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Development*of*a*STR*multiplex*kit*for*the*
individualisation*of*Hen*Harriers*(Circus'cyaneus)*
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! ! ! ! ! '''''''''Student:! ! Moniek!van!Hoppe! ! 'Internship'company:! ! University!of!Central!Lancashire!(GB)! ! ! ! ! ! ! ! Company'supervisor:! ! Dr.!Arati!Iyengar! ! ''Teacher'supervisor:! ! Ans!Arets! ! ! ! 'Internship'duration:! ! 28B01B2013!to!12B06B2013! ! '''''''''''''''Graduation:! ! July!2013! ! ! !Abstract
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The!hen!harrier!!(Circus!cyaneus)!is!an!endangered!bird!of!prey!species!in!England!(UK).!Their! numbers! declined! rapidly! because! of! habitat! loss! but! most! of! all! by! human! persecution.! Driven! red! grouse! shooting! is! a! popular! sport! in! Britain! and! hen! harriers! and! red! grouse! share!the!same!habitat.!This!results!in!hen!harriers!feeding!on!red!grouse!chicks.!With!the! numbers!of!red!grouse!declining!the!hen!harriers!are!shot!to!increase!the!population!size!of! the! red! grouse.! Despite! hen! harriers! having! full! legal! protection! they! are! still! being! killed! illegally.!The!purpose!of!the!multiplex!kit!is!to!contribute!to!the!prosecution!of!a!suspect!in! cases!of!killing!the!endangered!hen!harrier.!!
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In! this! study! six! microsatellites! markers! that! were! found! by! creating! a! genomic! library! enriched!for!tetranucleotide!repeats!have!been!screened!to!check!if!they!are!polymorphic.! Two! of! them! turned! out! to! be! monomorphic! and! could! therefore! not! be! used.! Another! marker! did! seem! polymorphic! but! showed! multiple! peaks! for! each! allele,! each! peak! 1! bp! different!in!size.!Additional!research!needs!to!determine!whether!this!marker!could!still!be! useful.!Two!markers!that!were!found!to!be!polymorphic!turned!out!to!be!the!same!locus!and! therefore!only!one!of!these!was!useful.!One!other!marker!turned!out!to!be!polymorphic!with! six!alleles!being!observed.!These!two!markers!that!were!polymorphic!and!a!different!marker! originally! found! in! a! related! bird! species! were! incorporated! into! a! multiplex! PCR! reaction! along!with!a!sexing!marker.!! ! This!STR!multiplex!can!be!used!as!a!starting!point!for!the!creation!of!a!multiplex!kit!suitable! for!individualisation!by!the!incorporation!of!additional!markers.! ! ! ! ! ! ! ! ! ! ! ! ! *
Contents
* ! 1.*Introduction* 5! 2.*Background*information* 6! 2.1! Hen!harriers!general!information! 6! 2.2!! Hen!harriers!in!the!United!Kingdom! 6! 2.3!! Importance!of!a!multiplex!kit!for!hen!harriers! 7! 2.4!! STR!multiplexes!for!birds!within!the!family!Accipitridae! 8! 2.4.1!! Ways!to!find!STR!markers!for!the!hen!harrier! 8! 2.5!! Primer!optimization! 8! 2.6! Estimation!of!allele!frequencies! 9! 2.6.1!! Creating!allele!frequency!databases! 9! 2.7!! Random!match!probability!calculations! 9! 2.8!! HardyBWeinberg!equilibrium! 10! 2.9!! Sizing!DNA!products!using!capillary!electrophoresis! 10! 2.10!! Biology!related!artefacts! 10! 2.10.1!! !Stutter!products! 10! 2.10.2!! NonVtemplate!addition! 11! 2.10.3!! Microvariants! 11! 2.10.4!! Null!alleles! 11! 2.11! Technology!related!artefacts! 12! 2.12!! Optimization!and!validation!of!multiplex!PCR! 13! 3.*Methods* 14! 3.1!! Sample!collection! 14! 3.2!! DNA!extraction! 14! 3.2.1!! Tissue! 14! 3.2.2!! Buccal!swabs! 14! 3.3!! DNA!quantification! 15! 3.3.1!! Tissue! 15! 3.3.2!! Buccal!swabs! 15! 3.4!! Primer!design!and!optimization! 15! 3.5!! Sequencing!PCR!products! 16! 3.6!! STR!analysis! 17! 3.7!! Multiplex!PCR! 17! 4.*! Results* 19! 4.1!! Microsatellite!markers!discovered! 19!4.2!! DNA!extraction!of!tissue!samples! 19! 4.3!! Primer!design! 20! 4.4!! Primer!optimization! 20! 4.5!! Sequencing!PCR!products! 21! 4.6!! STR!analysis! 23! 4.6.1!! Markers!HH13VG11!and!HH13VE12! 24! 4.6.2!! Markers!HH09VC1!and!HH09VE8! 25! 4.6.3!! Marker!HH11VG7! 27! 4.6.4!! Marker!HH11VD11! 28! 4.6.5!! HWE!and!PowerStatsV12! 28! 4.7!! Multiplex!PCR! 29! 5.*! Discussion* 31! 5.1!! Primer!design!and!optimization! 31! 5.2!! Selecting!samples!for!STR!analysis! 31! 5.3!! Hen!harrier!STR!markers! 31! 5.3.1!! HH13VE12!and!HH13VG11! 31! 5.3.2!! HH09VC1!and!HH09VE8! 32! 5.3.3!! HH11VD11! 32! 5.4!! Statistical!tests! 32! 5.5!! Multiplex!PCR! 33! 6.*! Conclusion* 34! 7.*! Future*research* 35! References* 36! Appendix* 40! Appendix!I:!DNeasy!Blood!&!Tissue!Kit!(Qiagen)! 40! Appendix!II:!QIAamp®!DNA!Blood!Mini!Kit!(Qiagen)! 43! Appendix!III:!MicroCLEAN!(Web!Scientific)!protocol! 46! Appendix!IV:!BigDye®!Terminator!v3.1!Cycle!Sequencing!Kit!(Applied!Biosystems)! 47! Appendix!V:!Raw!data!of!STR!markers! 48! !
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1.*Introduction*
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The!hen!harrier!(Circus!cyaneus)!is!a!bird!of!prey!species!that!can!be!found!in!the!United! Kingdom.! The! diet! of! hen! harriers! consist! of! red! grouse! chicks! (Lagopus! lagopus! scotica).! Driven! red! grouse! shooting! is! a! popular! sport! in! the! United! Kingdom,! but! because! hen! harriers! hunt! the! red! grouse! their! density! is! declining.! This! eventually! leads! to! economic! losses! for! the! grouse! moor! managers.! The! hen! harriers! were! given! full! legal! protection! in! 1954! because! of! rapidly! declining! numbers.! Despite! this! legislation! hen! harriers! are! still! being!killed!today,!leading!to!almost!extinction!in!England!with!only!one!breeding!attempt!in! 2012.!!
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The! goal! of! this! research! was! to! develop! a! PCR! based! STR! mutliplex! kit! for! the! individualisation!of!hen!harriers!using!microsatellites!previously!developed!at!the!University! of!Central!Lancashire.!The!primers!were!tested,!optimized!and!combined!into!a!multiplex!kit.! In! cases! of! illegal! killings! of! hen! harriers,! once! a! profile! has! been! obtained! and! a! match! probability!has!been!calculated!it!could!contribute!to!the!prosecution!of!a!suspect.!
2.*Background*information*
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2.1* Hen*harriers*general*information*
The!hen!harrier!(Circus!cyaneus)!is!a!bird!of!prey!species!of!the!order!‘Falconiformes’!within! the! family! ‘Accipitridae’.! Besides! the! United! Kingdom! hen! harriers! can! also! be! found! in! Europe!and!North!America.!Female!hen!harriers!are!brown/white/black!in!colour!while!the! males!are!grey!with!a!bit!of!black,!see!figure!2.1.!The!brown!colour!gives!the!female!bird! wellVneeded! camouflage! because! hen! harriers! prefer! to! nest! on! the! ground! in! open! landscapes! like! mature! heather! and! moorlands! (Cramp! et! al.! 1980).! The! diet! of! the! hen! harrier! consists! of! small! mammals! (rodents)! and! birds! like! meadow! pipits! and! red! grouse! (Lagopus! lagopus! scotica)! (Green! and! Etheridge! 1999).! In! the! months! of! April! and! May! a! female!bird!could!lay!four!to!six!eggs.! ! ! Figure*2.1*Male*and*female*hen*harrier.!Male!on!the!left!and!female!on!the!right!(RSPB,!2013).! !
2.2** Hen*harriers*in*the*United*Kingdom*
Hen!harriers!can!be!found!in!mature!heather!and!upland!moorlands!of!Scotland,!northern! England!and!the!Isle!of!Man!(RSPB,!2013).!Hen!harriers!used!to!breed!all!across!Britain,!but! became!limited!to!the!Orkney!Islands!(Scotland)!in!the!beginning!of!the!20th!century!due!to! habitat! loss,! persecution! and! eggVcollectors.! They! returned! to! England! in! the! 1960s! (Sharrock,!1976).!The!hen!harrier!is!currently!a!redVlisted!UK!bird!of!conservation!concern! and!got!full!legal!protection!in!1954.!!Research! into! hen! harrier! breeding! was! performed! for! several! years! by! Natural! England.! They!observed!a!total!of!127!nesting!attempts!in!the!period!2002!–!2008!in!England!of!which! 125!occurred!in!the!northern!uplands.!This!appears!to!be!quite!a!high!number,!but!when! looking! at! individual! years! these! is! no! more! then! 23! nesting! attempts.! Over! the! years! breeding! attempts! were! made! at! 12! locations,! but! by! far! the! most! were! made! in! the! Bowland! Fells! (83! out! of! 125).! Bowland! Fells! was! also! the! only! location! with! breeding! attempts!every!year.!Figure!2.2!shows!the!distribution!of!breeding!sites!over!the!period!2002!
