Microsatellites

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Microsatellites

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Microsatellites

  1. 1. Microsatellites
  2. 2. What is microsatellite • Simple Sequence Repeats (SSR) • 1-6 bp long
  3. 3. Classification of Microsatellites • Simple microsatelltes • Composite microsatellites
  4. 4. (GT)n (AC)n (AG)n Simple microsatellites contain only one kind of repeat sequences:
  5. 5. Composite microsatellites contain more than one type repeats
  6. 6. Molecular Basis of Microsatellite Polymorphism Different by 3 repeats • Slippage of DNA polymerase is believed to be the major cause of microsatellite variation • The mutation rate can be as high as 0.1 to 0.2% per generation
  7. 7. Abundant and Even Distribution
  8. 8. Abundant • Abundance varies with species, but all species studied to date have miocrosatellites • In well studied mammal species, one microsatellite exist in every 30-40 kb DNA.
  9. 9. Even distribution • • • • • • On all chromosomes On all segments of chromosomes With genes Often in introns In exons as well Trinucleotide repeats and human diseases: Huntington disease, fragile X, and other mental retardation-related human diseases
  10. 10. Small Locus sizes adapt them for PCR PCR 2 6 3 1
  11. 11. Microsatellites are co-dominant markers AB BC CD BC AD BD Allele A Allele B Allele C Allele D CC CD AC AB BD AC BD AB
  12. 12. Mendelian Inheritance of Microsatellites Microsatellites are inherited as codominant markers according to Mendelian laws Liu et al. 1999. Biochem. Biophys. Res Comm. 259: 190-194 Liu et al. 1999. J. Heredity 90: 307-311.
  13. 13. Advantages of Microsatellite Markers Abundant Evenly distributed Highly polymorphic Co-dominant Small loci
  14. 14. Development of microsatellite markers
  15. 15. Need • • SSR containing clones Sequences of the flanking regions of SSR
  16. 16. Microsatellites-enriched Small-insert DNA Libraries (I) Genomic DNA Digest with several 4-bp blunt enders Gel fraction of 300-600 bp Ligation to a phagemid vector insert insert insert insert micro Small insert 3.5 kb insert Small insert 3.4 kb Small insert Small insert Small insert Small insert 3.4 kb 3.4 kb 3.4 kb insert Small insert 3.4 kb insert Small insert 3.4 kb insert Small insert 3.4 kb 3.4 kb insert Small insert 3.4 kb
  17. 17. Microsatellites-enriched Libraries (II) micro insert insert Small insert plasmids 3.5 kb Small insert plasmids 3.5 kb insert in sert Small insert plasmids 3.5 kb Small insert plasmids 3.5 kb Using dut/ungCJ236 strain u u u Single-stranded phagemids 3.5 kb Conversion into single-stranded phagemids using helper phage u micro u u u u u u u u u Single-stranded phagemids Single-stranded phagemids Single-stranded phagemids 3.5 kb 3.5 kb Won’t be converted to ds will be degraded in WT host 3.5 kb
  18. 18. Microsatellite-enriched Libraries (III) micro Convert into ds micro u micro ds plasmids using (CA)15 (e.g.) Single-stranded phagemids 3.5 kb u Transform into WT E. coli u 3.5 kb 3.5 kb micro ds plasmids 3.5 kb According to Ostrander et al., 1992: PNAS 89:3419
  19. 19. Microsatellites-enriched Libraries CA GA TA CG CT GT CAA CAT CAG CAC CGG CGT CGC CGA ... 4 bp 5 bp
  20. 20. Characterization of Microsatellites • Isolate plasmid DNA; • sequence clones; • Identify clones with enough sequences for primer design.
  21. 21. PCR Optimization and PIC Analysis • PCR products best <200 bp • PCR conditions: annealing temperature, Mg++, pH, DMSO, etc. • Polymorphism information content • Polymorphism in reference families
  22. 22. Disadvantages of microsatellites • Previous genetic information is needed • Huge Upfront work required • Problems associated with PCR of microsatellites
  23. 23. The concept of Polymorphic information content • Measures the usefulness of a marker • Informativeness in specific families
  24. 24. Microsatellite Genotyping 1. AA x AA Not polymorphic 2. AA x BB No segregation 3. AØ x ØØ Only 1 allele segregating 1:1 4. AA x AB B segregates 1:1, A segregates with intensity 1:1 5. AA x BØ A not segregate B segregates 1:1 6. AØ x AB A segregates 3:1, B segregates 1:1 7. AB x AB A segregates 3:1, B segregates 3:1
  25. 25. Microsatellite Genotyping 8. AØ x BØ A segregates 1:1, B segregates 1:1 9. AB x ØØ A segregates 1:1, B segregates 1:1, A & B alternating 10. AA x BC 2 of the 3 alleles segregating 1:1 11. AØ x BC All 3 alleles segregating 1:1, 2 types with only 1 allele 12. AB x AC 2 of 3 alleles segregating 1:1, the other 3:1 with a single allele existing for some individuals 13. AB x CD All 4 alleles segregating 1:1
  26. 26. Polymorphic Information Content PIC) • PIC refers to the value of a marker for detecting polymorphism within a population • PIC depends on the number of detectable alleles and the distribution of their frequency. • Bostein et al. (1980) Am. J. Hum Genet. 32:314331. • Anderson et al. (1993). Genome 36: 181-186.
  27. 27. Polymorphic Information Content (PIC) n PICi = 1-∑ Pij2 j=1 Where PICi is the polymorphic information content of a marker i; Pij is the frequency of the jth pattern for marker i and the summation extends over n patterns
  28. 28. Polymorphic Information Content PIC) n PICi = 1-∑ Pij2 j=1 Example: Marker A has two alleles, first allele has a frequency of 30%, the second allele has a frequency of 70% PICa = 1- (0.32 + 0.72) = 1- (0.09 + 0.49) = 0.42
  29. 29. Polymorphic Information Content PIC) n PICi = 1-∑ Pij2 j=1 Example: Marker B has two alleles, first allele has a frequency of 50%, the second allele has a frequency of 50% PICb = 1- (0.52 + 0.52) = 1- (0.25 + 0.25) = 0.5
  30. 30. Polymorphic Information Content PIC) n PICi = 1-∑ Pij2 j=1 Example: Marker C has two alleles, first allele has a frequency of 90%, the second allele has a frequency of 10% PICc = 1- (0.92 + 0.12) = 1- (0.81 + 0.01) = 0.18
  31. 31. Polymorphic Information Content PIC) n PICi = 1-∑ Pij2 j=1 Example: Marker D has 10 alleles, each allele has a frequency of 10% PICd = 1- [10 x 0.12] = 1- 0.1 = 0.9
  32. 32. Allele frequency and Forensics • Say, we have 10 marker loci • We have done adequate population genetics to know each one have a 10% distribution • Test of each locus can define certain level of confidence as to what the probability is to obtain the results you are obtaining.
  33. 33. Allele frequency and Forensics • Locus 1, positive • You are included, but every one out of 10 people has the chance to be positive • locus 2, positive • You are included, but every one out of 100 people has the chance to be positive at both locus 1 and locus 2 • … • Locus 10, also posive • ...

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