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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.