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Figure*2.2*Distribution*of*hen*harrier*breeding*attempts*in*the*period*2002*–*2008.*Top!row!from! left!to!right:!2002!–!2003!–!2004!–!2005.!Bottom!row!from!left!to!right:!2006!–!2007!–!2008.!A!dot! stands!for!1!–!5!breeding!attempts,!a!triangle!for!6!–!10!and!a!square!for!11!–!15.! ! –!2008!from!the!study!by!Natural!England.!A!dot!stands!for!1V5!breeding!attempts,!a!triangle! for!6!–!10!and!a!square!for!11V!15.!A!total!of!72!out!of!127!nesting!attempts!were!successful,! resulting!in!225!young.!Persecution!(29!%),!predation!(23!%)!and!unknown!(18!%)!were!the! main! categories! described! as! the! cause! of! the! failed! breeding! attempts.! When! looking! at! Bowland!grouse!moors!the!ratio!was:!unknown!(37!%),!persecution!(15!%)!and!predation!(13! %).! On! grouse! moors! outside! Bowland! the! ratio! was! however:! persecution! (85! %)! and! unknown!(15!%)!(Natural!England,!2008).!!! The!distribution!of!hen!harriers!in!Britain!varies!a!lot.!The!hen!harrier!is!about!to!go!extinct!in! England!with!only!one!observed!breeding!attempt!in!2012!(RSPB,!2012)!while!high!numbers! are!found!in!the!Orkney!Islands!with!a!100!breeding!females!(RSPB,!2012).!Despite!full!legal! protection!humans!are!still!killing!hen!harriers.!In!June!2012!a!female!hen!harrier!was!found! dead!at!a!grouse!moor!in!Yorkshire.!The!bird!was!tagged!with!a!satellite!so!her!movements! could!be!studied.!It!was!later!confirmed!using!a!technique!new!to!UK!wildlife!crime!cases! that!the!bird!was!shot!(RSPB,!2012).!!*
2.3** Importance*of*a*multiplex*kit*for*hen*harriers*
Hen!harriers!and!red!grouse!have!the!same!habitat!making!the!red!grouse!chicks!easy!prey! for!hen!harriers.!Driven!red!grouse!shooting!is!a!popular!sport!in!Britain.!Gamekeepers!are! responsible! for! grouse! moorlands! used! during! the! shooting! season.! Because! hen! harriers! hunt!red!grouse,!their!numbers!decline!and!less!red!grouse!are!present!during!the!shooting! season!(Redpath,!1991).!This!means!a!smaller!number!of!grouse!can!be!shot.!Because!fewer! hunters! can! attend! the! event,! less! money! can! be! made.! This! causes! losses! in! the! local! economy! (Thirgood! &! Redpath,! 1997).! Eventhough! research! by! Etheridge! in! 1997! showed!that! most! hen! harriers! are! killed! on! grouse! moorlands! it! is! being! denied! this! is! done! to! increase!the!numbers!of!red!grouse.!In!order!to!stop!the!illegal!killing!of!hen!harriers!a!PCR! based!multiplex!kit,!consisting!of!microsatellites,!for!the!individualisation!of!hen!harriers!is! very! much! needed.! The! multiplex! kit! can! be! used! in! forensic! investigations! to! connect! an! item!belonging!to!a!suspect!to!a!dead!bird!found!in!moorland.!Blood!or!feathers!could!have! been! transferred! on! to! the! suspect.! Once! a! profile! is! obtained! and! a! match! probability! is! obtained!it!could!contribute!to!the!conviction!of!a!suspect.!!
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2.4** STR*multiplexes*for*birds*within*the*family*Accipitridae*
Within! the! family! Accipitridae! two! STR! multiplexes! have! been! reported.! Research! by! Bielikova!et!al.!2010!reports!the!development!of!a!multiplex!PCR!containing!13!microsatellite! loci!for!Aquila!chrysaetos!(the!golden!eagle).!Allele!frequencies!have!been!determined!for!all! 13!loci!using!50!wildVliving!individuals!and!89!captiveVbred!individuals.!Samples!from!50!wildV living!individuals!in!Slovakia!were!taken!during!a!10Vyear!monitoring!project!and!keepers!and! veterinarians! collected! samples! from! captiveVbred! individuals.! Allele! frequencies! made! it! possible!to!perform!statistical!calculations!for!individual!identification.!For!the!whiteVtailed! eagle! (Haliaeetus! albicilla)! four! STR! multiplexes! containing! two! to! four! microsatellite! markers!have!been!reported!by!Hailer!et!al.!2005.!! ! 2.4.1** Ways*to*find*STR*markers*for*the*hen*harrier* There!are!different!ways!to!create!a!STR!multiplex!for!the!hen!harrier.!First!of!all!the!readyV made!multiplexes!within!birds!of!the!family!Accipitridae!could!be!tested!on!the!hen!harrier! to!see!which!markers!are!also!polymorphic!in!the!hen!harrier.!Many!STR!markers!in!birds! from! the! family! Accipitridae! have! been! reported! (Busch! et! al! 2005,! Mira! et! al! 2002,! MartínezVCruz! et! al! 2002).! These! STR! markers! could! be! tested! on! the! hen! harrier! and! all! polymorphic! markers! can! be! combined! into! a! multiplex! reaction.! A! more! expensive! and! timeVconsuming! approach! is! to! create! a! partial! genomic! library! enriched! for! diV,! triV! or! tetranucleotide! probes! to! find! STR! markers! within! the! desired! species! (Van! Dongen! &! Mulder!2005).!!!!
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2.5** Primer*optimization*
A!total!of!22!microsatellites!have!already!been!found!for!the!hen!harrier!by!creating!a!partial! genomic! library! enriched! with! the! tetranucleotide! probes! ‘AAAG’! and! ‘AGAT’! (Van! Hoppe,! unpublished).! The! designed! primers! can! be! optimised! by! variation! of! PCR! reaction! components! and! the! thermal! cycling! conditions.! The! most! important! parameter! for! a! successful!amplification!is!the!annealing!temperature.!The!amount!of!magnesium!chloride! might!improve!annealing!of!the!primer.!The!amount!of!Taq!polymerase,!cycle!number!and! annealing!time!can!also!be!varied!to!improve!the!PCR!reaction.!Since!the!DNA!markers!are!
going!to!be!used!in!a!multiplex!reaction,!all!markers!need!to!work!at!the!same!annealing! temperature!with!the!same!conditions!(Moretti!et!al.!2002).!
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2.6* Estimation*of*allele*frequencies*
When! the! optimal! condition! is! determined! for! all! markers,! fluorescent! primers! can! be! ordered!making!it!possible!to!detect!PCR!products!on!a!genetic!analyser.!In!order!to!check!if! a! marker! is! polymorphic,! as! many! unrelated! individuals! have! to! be! screened! and! alleles! scored.!The!allele!frequency!is!calculated!by!the!number!of!a!specific!allele!found!divided!by! the!total!number!of!alleles!found!(Butler,!2005).!It!has!been!estimated!that!a!total!of!100V 150! individuals! need! to! be! screened! in! order! to! obtain! accurate! estimates! of! allele! frequencies!(Chakraborty!et!al.!1992,!Evett!and!Gill!1991).!When!a!particular!allele!is!only! found!once!or!twice,!an!inaccurate!estimate!of!the!allele!frequency!can!be!made.!A!report!by! the! National! Research! Council! (1996)! states! an! allele! must! have! been! found! at! least! five! times!in!order!to!make!reliable!calculations!with!allele!frequencies.!When!an!allele!is!found! less!than!five!times,!the!allele!frequency!can!be!calculated!with!the!formula:!5/2N,!with!N! standing!for!the!amount!of!individuals!tested.!
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2.6.1** Creating*allele*frequency*databases*
To! obtain! accurate! allele! frequency! databases! suitable! populations! need! to! be! used.! The! human! allele! frequency! databases! are! based! on! race.! For! the! PowerPlex®! 16! System! (Promega,!DC6531)!four!different!allele!frequency!databases!are!present:!AfricanVAmerican,! CaucasianVAmerican,! HispanicVAmerican,! and! AsianVAmerican.! Using! the! allele! frequency! from!the!wrong!population!can!result!in!major!consequences,!since!allele!frequencies!can! differ! a! lot! between! populations.! In! animals! allele! frequency! databases! are! based! on! geographical!regions!instead!of!race.!! !
2.7** Random*match*probability*calculations*
Once!the!allele!frequencies!for!each!STR!locus!have!been!determined!a!genotype!probability! for!an!individual!locus!in!the!STR!profile!can!be!calculated.!If!the!individual!is!heterozygous! the!probability!(P)!is!calculated!using:!P!=!2pq,!with!‘p’!and!‘q’!the!allele!frequencies!of!both! alleles.! The! probability! for! a! homozygous! individual! is! calculated! using:! P! =! p2.! When! the! probability! for! each! STR! locus! has! been! calculated! the! probability! for! the! complete! DNA! profile!(the!random!match!probability)!is!calculated!by!multiplying!all!probabilities!together.!! The! random! match! probability! (RPM)! is! the! probability! that! a! DNA! profile! matches! the! evidence!found!by!selecting!a!random!unrelated!person!(Koehler,!1995).!The!likelihood!ratio! is!the!probability!of!finding!the!evidence!if!the!defendant!is!guilty!divided!by!the!probability! of!finding!the!evidence!if!the!defendant!is!innocent.!The!probability!of!finding!the!evidence!if! the!defendant!is!guilty!is!1.!The!probability!to!find!the!evidence!if!the!defendant!is!innocent!is! the! random! match! probability.! This! results! in! the! formula:! LR! =! 1! /! probability! of! the! complete!DNA!profile!(Butler,!2005).!!
