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 Capillaries
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 Upper allele dropout – can check for this Null alleles
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. Effects of size homoplasy
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 FMBio II 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 Microsatellite DNA in fishes. Rev. Fish Biol. Fish. 7:
Goldstein and Schlotterer Microsatellites: evolution and application. Oxford University Press.