View
186
Download
1
Embed Size (px)
Citation preview
Dr.JariLouhelainen,MSc,PhDDocentofBiochemistry
SeniorLecturerofMolecularBiology
Applicationsofgenetictoolsforsolvingcrimes,including
medievalmurdersandthecaseofJackTheRipper
Karolinska Institutet, Sweden
University of Oxford
Cancer Research UK
Sanger Centre
Paterson Institute
University of Helsinki, Finland
- DNA/RNA work- Cold cases - Medieval bones - Dogs, Squirrels, Elephants!
- Gene expression- Korean FA, Liverpool FC- Psoriasis, rare blood
antigens, sports nutrition
- Health & Forensic applications
- Treatment, analysis
Cheshireexcavationsite
• Noupstandingremainsleft
• Associatedwith12-13thcenturyAbbeyandChapel
• Saxon,Romanandprehistoricelements
• Highproportionofthefarmlandistodayusedforpasturage
• Soilmostlystagnogley
Differencesinpreservationundersoil
2 feet
- Lots of variation in preservation of skeletons within just few feet distance (same layer)
WhyDNAanalysis• Proteinpolymorphisms
– Biologicalactivitygonesoonafterdeath– Testmaynotabletoresolvespeciesthataregeneticallysimilar
– Lotsofstartingmaterialneeded
• DNA– Canbeamplifiedfromminuteamounts(PCR)– Candistinguishspecies– Canbeisolatedfromsamplesevenhundredsofyearsold– DNApresentinallcells(except:redbloodcells)
DNAandDNAprofiling
FreshgenomicDNA DNAprofiling
• Forensics:theisolatedDNAcannotbeseenbynakedeyeornormallightmicroscope– Cellscanbeseenat400xmagnification
• TheDNAhastobeamplified– Severalmillionfoldneeded
– Nobelpricein1994
Where do we get DNA from?• Blood• Saliva• Semen• Feathers• Antlers• Hair roots and dandruff• Tissues/organs• Nail scrapings• Bones/ancient remains• Faeces• Urine
1) DNA isolation
2) DNA amplification and profiling
3) Compare the profile to suspect or database
1) g
DNAisolation
• First:collectthecellularmaterial
• Lysis ofcells– Bonemill– Mortarandpestle– Liquidnitrogen
• -196C(−321 F)• Nobiologicalactivitywhentemperatureislow
– Enzymes:proteinaseK
NormalHumanKaryotypeWe have:
- Two copies of each chromosome
- Two copies of each gene
- Often not exactly identical
- The variation is considered normal, unless linked to a disease, like cancer
Current DNA profilingMultiplex microsatellite analysis:
1) Sex of the victim/suspect2) Identity, unless identical twins or DNA old or in bad condition (fragmented)
Analysisofold,fragmentedorancientDNA
• GenomicDNAcanbevirtuallyfullydegradeinjustover10years.Deadend?Solution:mtDNA
• ManylaboratoriesanalyzeDNAupto10,000yearsold
• Bloodresidueshavebeenfoundfromtoolswhichhavebeendatedtobe100,000yearsold
GenomicDNAvs.mtDNA
• BecauseofthenatureofthemtDNA,itcannever,regardlessofcondition,giveasgoodmatchasthegenomicDNA.– mtDNA vs CODIS/SGM+/DNA17systems– Romanovfamily– RemainsofNikolasCopernicus
TVdocumentary:PrimeTimeSuspect:JackTheRipper(2011)
Robin Napper Russell Edwards
“Theshawl”
Over2mlongSilk200yearsold
Doubleswab/wetswabtechniqueusedinthe2011documentary
• Removesmaterialfromthesurfacefordrugand/orDNAprofiling
• Workswellwithhardmaterials
• Contaminationrisk
Newsamplingmethods
• Astheshawlhashistoricalvalue,non-invasivetechniqueshadtobeused– ChemicalsusedinDNAworkcanleavestainsanddissolvethefabric
– Cuttingandotherdestructivemethodscouldnotbeused
• Developnewtechniquesforsampling,forexampletoavoidsurfacecontamination
Selectivesampling• Crimelightanalysis• Identificationofforensicallyrelevantstains• Onlythesestainsweresampled• Surfacesamplingandvacuuming
Silk structure
Novelsamplevacuumingmethods
Newcommercialsystem(M-Vac,USA):
Custommadeforthisproject:
Only1-2dropletspersample
Considerably higher volumes of water in this system – would have ruined the shawl (the blue dye on the shawl is water soluble)
DNAofunknownorigin
• Whatspeciesdidthissamplecomefrom?–Areallthesesamplesfromthesamespecies?