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2.8** HardyXWeinberg*equilibrium*
The!HardyVWeinberg!equilibrium!is!based!on!the!assumptions!that!the!population!is!large,! there! is! random! mating,! and! no! mutation,! migration! and! natural! selection.! When! these! assumptions!apply!to!a!population!it!should!result!in!the!same!allele!frequencies!for!each! generation!with!the!genotype!frequencies!p2!(AA),!2pq!(Aa)!and!q2!(aa)!(Russell,!2010).!To! check!whether!markers!for!a!specific!population!are!in!HWE!an!exact!test!is!performed.!The! expected!genotype!frequencies!are!calculated!using!the!observed!allele!frequencies.!When! the!observed!and!expected!frequencies!are!similar!the!population!is!in!HWE.!PVvalues!>!0.05! show!no!departure!from!HWE.!It!is!important!that!STR!markers!used!for!DNA!profiling!are!in! HWE!because!otherwise!the!rules!mentioned!do!not!apply.!! !
2.9** Sizing*DNA*products*using*capillary*electrophoresis*
When!individuals!are!screened!for!alleles!it!is!very!important!that!the!PCR!products!are!sized! accurately! in! each! run.! An! internal! size! standard! is! run! together! with! every! sample! to! calculate!the!size!of!the!PCR!product(s).!The!ROXV500!internal!size!standard!is!often!used!and! contains! 16! DNA! fragments! ranging! from! 35V500bp.! The! global! southern! method! is! then! used!to!produce!a!calibration!curve!(Y!=!ax!+!b)!based!on!all!16!peaks.!The!linear!function!is! used!to!calculate!the!size!of!the!PCR!product!using!the!run!time!as!‘x’!in!the!linear!function.! When!the!calibration!curve!is!not!reliable!the!size!of!the!PCR!products!are!incorrect.!When! the! internal! size! standard! is! degraded! or! when! it’s! out! of! date! the! calibration! curve! can! become!unreliable!(Butler,!2005).! !2.10** Biology*related*artefacts*
! 2.10.1***Stutter*products* Stutter!products!are!small!peaks!often!one!repeat!shorter!than!the!real!allele!peak!and!are! common! artefacts! during! PCR! amplification! of! STR! markers.! Stutter! products! can! be! one! repeat!unit!larger!in!size,!but!this!is!very!rare!using!tetranucleotide!STRs.!Stutter!products! can!be!explained!using!the!slippedVstrand!mispairing!model!(Hauge!and!Litt,!1993).!During! the! PCR! reaction! the! two! DNA! strands! come! apart,! but! because! there! are! many! identical! repeat! units! the! DNA! strands! can! anneal! incorrectly! causing! the! size! difference.! Tetranucleotide!repeats!often!have!a!stutter!percentage!below!15!%.!Stutter!products!are! reduced!when!pentanucleotide!repeats!are!used!or!when!alleles!contain!imperfect!repeats! (Walsh!et!al,!1996).!!2.10.2**NonXtemplate*addition*
During!PCR!amplification!Taq!polymerase!adds!an!additional!nucleotide,!mainly!adenine!‘A’,! to! the! 3’Vend! of! the! template! DNA! (Clark,! 1988).! This! process! is! known! as! nonVtemplate! addition!or!adenylation.!Because!an!extra!nucleotide!is!added,!the!PCR!product!is!one!base! pair!longer!than!its!actual!size.!When!partial!adenylation!occurs,!a!mixture!will!be!formed!of! PCR!products!that!contain!the!extra!adenine!(+A!peaks)!and!PCR!products!without!the!extra! adenine! (VA! peaks).! Shoulder! or! split! peaks! will! then! be! visible,! see! figure! 2.3.! When! the! amount! of! adenylation! varies! between! samples! at! a! specific! allele,! it! can! be! difficult! to! analyse! the! data.! In! order! to! produce! PCR! products! only! with! +A! peaks,! a! final!! extension!time!of!45!to!60!minutes!at!60!°C!or!72!°C!is!used!to!give!Taq!polymerase!enough! time!to!complete!adenylation!for!each!PCR!product!(Kimpton!et!al,!1993).!When!too!much! DNA! is! added! to! the! PCR! reaction! it! can! result! in! split! peaks.! It! is! therefore! important! to! determine!the!optimal!amount!of!DNA!needed!for!the!PCR!reaction!so!complete!adenylation! occurs!(Butler,!2005).!!
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2.10.3**Microvariants*
During!the!screening!of!a!population!alleles!can!sometimes!be!found!that!are!one!or!two! base! pairs! bigger! or! smaller! than! the! common! alleles.! Variation! between! alleles! can! be! caused! by! insertions,! deletions! or! nucleotide! changes.! Alleles! that! contain! any! of! these! variations! are! called! microvariants.! Because! microvariants! are! often! not! present! in! allelic! ladders,!they!are!also!called!‘offVladder’!alleles!(Gill!et!al,!1996).!Microvariants!have!not!been! described!in!bird!species.!! ! 2.10.4**Null*alleles* Sequence!polymorphisms!like!SNPs,!insertions!or!deletions!have!been!reported!within!the! STR!repeat,!in!the!flanking!region!or!at!the!primerVbinding!site!(Callen!et!al.!1993).!A!PCR! product!will!still!be!formed!when!sequence!polymorphisms!occur!within!the!STR!repeat!or! Figure*2.3!Schematic!example!of!incomplete!adenylation!resulting!in!shoulder!and!split!peaks!(Butler,! 2005)!
the! flanking! region,! but! variation! in! the! primerVbinding! site! disrupts! the! primer! from! annealing! and! results! in! amplification! failure.! This! is! called! null! allele! or! allele! dropout! (Butler,! 2005).! There! are! a! few! solutions! when! allele! dropout! is! observed.! First! of! all! the! primer! responsible! for! the! allele! dropout! could! be! redesigned! so! it! doesn’t! anneal! at! the! sequence! polymorphism! site! anymore.! This! solution! is! very! timeVconsuming! since! a! new! optimization!might!be!necessary!with!a!new!primer.!A!more!radical!solution!is!to!remove!the! STR! locus! from! the! multiplex! kit.! This! is! only! likely! to! be! done! in! early! stages! of! the! development!of!a!multiplex!kit.!Another!solution!is!to!add!an!extra!primer!that!contains!the! sequence! polymorphism.! This! way! amplification! of! the! allele! still! occurs! even! though! a! variation!in!the!primerVbinding!site!is!present.!When!the!sequence!variation!is!very!common! in!individuals!it!is!worth!it!to!use!this!solution.!LargeVscale!screening!may!indicate!presence! of!null!alleles!e.g.!imbalanced!allele!peak!heights!could!indicate!a!sequence!polymorphism!is! present!at!the!primerVbinding!site!(Butler,!2005).!! !
2.11* Technology*related*artefacts*
Besides!biology!related!artefacts!the!technology!used!can!also!produce!artefacts.!A!‘pull!up’! is!visible!when!the!dye!colours!cannot!properly!be!distinguished!because!of!spectral!overlap.! A! small! peak! is! being! ‘pulledVup’! at! another! colour! on! the! same! spot! of! the! allele! peak.! Another! artefact! called! dye! blobs! are! present! when! the! fluorescent! dye! comes! off! the! labelled!primer.!Dye!blobs!are!observed!as!wide!peaks.!A!sharp!peak!(spike)!that!is!visible!at! all!colours!could!be!caused!by!air!bubbles!or!voltage!spikes.!When!the!sample!is!reVinjected! the!spikes!should!be!gone!or!appear!on!a!different!location!since!they!are!not!reproducible.! Figure!2.4!shows!a!schematic!example!of!all!three!artefacts!(Butler,!2005).!! ! ! ! ! ! ! ! !*
*
! Figure*2.4!schematic!examples!of!dye!blobs,!pullVups!and!spikes!(Butler,!2005)!2.12** Optimization*and*validation*of*multiplex*PCR*
Once!all!STR!markers!have!been!optimized!in!singleplex!PCRs,!two!primer!pairs!can!be!tested! in!one!single!reaction!creating!a!duplex!PCR.!When!using!two!different!primer!sets!in!a!single! reaction!it!is!important!that!both!loci!are!amplified!equally.!This!can!be!achieved!by!changing! the! amount! of! primers! used! in! the! reaction.! For! small! loci! less! primer! is! needed! because! they! are! amplified! more! successfully! than! larger! products.! Once! the! duplex! is! optimized! another! primer! pair! can! be! added! to! the! reaction! till! all! primers! pairs! work! in! a! single! reaction! (Merdinoglu! et! al.! 2005).! Once! the! multiplex! has! been! developed,! two! types! of! validation!are!required:!developmental!and!internal.!The!developmental!validation!includes:! characterization!of!genetic!markers,!species!specificity,!sensitivity!studies,!stability!studies,! reproducibility,! caseVtype! samples,! population! studies! and! mixture! studies! (SWGDAM! validation!guidelines,!dec!2012).!Characterization!of!genetic!markers!includes:!inheritance!of! DNA!marker!shown!through!family!studies,!genomic!location!of!the!marker,!technology!used! to! identify! marker! and! the! type! of! polymorphism! it! shows.! Species! specificity! includes! investigation! into! the! possibility! to! obtain! genetic! information! from! nonVtargeted! species.! The!sensitivity!can!be!tested!using!different!DNA!dilutions!and!testing!different!sample!types! of!varying!quality.!An!upper!and!lower!DNA!range!can!be!determined!that!still!give!reliable! results! (Howard! et! al.! 2008).! The! multiplex! reaction! is! reproducible! when! someone! else! using!different!instruments!obtains!the!same!results!(Daniels!et!al.!2004).!!
