x 1/65 = 1/17,850,625
  • VNTR Markers
    • How sure does one need to be?
    • Paternity - 1/10000 (99.9% likely)
    • Guilt - 1/10,000,000
    • Problems - ethic homogeneity
    • Sample size
    • Contamination
  • STR Markers
    • STR = Sequence tagged repeats
    • Related to VNTR
    • PCR based
    • Requires sequence knowledge of the VNTR regions
    • Regions flanking the VNTRs are often highly conversed re: DNA sequence
  • STR Markers
    • Specific primers for PCR are designed to the conserved flanking regions
    • PCR reactions amplify the VNTR region
    • Reaction products are run on gel
    • Different number of repeats = different length PCR products
  • STR Analysis
  • STR Analysis
  • STR Markers
    • Faster than Southern blotting
    • Can used much less sample
      • 50 blood cells vs 5000 cells
    • Can use partially degraded samples
    • Not all VNTRs can be converted to STR
    • Small samples can skew results
    • More susceptible to contamination
  • SSR Markers
    • Similar to STRs but uses microsatellite sequences
    • Microsatellites are repeating units of 2 - 6 bp in size
    • Much more than VNTRs in genomes
    • Are converted to PCR based markers using the conserved flanking regions
  • RAPD Markers
    • Random amplification of DNA
    • Uses 10 base primers for PCR
    • Only 1 primer per reaction
    • Short primers bind randomly on the chromosomes
    • When a primers bind on complimentary strands within a 1000 bp and fragment is amplified
  • RAPD Markers
    • PCR reaction products separated by electrophoresis: Differences may be produced between samples
    • Used mainly to develop PCR markers for genetic traits
    • Problems are the low annealing temperatures reduce reproducibility
    • “ Random Artifiacts Produced Daily”
  • RAPD Marker