1 / 17

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

wesley-acosta
Télécharger la présentation

What is a microsatellite?

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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.

More Related