! ! !
3.*Methods!
!
3.1** Sample*collection*
Licensed! field! workers! collected! buccal! swabs! from! hen! harrier! nestlings! during! routine! ringing!in!the!period!2006!–!2008!at!various!sites!in!the!United!Kingdom.!The!samples!from! various!sites!in!England!were!collected!by:!Steve!Downing,!Steve!Murphy!and!Dave!Sowter.! In!Scotland!samples!were!collected!by:!Brian!Etheridge,!Alan!Leitch,!E.R.!Meek,!B.!Ribbands,! Andrew! Sanderman,! Geoff! Sheppard,! B.! Taylor,! E.J.! Williams! and! Jim! Williams.! The! Wales! samples!were!collected!by:!J.!Roberts!and!Ian!M.!Spence.!Chris!Sharp!collected!the!samples! from! the! Isle! of! Man.! When! a! nest! with! young! chicks! was! found,! the! chicks! were! visually! sexed,! ring! tagged! and! their! mouths! swabbed! using! a! cotton! swab.! Most! of! the! collected! swabs!were!placed!in!a!1.5!ml!Eppendorf!tube!containing!400!µl!Puregene!Cell!lysis!solution! (Qiagen)! before! they! were! sent! to! the! University.! Some! of! the! collected! swabs! that! were! sent!to!the!University!were!stored!in!their!plastic!casing!without!lysisbuffer.!Upon!arrival!all! samples!have!been!stored!at!V40!°C.!! !
3.2** DNA*extraction*
! 3.2.1** Tissue*Approximately! 25! mg! of! muscle! tissue! was! taken! from! the! back! of! a! frozen! female! hen! harrier!using!a!scalpel.!A!DNA!extraction!was!performed!using!the!DNeasy!Blood!&!Tissue! Kit:! Purification! of! Total! DNA! from! Animal! Tissue! (SpinVColumn! Protocol)! (Qiagen,! 69504)! according! to! the! manufacturer’s! instructions! (see! appendix! I).! The! following! modifications! were!made:!Before!adding!the!proteinase!K!at!step!2!the!tissue!was!crushed!using!a!teflon! pestle.!The!content!was!incubated!overnight!at!56!°C.!At!step!7!only!100!µl!AE!buffer!was! pipetted!directly!on!the!membrane!and!the!contents!was!left!to!incubate!four!minutes!at! room!temperature!prior!to!elution.! ! 3.2.2** Buccal*swabs*
A! DNA! extraction! was! performed! on! the! collected! hen! harrier! buccal! swabs! using! the! QIAamp®!DNA!Blood!Mini!Kit:!DNA!Purification!from!Buccal!Swabs!(Spin!Protocol)!(Qiagen,! 51104)! according! to! the! manufacturer’s! instructions! (see! appendix! II).! The! following! modifications!were!made:!At!step!1!no!PBS!was!added!since!the!collected!hen!harrier!buccal! swabs! were! stored! in! a! 1.5ml! eppendorf! tube! with! approximately! 400ul! lysisbuffer! (Tris! 100mM! pH! 8,! EDTA! 10! mM! pH! 8,! NaCl! 250! mM,! 1%! SDS).! If! the! tube! did! not! contain! lysisbuffer,! 400! µl! ATL! buffer! was! added.! At! step! 2,! 10! µl! protease! (7.5! AU,! Qiagen)! was! added!and!no!additional!AL!buffer.!The!contents!was!incubated!for!2,5!hours!at!56!°C!at!step! 3.!After!the!incubation!1!µl!carrier!RNA!(QIAamp!DNA!Micro!Kit,!Qiagen,!56304)!was!added!
and!the!contents!was!briefly!vortexed!and!centrifuged.!The!protocol!was!then!continued!at! step!4!adding!400!µl!ethanol!96!%.!At!step!10,!100!µl!AE!buffer!was!added!to!the!column! (QIAquick! spin! column,! Qiagen).! The! content! was! incubated! at! room! temperature! for! 10! minutes.! After! elution! 25! µl! of! DNA! was! transferred! to! a! new! tube.! The! extracted! DNA! samples!were!stored!at!V20!°C.!!
!
3.3** DNA*quantification*
Quantification!of!the!extracted!DNA!samples!was!done!using!the!Nanodrop!2000!(Thermo! Scientific)! and! agarose! gel! electrophoresis.! Lambda! DNA! dilutions! were! made! and! run! together!with!the!DNA!samples!on!a!1!%!agarose!gel!stained!using!SafeView!Nucleic!Acid! Stain! (NBS! Biologicals! LTD;! Cambridgeshire,! UK)! in! order! to! determine! the! DNA! concentration.!A!BioDocVit!transilluminator!was!used!to!view!the!agarose!gel.!
!
3.3.1** Tissue*
Four!Lambda!DNA!dilutions!for!comparison!were!made!from!the!stock!solution!at!300!ng/µl:! 80! ng/µl,! 60! ng/µl,! 40! ng/µl! and! 20! ng/µl.! From! both! the! extracted! DNA! samples! and! Lambda!dilutions,!1!µl!DNA!was!mixed!with!4!µl!H2O!and!1!µl!6x!loading!dye!prior!to!loading.! ! 3.3.2** Buccal*swabs* Five!Lambda!DNA!dilutions!for!comparison!were!made!from!the!stock!solution!at!300!ng/µl:! 20!ng/µl,!10!ng/µl,!5!ng/µl,!2.5ng/µl!and!1.25!ng/µl.!From!each!Lambda!dilution!2!µl!DNA! with!0.5!µl!6x!loading!dye!was!run!on!gel.!This!created!the!following!final!concentrations:!40! ng,!20!ng,!10!ng,!5!ng!and!2.5!ng.!From!the!extracted!hen!harrier!DNA!3!µl!of!DNA!with!0.5!µl! 6x!loading!dye!was!run!on!gel.!! !
3.4** Primer*design*and*optimization*
Primers!were!designed!manually!for!the!flanking!region!of!a!microsatellite.!The!primers!were! checked! for! Tm,! hairpin! formation! and! selfVdimerisation! using! OligoAnalyzer! 3.1! from!Integrated! DNA! Technologies! (available! at!
http://www.idtdna.com/analyzer/applications/oligoanalyzer/).!A!Tm!of!approximately!55!°C! was!used!as!a!guideline!for!all!primers!in!order!for!them!to!work!in!a!possible!multiplex!PCR.!! !
The!designed!primer!pairs!were!tested!on!the!tissue!DNA!samples!using!a!gradient!PCR!with! the! following! MgCl2! concentrations:! 1.5! mM! and! 3.0! mM.! A! PCR! was! performed! using! 2x! Reddymix!(ABgene),!10!ng!hen!harrier!DNA!and!PCR!grade!water!to!get!a!reaction!volume!of! 10!µl:! !
2x!Reddymix! ! ! 5.0!µl! ! 10!µM!Forward!primer! 0.5!µl!
! 10!µM!Forward!primer! 0.5!µl!
! 25!mM!MgCl2!!! ! 0.6!µl!(only!for!3.0!mM!MgCl2,!no!MgCl2!for!1.5!mM)!! !
Based!on!the!Tm!of!the!primer!pair!the!following!annealing!temperatures!were!chosen:!Tm!–! 5!°C!and!Tm!–!7!°C.!The!PCR!conditions!used!were:!95!°C!for!3!min,!25!cycles!of!95!°C!for!1! min,!50!–!55!°C!for!1!min,!72!°C!for!1!min!and!a!final!extension!of!72!°C!for!15!min.!Hold!at! 12!°C.!For!primer!pair!HH02VB1,!a!touchdown!PCR!from!62!–!52!°C!dropping!1!°C!per!cycle! was! performed.! The! same! reaction! mix! was! used.! PCR! products! were! checked! on! a! 2! %! agarose!gel!stained!with!SafeView.! !
3.5** Sequencing*PCR*products*
In!order!to!sequence!PCR!products!for!confirmation!of!specific!amplification,!a!larger!volume! PCR!was!performed!on!nine!primer!pairs!using!10!ng!hen!harrier!DNA!and!PCR!grade!water! to!get!a!reaction!volume!of!25!µl:! ! 2x!Reddymix! ! ! 12.5!µl! ! 10!µM!Forward!primer! 1.25!µl! ! 10!µM!Reverse!primer! 1.25!µl!Previously! described! cycle! conditions! in! section! 3.4! were! used! with! the! following! adjustment:! Ta! of! 55! °C.! PCR! products! were! checked! on! a! 2! %! agarose! gel! stained! with! SafeView.!!
MicroCLEAN! (Web! Scientific,! Crewe,! UK)! was! used! according! to! the! manufacturer’s! instructions!to!purify!the!PCR!products!(see!appendix!III).!The!following!changes!were!made:! the!samples!were!left!at!room!temperature!for!10!minutes!after!adding!MicroCLEAN!to!the! PCR!product.!The!final!pellet!was!dissolved!in!the!same!volume!of!PCR!product!that!was!used! for!purification.!
Cycle! sequencing! reaction! (BigDye! Terminator! v3.1! Cycle! Sequencing! Kit,! Applied! Biosystems)! was! performed! using! 15! ng! template! and! PCR! grade! water! to! get! a! reaction! volume!of!10!µl:! ! Reaction!Mix! ! ! 0.50!µl! ! 5x!sequencing!buffer!! 1.70!µl! ! 10!µM!primer!! ! 0.32!µl! Cycle!conditions!are!described!in!appendix!IV.! ! The!products!from!the!cycle!sequencing!reaction!were!purified!using!ethanol!precipitation!as! follows:!1!μl!cold!NaOAc!(3!M!pH!4.6),!1!μl!glycogen!(20!μg/μl),!1!μl!EDTA!(100!mM)!and!30! μl!cold!96!%!ethanol!were!added!and!the!samples!were!incubated!either!overnight!at!room! temperature! or! four! hours! at! room! temperature.! The! samples! were! centrifuged! at! 13000!