– Fromhuman?
• Arethesesamplesfromthesameindividual?
• Here:testingifthestainswerefaked,say,withhorse,pigorcowbloodDNA
DNAdegradeswithtime
1 agaaggggcc ttctgaggtt tgggggctgt agggccatgg gcctcagggc cagaggtggt61 tgttagcctg gcaagacagg tctgggcaac atgcatgccc agaggcagtt cgttgtagct121 gcagtgagag cagaagtcag acgacatgag gtggccaagc aggctctaaa ccgcctcagg181 aagctggcag agagggtgga cgaccccgaa ctccaggaca gcatccaggc ctcattggac241 agcattcgag aggctgtgat caatagccag gaatggactt tgagccgctc cattcctgaa301 ctgcgcctgg gtgtgctggg cgatgccagg agtgggaagt catcgctcat ccaccgattc361 ctgactggct cataccaggt gctggagaag acagagagtg agcagtacaa gaaagaaatg421 ttggtggatg gacagacaca tctggtgcta atccgagagg aagctggggc acctgatgcc481 aagttctcag gctgggcaga tgctgtgatc ttcgtcttca gcctggagga tgagaacagt541 ttccaggctg tgagccgtct ccatgggcag ctgagttccc ttcgcgggga gggacgagga601 ggcctggcct tggcactggt ggggacacaa gacaggatca gtgcttcctc ccctcgggtg661 gtgggagatg ctcgtgccag agctctgtgc gcggacatga aacgctgcag ctactatgag721 acttgtgcaa cctatgggct caatgtggat cgggtcttcc aggaggtggc
Fragment 1:
1 agcattcgag aggctgtgat caatagccag gaatggactt tgagccgctc61 cattcctgaactgcgcctgg gtgtgctggg cgatgccagg agtgggaagt121 catcgctcat ccaccgattcctgactggct
Fragment 2:
601 ggcctggcct tggcactggt ggggacacaa gacaggatca gtgcttcctc ccctcgggtg661 gtgggagatg ctcgtgccag agctctgtgc gcggacatga aacgctgcag ctactatgag721 acttgtgcaa cctatgggct caatgtggat cgggtcttcc aggaggtggc
Etc.
Heavily degraded DNA sample:
Good quality, freshly isolated DNA:
mtDNA analysis
• “Old”mtDNA extractedfromtheshawl• Multiplesamplingsfromindividualforensicallyimportantstains,linkedtothevictim
• DNAprofilingtooksometimebutfinallysuccessful
• But:noprofiletocompareitwith
Backandforth• ReceivedatextmessagefromRussellEdwards“Ifoundher!!”
• Nextproblem:– Consent;permissiontouseherDNA– HumanTissueAct/ethicalapprovalforsampling,asthiswasnotacurrentpolicecase
Catherine Eddowes
- Ethical approval for the project accepted (2nd round)
- Descendant happy to provide a DNA sample- She is a police officer!
- DNA sequencing
- Profile match!
- What does it prove?
DNA• Betweenindividualsweshare99.5%oftheDNAsequence
• DNAismadeoffournucleotides:A,G,CandT
123456789101112
Advanced stain mapping• Previously missed stains – semen-type
fluorescence, not blood– Modus operandi!
• Surely the semen in these stains must have degraded in 126 years? PSA negative.
“Heywethinkyourgreatgreat grandfatherisa
murdered,canwehaveyourDNAsampletoverifyit?”
Goodluck…
CatherineEddowesAaronKosminsky
12345678910111213
1234567891011
Lane7=ReferenceLane8=Victim
Lanes1-6and9-13correspondtomainsuspect
Thisisapartialhaplotype,containingmultiplemtDNA “markers”.Forexample,lane7and8
WholeGenomeAmplification(WGA)fromsinglecells
• Justafewofnucleatedcells• Optimisation ofthereaction• Thisopenednewavenuesopenedforthesuspectsprofiling:• Eyecolour• Haircolour• Skincolour
DNA analysis for phenotypic characteristics
- No single gene or variation determines our
eye colour
- 12-13 specific single nucleotide changes.
- Linked to our hair and skin colour as well
SNPs: Frequently OccurringGenetic Variants
Most of the population
Functional protein
Variant sequence
Common sequence
SNP site
G to C
At least 1 percent of the population
Functional protein
Redhair
Blueeyes
Browneyes
Tanning
Lighthair
Etc.
