What is a microsatellite Tandemly repeated DNA may

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

Visualization • Slab gels – Thin layer (1 mm 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 mega. BACE 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

Mega. BACE vs FMBio II • Mega. BACE – – 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: 331363 • Goldstein and Schlotterer. 1999. Microsatellites: evolution and application. Oxford University Press.