RPM!for!30!minutes!at!4!°C!and!the!supernatant!was!removed!by!pipetting.!To!the!pellet!100! µl!70!%!ethanol!(freshly!prepared)!was!added.!The!samples!were!centrifuged!at!13000!RPM! for!15!minutes!at!4!°C!and!the!supernatant!was!removed!by!pipetting.!The!wash!step!with!70! %!ethanol!was!repeated.!The!supernatant!was!removed!by!pipetting!and!the!pellet!was!airV dried!using!a!thermal!cycler!at!50!°C!for!15!minutes,!but!keeping!the!tubes!open.!Just!before! running! the! samples! on! a! genetic! analyser,! 13! µl! HiVDi! formamide! was! added! and! the! contents! was! transferred! to! a! 96! well! plate! and! run! on! an! ABI! 3500! Genetic! Analyzer.! Viewing! and! editing! of! sequences! was! done! using! the! software! program! ‘BioEdit’! version! 7.1.7!(Hall,!2005).!!
*
*3.6** STR*analysis*
A! total! of! 24! samples,! extracted! as! described! in! section! 3.2.2,! were! selected! for! testing! selected!STR!markers.!In!order!to!have!unrelated!individuals!only!one!chick!from!a!particular! nest!was!used,!so!as!to!exclude!brothers!and/or!sisters.!As!many!individuals!from!all!over!the! United! Kingdom! were! chosen.! A! PCR! was! performed! with! 2! ng! hen! harrier! DNA! and! PCR! grade!water!to!get!a!reaction!volume!of!10!µl:! ! 2x!Reddymix! ! ! ! 5.0!µl! ! 10!µM!Labelled!forward!primer! 0.5!µl! ! 10!µM!Reverse!primer! ! 0.5!µl! The!PCR!conditions!used!were:!95!°C!for!3!min,!25!cycles!of!95!°C!for!1!min,!55!°C!for!1!min,! 72!°C!for!1!min!and!a!final!extension!of!60!°C!for!60!min.!Hold!at!12!°C.!PCR!products!were! checked!on!a!2!%!agarose!gel!stained!with!SafeView.!! To!1!µl!PCR!product,!13!µl!HiVDi!formamide!and!0.2!µl!ROXV500!(Applied!Biosystems)!was! added.!The!samples!were!then!run!on!an!ABI!3500!Genetic!Analyzer.!The!data!was!viewed! using! the! software! program! GeneMapper®! IDVX! 1.2! (Applied! Biosystems).! The! loci! were! tested! for! HardyVWeinberg! equilibrium! and! linkage! using! GENEPOP! (available! at! http://wbiomed.curtin.edu.au/genepop/index.html! (Raymond! &! Rousset! 1995)).! PowerStatsV12! was! used! to! obtain! allele! frequencies! and! other! forensic! parameters! (Brenner!&!Morris,!1990).!!
!
3.7** Multiplex*PCR*
For! the! multiplex! PCR! a! 10! µl! PCR! was! performed! using! 8! ng! hen! harrier! DNA! and! four! primer!pairs!(three!STR!loci!and!a!sexing!marker):! ! 2x!Reddymix! ! ! ! ! ! ! ! 5.0!µl! ! 10!µM!Labelled!forward!primer/reverse!primer!CHD1! ! 0.7!µl! ! 10!µM!Labelled!forward!primer/reverse!primer!HH09VC1! ! 0.2!µl! ! 10!µM!Labelled!forward!primer/reverse!primer!HH11VG7! ! 0.5!µl!! ! 10!µM!Labelled!forward!primer/reverse!primer!D220! ! 0.7!µl!
PCR!products!were!checked!on!a!3!%!agarose!gel!stained!with!SafeView.!To!1!µl!PCR!product,! 13!µl!HiVDi!formamide!and!0.35!µl!ROXV500!was!added.!The!samples!were!then!run!on!an! ABI!3500!Genetic!Analyzer.!The!data!was!viewed!using!the!program!GeneMapper®!IDVX!1.2.! !!
!
4.** Results*
!4.1** Microsatellite*markers*discovered*
By!creating!a!genomic!library!enriched!for!AGAT!and!AAAG!tetranucleotide!repeats!a!total!of! 22!microsatellites!were!found!in!the!hen!harrier,!see!table!4.1!(Van!Hoppe,!unpublished).!! ! Table*4.1!Microsatellites*discovered*in*the*hen*harrier.!The!repeat!motif,!number!of!repeats!and! which!microsatellites!have!been!used!for!primer!design! Probe* Used*for* primer*design* Repeat*Motif* Number*of*Repeats* For.*primer* Rev.*primer*
AGAT! ! (GGA)3(GAG)(GGA)! 5! V! V! AAAG! ! TTTC! 4! V! V! AAAG! ! GAAA! 4! V! V! AAAG! ! TTTC! 4! 22!nt! V! AAAG! ! AAAG! 5! V! V! AAAG! ! AAAG! 5! V! V! AAAG! ! (TCCC)2(TCCCC)(TCCC)2! 5! V! V! AGAT! ! TATC! 5! V! V! AGAT! ! ATAG! 5! V! V! AAAG! ! TTTC! 5! 22!nt! V! AAAG! ! (CTTT)4(ATTT)2! 6! 28!nt! V!
AGAT! Yes! AGAT! 6! V! V!
AAAG! Yes! AAAC! 7! V! V!
AAAG! Yes! TTTG! 9! V! V!
AGAT! Yes! ATAG! 9! 22!nt! V!
AAAG! Yes! CTTT! 11! V! V!
AGAT! ! ATAG! 12! V! V!
AGAT! Yes! (CAGAA)4(CAAAC)3! 7! V! V!
AAAG! Yes! GAAAGGAA! 7! V! V!
AAAG! Yes! CTTTT! 7! V! V!
AAAG! Yes! CAGCTTTCTTT! 10! V! V!
AGAT! Yes! ATAGA! 20! 21!nt! V!
V! Good!quality!sequence!available!
! Number! of! nucleotides! between! vector! and! beginning! of! microsatellite/good! quality! reverse!sequence!not!available! ! !
4.2** DNA*extraction*of*tissue*samples*
The!five!extracted!DNA!samples!from!the!same!individual!are!shown!in!figure!4.1.!All!five! samples!show!a!clear!high!molecular!weight!(HMW)!fragment.!! !!
!
Figure*4.1*Picture*of*a*1*%*agarose*gel*containing*five*extracted*hen*harrier*samples.*Lanes!2!to!5!
show! the! Lambda! DNA! concentrations:! 80! ng,! 60! ng,! 40! ng! and! 20! ng.! Lanes! 6! to! 10! show! the! extracted!DNA!samples!
!
4.3** Primer*design*
Primers!were!designed!for!the!microsatellites!shown!in!table!4.1!that!contained!six!or!more! consecutive!repeat!units.!The!designed!primers!are!shown!in!table!4.2.!When!designing!the! primers,! a! Tm! of! 55! °C! was! used! as! a! guideline! in! order! for! them! to! work! in! a! possible! multiplex! PCR! reaction.! All! primer! pairs! except! for! HH02VB1! and! HH13VG11! have! a! Tm! of! approximately!55!°C,!but!these!microsatellites!only!had!21!and!22!nucleotides!that!could!be! used!for!designing!the!forward!primer.!
!
4.4** Primer*optimization*
A! singleplex! gradient! PCR! was! performed! on! all! ten! markers.! This! resulted! in! successfully! amplified!PCR!products!at!nine!out!of!10!markers,!see!table!4.3.!Green!colour!represents!a! successful! amplification.! Orange! colour! indicates! that! a! product! was! observed! but! it! was! very!faint!and!red!colour!represents!no!amplification.!Marker!HH02VB1!did!not!amplify!the! expected! fragment! but! showed! very! faint! nonVspecific! product.! The! nine! successfully! amplified!markers!were!then!tested!in!singleplex!PCRs!with!an!annealing!temperature!of!! ! Table*4.3*Results*of*the*optimization*PCR.'Green!colour!represents!successful!amplification.!Orange! colour!indicates!a!very!faint!product!was!observed!and!red!colour!represents!no!amplification.! ! ! ! ! ! * * * ! HH0 2V B1 ! HH0 9V C1 ! HH0 9V C3 ! HH0 9V E8 ! HH1 0V B6 ! HH1 1V B2 ! HH1 1V D1 1! HH1 1V G7 ! HH1 3V E12 ! HH1 3V G1 1! MgCl2!1.5!mM! 50! 52! 53! 53! 52! 51! 53! 52! 52! 50! 52! 54! 55! 55! 54! 53! 55! 54! 54! 52! MgCl2!3.0!mM! 50! 52! 53! 53! 52! 51! 53! 52! 52! 50! 52! 54! 55! 55! 54! 53! 55! 54! 54! 52! !!!!!!!!!!!80!ng!!!60!ng!!40!ng!!20!ng!!!!CC1!!!!CC2!!!!!!CC3!!!!!CC4!!!!!CC5!!!!Neg!
!
Table*4.2.*Sequence,*Tm*and*PCR*product*size*for*10*designed*primer*pairs.*
!
Loci*name* Repeat* Primer* Sequence*(5’*–*3’)* Tm*
PCR* product*
Size* 1* HH02VB1! ATAGA(20)! For.! CAATACCTTGTTTCCAGTCC! 50.9! 166!bp!
