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– 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– 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

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– 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.