biological markers

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biological markers

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biological markers

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

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