Rev.! CAGTTGGACTAGATGACCG! 52.2! 2* HH09VC1! TTTG(9)! For.! CAGTGTTAGGATTAACCTAAGCTG! 53.1! 265!bp! Rev.! GGAAGCAGACTAAGTAACCTTG! 53.0! 3* HH09VC3! CTTTT(7)! For.! AGATGCTGGTCTGAAGTTTGC! 55.5! 237!bp! Rev.! CTGAACACAGGTTAGAATTTGATTTGC! 55.1! 4* HH09VE8! AAAC(7)! For.! CACTCCCATTCATCTTCAGAGAC! 55.0! 197!bp! Rev.! GGATTAACCTAAGCTGCATCTCC! 55.3! 5* HH10VB6! CAGAA(4)CAAAC(3)! For.! CTGACCTGCCATTCCAGAAC! 55.9! 145!bp! Rev.! GCTCATTTCCTCGGAGACAC! 55.5!
6* HH11VB2! GAAAGGAA(7)! For.! TACATCTATTCAGGATGGAGGC! 53.6! 257!bp!
Rev.! GGAACATTAGCTTGTGATTGC! 52.3!
7* HH11VD11! CTTT(11)! For.! GGATGACTCCATACCTTACCC! 54.2! 260!bp!
Rev.! GAGCCACAGCCAACTTTATTG! 54.4!
8* HH11VG7! CAGCTTTCTTT(10)! For.! TGTATATTCCACTGAGACAGCTG! 54.1! 192!bp!
Rev.! CAGTAATGGTGCCTTGGTG! 53.7!
9* HH13VE12! AGAT(6)! For.! TCAAAAGTTGTGAGTGAGGCTAC! 55.0! 161!bp!
Rev.! GTGATTCAGATGCTCCCTGG! 55.4!
10* HH13VG11! ATAG(9)! For.! CCTCTATCCCAAACTGCA! 51.5! 118!bp!
Rev.! GCCATTACTAACTCTTGGAATTC! 51.6! ! =!Sequence!is!blocked!for!public!sharing!
! !
55!°C! and! 1.5! mM! MgCl2.! A! touchdown! PCR! from! 62! V! 52! °C! was! performed! for! marker! HH02VB1.! The! results! are! shown! in! figure! 4.2.! All! nine! markers! using! a! Ta! of! 55! °C! successfully! amplified! the! expected! fragment.! Marker! HH02VB1! shows! a! smear! from! approximately! 120! bp! –! 250!bp! using! 1.5! mM! MgCl2! in! lane! 13! and! a! smear! from! approximately! 120! bp! –! 200! bp! using! 3.0mM! MgCl2! in! lane! 14.! Therefore,! the! expected! fragment!of!166!bp!cannot!be!clearly!distinguished.!A!new!reverse!primer!was!designed!for! marker!HH02VB1,!but!no!fragment!could!be!amplified.!Because!no!new!forward!primer!could! be!designed,!research!was!continued!with!the!other!nine!markers.!
!
4.5** Sequencing*PCR*products*
For! all! nine! markers! both! the! forward! and! reverse! primer! was! used! to! sequence! the! PCR! product.! Markers! HH09VC1,! HH09VC3,! HH09VE8,! HH11VB2,! HH11VD11,! HH11VG7! and! HH13V E12!have!been!confirmed!by!sequencing!to!amplify!specific!products.!Markers!HH10VB6!and!
!
HH13VG11!still!need!to!be!confirmed!by!sequencing!because!there!was!either!no!sequence! data! available! or! the! quality! of! the! sequence! was! really! poor! and! could! not! be! used! for! confirmation.!Figure!4.3!shows!the!cloned!vector!sequence!of!marker!HH11VB2!(A)!and!the! sequence!of!the!PCR!product!generated!using!hen!harrier!specific!primer!(B).! * * Figure*4.2*Picture*of*2*%*gel*showing*the*PCR*products*for*all*10*markers.'Lanes!1!to!5!and!7!to!10! show!the!nine!markers!with!a!Ta!of!55!°C!and!1.5!mM!MgCl2.!Lanes!13!and!14!show!marker!HH02VB1! with!a!touchdown!PCR!of!62!–!52!°C!and!the!MgCl2!concentrations!1.5!mM!and!3.0!mM.! ! ! Figure*4.3*Confirmation*of*a*marker*by*sequencing.!(A)!Shows!the!flanking!region!and!the!start!of!
the! microsatellite! in! the! cloned! vector.! (B)! Shows! the! flanking! region! and! the! start! of! the!
(A)*
!
4.6** STR*analysis*
A!total!of!six!markers,!divided!into!two!possible!triplexes,!have!been!tested.!The!size!of!the! markers!and!the!fluorescent!dye!label!used!is!shown!in!table!4.4.! ! ****Table*4.4*Overview*of*available*markers*with*a*fluorescentXlabelled*forward*primer.* ! Triplex*one* Triplex*two*Marker* HH13VG11! HH09VE8! HH09VC1! HH13VE12! HH11VG7! HH11VD11!
Dye* FAM! HEX! FAM! FAM! HEX! JOE!
Size* 118!bp! 197!bp! 265!bp! 161!bp! 192!bp! 260!bp!
!
Between! 22! and! 24! unrelated! individuals! were! used! to! determine! polymorphism! and! the! allelic!range!of!each!marker.!Figure!4.4!shows!a!map!of!the!UK!that!indicates!the!locations!of! the!different!individuals.!Grid!references!were!used!when!available,!otherwise!the!location! on! the! buccal! swab! label! was! used.! One! location! (circled)! may! be! incorrect! since! it! is! not! likely!that!hen!harriers!nest!in!that!location.!
Figure*4.4*Map*of*the*United*Kingdom*showing*the*locations*of*the*chicks*used*for*STR*analysis.*It!
is!unknown!whether!the!circled!location!in!the!south!of!England!is!correct!since!hen!harriers!have!not! been!recorded!as!nesting!here.!
!
The!raw!data!of!the!STR!analysis!can!be!found!in!Appendix!V.!The!bins!for!each!allele!have! been! calculated! using! a! 99! %! confidence! interval! (CI).! The! standard! deviation! (SD)! was! multiplied!by!3!and!this!value!was!done!plus!and!minus!the!product!size.!
!
4.6.1** Markers*HH13XG11*and*HH13XE12*
Table! 4.5! and! 4.6! show! the! STR! analysis! results! for! markers! HH13VG11! and! HH13VE12.! At! both!markers!all!22!individuals!possessed!the!same!sized!PCR!product.!No!alleles!were!found! being!out!of!bin.!The!electropherograms!of!two!individuals!at!marker!HH13VG11!(A)!and!(B)! are!shown!in!figure!4.5!and!two!individuals!at!marker!HH13VE12!(C)!and!(D)!are!shown!in! figure!4.6.! ! Table*4.5*STR*analysis*results*of*marker*HH13XG11* HH13XG11*
HeteroXzygotes* Alleles* Samples* Repeat*motif*
V! 1! 22! (ATAG)9!
!!
Allele* size*(bp)* σ*(SD)*Product* Bin*(bp)* Number*found* Freq.*
9! 116.14! 0.029! 116.06! 116.22! 44! 1.0!
* *
Table*4.6*STR*analysis*results*of*marker*HH13XE12*
HH13XE12*
HeteroXzygotes* Alleles* Samples* Repeat*motif*
V! 1! 22! (AGAT)6!
!!
Allele* size*(bp)* σ*(SD)*Product* Bin*(bp)* Number*found* Freq.*
6! 161.16! 0.037! 161.05! 161.27! 44! 1.0!
* *
****Figure*4.5*Electropherograms*of*two*individuals*(A)*and*(B)*at*marker*HH13XG11!! !
! !!!!Figure*4.6*Electropherograms*of*two*individuals*(C)*and*(D)*at*marker*HH13XE12.! ! ! 4.6.2** Markers*HH09XC1*and*HH09XE8* Table!4.7!and!4.8!show!the!STR!analysis!results!for!markers!HH09VC1!and!HH09VE8.!A!total!of! four!alleles!were!observed!at!both!markers,!with!allele!6!being!found!twice!and!alleles!7,!8! and!9!between!12!and!16!times.!This!resulted!in!the!allele!frequencies:!0.045,!0.364,!0.318! and!0.273.!No!alleles!were!found!being!out!of!bin.!Figure!4.7!shows!four!individuals!(E),!(F),! (G)!and!(H)!at!marker!HH09VC1!and!figure!4.8!shows!four!individuals!(I),!(J),!(K)!and!(L)!at! marker!HH09VE8.! ! Table*4.7*STR*analysis*results*of*marker*HH09XC1* HH09XC1*
HeteroXzygotes* Alleles* Samples* Repeat*motif*
59.1%! 4! 22! (TTTG)9!
!!
Allele* size*(bp)* σ*(SD)*Product* Bin*(bp)* Number*found* Freq.*
6! 254.70! 0.021! 254.64! 254.76! 2! 0.045! 7! 258.71! 0.050! 258.56! 258.86! 16! 0.364! 8! 262.68! 0.029! 262.60! 262.76! 14! 0.318! 9! 266.70! 0.037! 266.59! 266.81! 12! 0.273! * Table*4.8*STR*analysis*results*of*marker*HH09XE8! HH09XE8*
HeteroXzygotes* Alleles* Samples* Repeat*motif*
59.1%! 4! 22! (AAAC)7!
!!
Allele* size*(bp)* σ*(SD)*Product* Bin*(bp)* Number*found* Freq.*
6! 191.98! 0.049! 191.83! 192.12! 2! 0.045! 7! 195.82! 0.050! 195.67! 195.97! 16! 0.364! 8! 199.89! 0.041! 199.77! 200.01! 14! 0.318! 9! 203.91! 0.052! 203.76! 204.06! 12! 0.273!
