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What is a microsatellite?

What is a microsatellite?. Tandemly repeated DNA ( may see in the literature as STRs - Short tandem repeats) Poly A/T most common 1-10 bp tandemly repeated = ‘micro’ satellite >10 = ‘mini’ satellite Types of microsats Di, tetra and tri nucleotide ( used in that order ) Perfect

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What is a microsatellite?

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  1. What is a microsatellite? • Tandemly repeated DNA (may see in the literature as STRs - Short tandem repeats) • Poly A/T most common • 1-10 bp tandemly repeated = ‘micro’ satellite • >10 = ‘mini’ satellite • Types of microsats • Di, tetra and tri nucleotide (used in that order) • Perfect • Imperfect/interrupted • Compound • Varying levels of variation associated with each type • Difficulty in scoring

  2. Microsatellite mutation • Rates between 10-3 and 10-6 per locus per generation • Mutation models • Slipped strand mispairing • Recombination – unequal crossing over • IAM or KAM, SSM in microsatellite analysis

  3. Microsatellite PCR • Long extensions for A-adenylation problems • PCR multiplexing • Multiple loci PCR amplified at once • Tricky and time consuming to develop • Post-PCR multiplexing • Amplify each locus individually • Run together on one gel

  4. Visualization • Alleles are generally small 90-400bp • Alleles generally differ by 1 repeat unit (2-4bp) • Acrylamide gels provide required resolution • Slab gels – automated/manual • Capillary – automated sequencers

  5. Visualization • Slab gels • Thin layer (1mm or less) of polymerized acrylamide between two glass plates • Capillaries • Hair-thin glass capillary filled with polymerized acrylamide

  6. Visualization • Manual method 1 (staining) • Run DNA for some time • DNA entrained in gel • Stain gel – ethidium, or in this lab SYBRgreen • Visualize on lightbox or some sort of scanner – FMBio – gel image

  7. Visualization • Manual method 2 (fluorescent dyes) • PCR using primers labeled with fluorescent dyes • Run DNA for some time • DNA entrained in gel • NO STAINING • Scan gel on scanner (lightbox wont work) – FMBio – gel image

  8. Visualization • Automated method (slab gel or capillary) • Combines electrophoresis and scanning • PCR using primers labeled with fluorescent dyes • Run DNA past scanning laser (all DNA eventually exits gel) • Computer records information – electropherogram

  9. Automated Sequencers/Scanners • Laser excites chemical dye • Filter filters out noise (esp. with more than one dye) • Specific filters for different dyes • Each dye emits a different spectra of light wavelengths when excited by a laser • Computer collects and compiles information

  10. Microsatellite practical problems • Stutter • Inversely related to repeat number (as repeat # goes up, stutter goes down) • Positively related to allele size (as allele size goes up, stutter gets worse) • Large allele dropout • Mostly a PCR problem – small alleles are favored • Also a megaBACE problem – electrophoretic injection • Null alleles • Mutations at priming site

  11. Fixes • Stutter • Binning • Change loci to higher repeat • Redesign primers for shorter alleles • Upper allele dropout – can check for this • Change PCR conditions • Reamplification of samples • Null alleles • Redesign primers

  12. Other practical problems • Sizing • Molecular ladders • Labeled ladder expensive • Standardization between labs • Different visualization platforms • Different molecular ladders • Binning • Variation in allele sizing

  13. Effects of practical problems • Depends on type of analysis • Deviations from Hardy-Weinberg • Most population differentiation analysis models assume H-W • Mismatch of parents to offspring • No real problems in genome mapping • Some extra analysis

  14. Theoretical problems • Size homoplasy • Alleles identical in state, not by descent Effects of size homoplasy • Incorrect data and conclusions

  15. Size homoplasy fix? • No easy fix • Can attempt to estimate by sequencing lots of alleles • Expensive and time consuming

  16. MegaBACE vs FMBio II • MegaBACE • Semi-automated allele calling • Expensive • Have to use Genetic Profiler – not user friendly • Interprets electropherograms and allows automatic allele size calling • FMBio II • System not worked out in our lab • Cheap • Easy, in theory

  17. Sources • O’Connell and Wright. 1997. Microsatellite DNA in fishes. Rev. Fish Biol. Fish. 7:331-363 • Goldstein and Schlotterer. 1999. Microsatellites: evolution and application. Oxford University Press.

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