!
Figure*4.7*Electropherograms*of*four*individuals*(E),*(F),*(G)*and*(H)*containing*all*four*alleles*of* marker*HH09XC1.*
!
4.6.3** Marker*HH11XG7*
At!marker!HH11VG7!with!a!repeat!unit!of!11!bp!a!total!of!six!alleles!have!been!observed,!see! table!4.9.!!Alleles!11!and!12!were!only!found!once!and!twice.!The!other!four!alleles!were! found! between! four! and! 18! times.! This! resulted! in! the! allele! frequencies:! 0.391,! 0.283,! 0.174,!0.087,!0.022!and!0.043.!No!alleles!were!found!being!out!of!bin.!Four!individuals!(M),! (N),!(O)!and!(P)!are!shown!in!figure!4.9! ! ! Table*4.9*STR*analysis*results*of*marker*HH11XG7! HH11XG7*
HeteroXzygotes* Alleles* Samples* Repeat*motif*
60,9%! 6! 23! (CAGCTTTCTTT)10!
!!
Allele* size*(bp)* σ*(SD)*Product* Bin*(bp)* Number*found* Freq.*
7! 157.21! 0.024! 157.14! 157.28! 18! 0.391! 8! 168.23! 0.050! 168.08! 168.38! 13! 0.283! 9! 179.26! 0.043! 179.13! 179.38! 8! 0.174! 10! 190.30! 0.029! 190.21! 190.38! 4! 0.087! 11! 201.39! V! V! V! 1! 0.022! 12! 212.93! 0! V! V! 2! 0.043! ! * Figure*4.9*Electropherograms*of*four*individuals*(M),*(N),*(O)*and*(P)*containing*all*six*alleles*of* marker*HH11XG7.* ! !
! 4.6.4** Marker*HH11XD11* Figure!4.10!shows!electropherograms!of!four!individuals!(Q),!(R),!(S)!and!(T)!at!marker!HH11V D11.!Individuals!(Q),!(R)!and!(T)!are!heterozygous!and!individual!(S)!seems!homozygous.!All! alleles!contain!multiple!peaks!all!1!bp!different!in!size.!It!was!not!possible!to!analyse!the!data! for!this!marker!since!it!is!unknown!which!peak!is!the!real!allele.!However!the!marker!does! seem!to!be!polymorphic!with!an!estimated!four!alleles.! * * Figure*4.10*Electropherograms*of*four*individuals*(Q),*(R),*(S)*and*(T)*at*marker*HH11XD11* * * 4.6.5** HWE*and*PowerStatsV12* Because!markers!HH09VC1!and!HH11VG7!could!be!used!for!a!possible!multiplex!PCR!the!two! markers! were! tested! for! HWE! and! linkage! disequilibrium.! Both! markers! are! in! HardyV Weinberg!equilibrium!and!linkage!equilibrium!(P!>!0.05).!PowerStatsV12!was!used!on!both! markers!to!check!the!forensic!and!paternity!parameters,!see!table!4.10.!! ! Table*4.10*PowerStatsV12*results*of*markers*HH09XC1*and*HH11XG7* ! HH09XC1* HH11XG7* Forensic* ! ! Matching!Probability! 0.165! 0.138! !!!!!!!!!Expressed!as!1!in!…! 6.1! 7.2! Power!of!Discrimination! 0.835! 0.862! PIC! 0.63! 0.68! Paternity* ! ! Power!of!Exclusion! 0.280! 0.301!
!
4.7** Multiplex*PCR*
As!a!preliminary!trial,!a!multiplex!PCR!was!performed!in!duplicate!on!two!individuals:!the! adult!female!(sample!CC5)!and!a!buccal!swab!(sample!5!from!Strath!Fleet,!Scotland)!using! the!loci!HH09VC1,!HH11VG7,!D220!and!the!sexing!marker!CHD1!(Henderson!at!al,!2012).!The! D220!locus!was!characterised!and!optimized!by!Le!Roux!(unpublished).!Table!4.11!shows!the! size!and!fluorescent!dye!for!each!marker.!! ! *****************************Table*4.11*Overview*of*used*markers*showing*size*and*dye*colour! Marker* CHD1* HH09XC1* HH11XG7* D220*Dye* FAM! FAM! HEX! HEX!
Size* 212/219!bp! 265!bp! 192!bp! 295!bp! !
PCR!products!were!checked!on!a!3!%!agarose!gel,!see!figure!4.11.!Locus!D220!shows!two! fragments! of! approximately! 320! bp! and! 400! bp! for! sample! 5! (SNWHL)! and! one! thick! fragment! of! approximately! 300! bp! for! sample! CC5.! Locus! HH09VC1! shows! a! fragment! of! approximately! 250bp! in! all! four! samples.! The! bright! fragments! observed! at! approximately! 210! bp! in! all! four! samples! belong! to! the! sexing! marker! CHD1,! which! has! a! size! of! 212! /! 219!bp.! A! very! faint! product! of! approximately! 180! bp! is! visible! at! sample! CC5,! but! two! fragments! of! approximately! 170! bp! and! 190! bp! are! visible! for! sample! 5! (SNWHL).! These! fragments! belong! to! marker! HH11VG7,! which! has! a! size! of! 192! bp.! Figure! 4.12! shows! the! electropherograms! of! both! samples! and! table! 4.12! shows! the! size! of! each! peak.! Different! alleles!have!been!observed!for!each!sample!at!the!loci!HH09VC1,!HH11VG7!and!D220.! !! ! ! Figure*4.11*Picture*of*a*3*%*agarose*gel*showing*the*PCR* products*of*the*multiplex*PCR.*Lane!1!contains!a!100!bp! sizing!standard.!Sample!5!(SNWHL)!is!shown!in!lane!2!and! sample! CC5! is! shown! lane! 3.! The! negative! control! is! shown! in! lane! 4.! The! markers! have! the! following! expected! size:! D220! (295bp),! HH09VC1! (265bp),! Sexing! (211/218!bp)!and!HH11VG7!(192!bp).!
!
!
Figure' 4.12' Electropherogram' showing' the' loci' HH09;C1' (FAM),' HH11;G7' (HEX),' D220' (HEX)' and' the' sexing' marker' CHD1' (FAM)' for' the' samples' 5' (SNWHL)'and'CC5.!! ! ! ! ! ! ! ! Table'4.12'Sizes'of'the'peaks'in'the'electropherogram' ! CHD1! HH09)C1! HH11)G7! D220! 5!(SNWHL)! 211.26! 218.89! 254.72! 258.73! 157.37! 179.42! 325.25! 394.45! CC5! 211.29! 218.88! 258.73! 266.64! 168.37! 190.44! 310.08! 330.31! !
!
5.## Discussion#
!5.1## Primer#design#and#optimization#
Primers!were!designed!for!microsatellites!with!a!minimum!of!six!consecutive!repeat!units!to! increase!the!likelihood!that!the!locus!would!be!polymorphic!(Weber!&!May,!1989).!It!was! not! always! possible! to! design! optimal! primers! when! only! 22! nucleotides! were! present! between! the! end! of! the! vector! and! the! beginning! of! the! microsatellite.! A! wellHdesigned! primer!should!work!at!a!range!of!different!annealing!temperatures.!It!was!hoped!this!would! be! the! case! for! markers! HH02HB1! and! HH13HG11.! Marker! HH02HB1! did! show! signs! of! amplification!but!resulted!in!many!nonHspecific!fragments.!Therefore!a!touchdown!PCR!was! performed.!This!PCR!reaction!starts!with!a!higher!annealing!temperature,!which!is!dropped! by! a! few! degrees! each! cycle! until! the! optimal! annealing! temperature! is! reached.! This! can! help!prevent!nonHspecific!binding!of!the!primers!(Don!et!al.!1991).!When!the!touchdown!PCR! and!a!new!reverse!primer!did!not!result!in!amplification!and!no!new!forward!primer!could! be!designed,!the!marker!was!abandoned.! !5.2## Selecting#samples#for#STR#analysis#
The!samples!selected!for!the!initial!screening!originated!from!various!locations!across!the! United! Kingdom! to! ensure! all! possible! alleles! for! a! particular! marker! would! be! observed.! Selecting!only!one!chick!from!a!single!nest!avoided!related!individuals.!In!this!study!between! 22!and!24!samples!have!been!used!to!check!whether!a!marker!is!polymorphic.!This!is!not! enough!to!give!an!accurate!allele!frequency!but!does!indicate!the!allelic!range!of!a!marker! that!is!needed!when!designing!a!multiplex!PCR!reaction.!Further!genotyping!will!be!carried! out!once!all!loci!have!been!characterised!and!optimised!for!this!purpose.!! !5.3## Hen#harrier#STR#markers#
! 5.3.1## HH13AE12#and#HH13AG11#The! markers! HH13HE12! and! HH13HG11! both! turned! out! to! be! monomorphic! even! though! they! contained! six! and! nine! repeats.! Therefore! these! markers! cannot! be! used! for! individualisation!purposes.!Marker!HH13HE12!has!been!confirmed!by!sequencing!to!amplify! the!specific!microsatellite!region,!but!marker!HH13HG11!has!not!been!confirmed!yet.!Marker! HH13HG11!could!amplify!a!different!region!instead!of!the!microsatellite!region.!This!could!be! observed!as!double!bands!or!fuzzy!bands.!A!fuzzy!band!seems!to!be!present!at!figure!4.2.!A! fragment!of!the!right!size!is!being!amplified!with!the!expected!product!size!of!118!bp!and! the!electropherogram!showing!peaks!of!116!bp.!Perhaps!the!markers!HH13HE12!and!HH13H G11!do!not!show!polymorphism!within!the!hen!harrier!but!do!show!signs!of!polymorphism! in!another!birds!of!the!family!Accipitridae.!Research!by!Busch!et!al.!2005!revealed!a!locus!
!
was! monomorphic! in! the! species! Aquila' heliacal,! but! six! alleles! were! found! in! Haliaeetus' albicilla.! Two! of! the! six! tested! microsatellite! markers! were! monomorphic,! this! is! equal! to! 33!%.!An!inbred!population!could!be!the!cause.!When!the!population!is!inbred!fixed!alleles! are!being!observed!at!microsatellite!markers.!! ! 5.3.2## HH09AC1#and#HH09AE8# The!markers!HH09HC1!and!HH09HE8!have!been!confirmed!by!sequencing.!However!an!error! was!made!and!the!markers!turned!out!to!be!the!same.!For!marker!HH09HC1!a!total!of!four! alleles! were! observed.! This! is! a! small! number! of! alleles! but! a! multiplex! kit! developed! by! Bielikova!et!al.!2010!for!Aquila'chrysaetos!also!used!markers!with!three!and!four!observed! alleles!within!wildHliving!and!captiveHbred!individuals.!!!
!
5.3.3## HH11AD11#
Marker!HH11HD11!seemed!to!be!polymorphic!but!could!not!properly!be!analysed!because!of! multiple! peaks,! all! 1! bp! different! in! size,! for! each! allele.! The! PCR! product! looks! like! a! mononucleotide!marker!even!though!the!main!microsatellite!sequence!is!a!tetranucleotide.! Stutter! products! are! normally! 4! bp! smaller! in! size! than! the! actual! allele! in! case! of! a! tetranucleotide!repeat.!The!additional!peaks!in!between!could!be!explained!by!the!THstretch! that!is!present!in!this!marker!immediately!after!the!‘CTTT’!repeats.!During!the!PCR!reaction! T’s!can!be!added!or!deleted,!creating!multiple!products!each!1!bp!different!in!size.!Because! the!THstretch!is!present!immediately!behind!the!microsatellite!it!is!impossible!to!design!new! primers!that!do!not!include!the!THstretch.!If!for!instance!allele!10!always!contains!the!same! number!of!peaks!with!the!exact!same!lengths!in!bp,!it!might!be!possible!to!determine!allele! 10!in!all!individuals.!A!software!program!called:!Peakseeker!(Thompson!&!Salipante,!2009)! makes!it!possible!to!determine!genotypes!of!mononucleotide!repeats.!Eventhough!signs!of!a! mononucleotide! repeat! is! present! at! this! marker! it! is! doubtful! whether! genotypes! can! be! determined! with! this! program.! ! The! accuracy! of! the! program! declines! when! alleles! are! separated!in!length!by!2!bases!and!this!marker!should!have!differences!of!4!bases.! !
5.4## Statistical#tests#
The!markers!HH09HC1!and!HH11HG7!have!been!tested!for!HardyHWeinberg!equilibrium!and! linkage!disequilibrium.!The!tests!concluded!there!is!no!significant!departure!from!HWE!and! linkage!disequilibrium.!Once!all!loci!have!been!characterised!more!samples!will!be!used!to! check!for!HWE.!For!marker!HH09HC1!and!HH11HG7!a!matching!probability!below!0.17!was! found! resulting! in! power! of! discrimination! values! higher! then! 0.83.! A! high! power! of! discrimination! results! in! higher! chances! two! individuals! will! contain! different! genotypes.! Both!markers!are!informative!with!PICs!of!0.63!and!0.68.!The!found!values!in!this!research!!
differ!only!a!bit!with!the!values!found!by!Mpoloka!et!al.!2008!and!they!could!be!used!for! identification!purposes.!
!
5.5## Multiplex#PCR#
A! breakthrough! was! achieved! when! a! multiplex! PCR! reaction! using! 3! loci! and! the! sexing! marker!amplified!successfully.!The!electropherogram!and!the!agarose!gel!of!the!multiplex! PCR,! however,! did! show! some! unexpected! differences.! A! clear! difference! in! fragment! intensity! is! visible! on! agarose! gel! between! marker! HH11HG7! and! CHD1.! The! fragment! intensity!of!CHD1!is!much!higher,!however!when!looking!at!the!electropherogram!the!peak! heights!of!HH11HG7!are!not!lower!then!those!of!CHD1.!In!fact!it!is!the!opposite.!Perhaps!the! fluorescent! dye! of! the! sexing! marker! started! to! degrade! by! exposure! to! light.! This! would! results!in!lower!peaks!on!a!genetic!analyser,!but!a!normal!intensity!product!would!be!visible! on!gel.!!
!
The!211!bp!product!of!the!sexing!marker!shows!an!additional!peak,!which!looks!like!a!stutter! product.!This!should!not!be!the!case!since!the!sexing!marker!is!not!a!microsatellite.!Stutter! products! only! occur! at! STRs.! ! An! explanation! for! the! additional! peak! could! be! partial! adenylation.!The!cycling!conditions!for!the!multiplex!reaction!uses!a!final!extension!time!of! 60! minutes! of! 60! °C.! This! should! be! long! enough! in! order! to! obtain! full! adenylation.! Therefore! the! reverse! primer! of! CHD1! might! need! ‘PigHtailing’! at! the! 5’Hend.! This! should! result!in!full!adenylation!(Brownstein!et!al.!1996).!The!218!bp!product!of!the!sexing!marker!is! significantly!lower!in!height!compared!to!the!211!bp!product!in!both!individuals.!Both!peaks! should!be!similar!in!height.!! ! The!peaks!of!the!other!three!markers!look!good.!It!is!clearly!visible!that!different!profiles! have!been!obtained!for!both!individuals.!Because!this!multiplex!reaction!was!a!first!test!no! optimization!has!been!performed!yet.!The!primer!concentrations!of!the!sexing!marker!and! D220!should!be!increased!in!order!to!obtain!similar!peak!heights!for!all!markers.!! !! ! !!! !!
!
6.## Conclusion#
!
The! goal! of! this! research! was! to! develop! a! multiplex! PCR! for! the! individualisation! of! hen! harriers!that!could!be!used!to!contribute!to!the!prosecution!of!a!suspect!in!cases!of!illegal! killings.! From! the! six! characterized! microsatellites! the! markers! HH13HE12! and! HH13HG11! turned! out! to! be! monomorphic! and! markers! HH09HC1! and! HH09HE8! turned! out! to! be! the! same!locus.!Marker!HH11HD11!showed!signs!of!polymorphism!but!due!to!additional!peaks!at! each! allele! it! needs! further! optimisation! before! it! can! be! used! successfully.! HH09HC1! and! HH11HG7!were!found!to!be!polymorphic!with!four!and!six!alleles!observed!respectively.!Both! markers!showed!reasonable!power!of!discrimination!and!informativeness.!
!
A!first!step!towards!achieving!the!main!goal!of!this!research!was!made!when!a!multiplex!PCR! reaction! containing! the! markers:! HH09HC1,! HH11HG7,! D220! and! the! sexing! marker! CHD1! amplified! successfully.! Two! different! profiles! were! established! for! two! individuals.! After! optimisation,!this!multiplex!PCR!reaction!can!be!used!as!a!starting!point!for!a!multiplex!STR! kit! for! hen! harriers.! Additional! markers! can! be! added! until! a! reaction! is! created! with! the! desired!number!of!markers.!!
!
7.## Future#research#
!
Of! the! 10! designed! primer! pairs,! markers! HH10HB6! and! HH13HG11! have! not! yet! been! confirmed!to!amplify!specific!products!by!sequencing.!Once!HH10HB6!has!been!confirmed,! fluorescent!primers!could!be!ordered!and!tested!on!a!minimum!of!30!unrelated!individuals.! The! forward! and/or! reverse! primer! of! the! sexing! marker! CHD1! have! to! be! reHdesigned! to! check!if!that!solves!the!double!peak!problem.!!
A! set! of! unrelated! individuals! (30! to! 50)! that! represent! a! real! population! in! the! United! Kingdom!have!to!be!screened!using!all!polymorphic!markers!in!order!to!check!if!they!are!in! HWE.!!!
At! the! moment! a! multiplex! PCR! reaction! is! working! that! incorporates! 3! loci! and! a! sexing! marker!CHD1.!A!schematic!overview!of!the!multiplex!is!given!in!figure!7.1.!The!markers!with! a!black!edge!are!currently!incorporated!in!the!multiplex!PCR.!At!the!time!of!writing!five!new! fluorescentHlabelled!markers!were!ordered:!two!FAM,!one!HEX!and!two!NED!shown!in!the! figure! without! a! black! edge.! If! these! markers! turn! out! to! be! polymorphic! a! multiplex! PCR! could!be!created!with!eight!loci!and!a!sexing!marker.!The!figure!shows!there!is!still!space!left! for! more! markers:! a! FAM! labelled! marker! bigger! then! 265! bp,! a! HEX! labelled! marker! between!225!bp!and!260!bp!and!NED!labelled!markers!bigger!then!170bp.!The!four!markers! that!have!not!been!tested!for!polymorphism!yet!could!be!incorporated!into!these!regions! along!with!others!that!are!currently!being!developed!(Le!Roux!et!al.,!unpublished).!Once!an! appropriate! number! of! markers! has! been! incorporated,! the! PCR! reaction! needs! to! be! optimised!so!all!markers!are!amplified!optimally.!After!optimisation!the!multiplex!kit!needs! to!be!validated.!!
!
!
Figure# 7.1# Schematic# overview# of# markers# used# in# the# multiplex# PCR# (black# edge)# and# ordered# markers.##
FAM#
HEX# NED#
!
References!
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Appendix##
!Appendix#I:#DNeasy#Blood#&#Tissue#Kit#(Qiagen)#
! !!