Summary of the findings
1) Mitochondrial DNA profile from the forensic stains related to the victim matched the haplotype of the maternal relative
2) DNA profile from the semen cells matched the maternal descendant of the suspect
3) Both profiles found from the same piece of evidence raises the statistical power of the findings
Summary of the findings
1) The hair/eye/skin colour characteristics from genomic DNA matched the only reliable eyewitness report of Jack the Ripper
1) Additional circumstantial evidence, like shawl characteristics (water-soluble blue dye, chemical tests, stain patterns etc.)
DNAworkbyothers• ProfessorIanFindlay
– Cellsfromthestampglueetc.– Sexdeterminationusingamelogenin genemarkers(XandYchromosomes)
• Peerreviewed? No• Dataavailable? No• Problems:Widelyknownsocalled“Alleledropout”problem=>oneoftheallelesamplifiesmorereadilywhenDNAisold/damaged.Andrareanomalies:
Ripperletters• Mostofthem,ifnotall,havebeenprovedtobefakes
• Twopeoplehavebeencaughtmanufacturingthem– Convicted– Botharefemales
• Evenifreal,postofficepersonnelmighthaveattachedthestamps,notJack?
DNAworkbyothers(cont.)• PatriciaCornwell
– Crimewriter– TestbasedonmtDNA fromletters– Unknownhiredscientists?– 1%matchwithpopulationinUK
• Estimated3-6millionpeopleinLondon=>matchwith30-60,000people
– Peerreviewed?No– Dataavailable?No– “Idobelieve100%thatWalterRichardSickertcommittedthesecrimes”
TheBookandTheMediaCircus
• EveryyearnewRipperbooksarepublished– Usuallytheygounnoticed
• Weweretoldtwoweeksbeforethat“youmightgettheDailyMailcover”– Weekafter:noyouwont,justatwo-pagespreadsomewhereinthemiddle
• TwodaysbeforeweweretoldthatBBCBreakfastmightbeinterested– Unlesstheyhavesomethingelse!
• Targetaudience:generalpublic– Notscientists
• However,treatedby(some)mediaasfull-blownscientificpaper
• Beforepublishing,somescientificdatahadtobeleftoutasthebookwasbecoming“tooscientific”accordingtothepublisher– Feedbackfromsomereadersaswell
• OnemarkerfromthemtDNA haplotypewasusedasanexample– reminder>:aDNAprofileisneverjustonemarker
• However,wehavethehaplotypeofthevictimand thesuspect=>p-value
TVandradio:BBCLondon
ITVITNCNNABCCBS
Al-JazeeraFoxNewsChicagoTVSpanishTVFinnishTVPolishTV
RadioIsraelRadioJapanRadioChina
RadioAustraliaRadioToronto
etc.Over100interviews,andovertwoyearslater…stillgoingon
River
Sea
Tap
Marie
Bottled
Bacteria 1Bacteria 2Bacteria 3Bacteria 4Bacteria 5Bacteria 6
Green=highrelativeabudanceRed=lowrelativeabundanceWhite=nochange
Tissue type profiling
• Not based on DNA but RNA– mRNA or miRNA
• Forensic science:– Can distinguish different body fluids– Can give support for witness statements, or
support victims/suspects statement• Medicine:
– Biobank quality control– Tracing cancer metastasis
Gene structure of eukaryotes
UTR = UnTranslated Region
‘upstream’
exon intron exon
‘downstream’
‘open reading frame’
Start codon Stop codon
5’ 3’
promoter
a
R
Noncoding region
Noncoding Regions
Exon
Gene 2
Gene 1
Noncodingintron
Protein 1
Protein 2
Noncoding region
End result: “signature” of a tissueSa
mpl
e 1
Sam
ple
2Sa
mpl
e 3
Sam
ple
4Sa
mpl
e 5
Sam
ple
6Sa
mpl
e 7
Sam
ple
8Sa
mpl
e 9
Sam
ple
10Sa
mpl
e 11
Sam
ple
12
Sam
ple
1Sa
mpl
e 2
Sam
ple
3Sa
mpl
e 4
Sam
ple
5Sa
mpl
e 6
Sam
ple
7Sa
mpl
e 8
Sam
ple
9Sa
mpl
e 10
Sam
ple
11Sa
mpl
e12
Marker 1Marker 2Marker 3Marker 4Marker 5Marker 6Marker 7Marker 8Marker 9Marker 10Marker 11Marker 12Marker 13Marker 14
Red = highly expressionGreen = low expressionBlack = not expressed
For example, we might expect markers 1-10 to be all red for a skin sample. Thus, samples 4-10 are not skin
Project Basics
• Development of Point-of-Care (POC) system• Health• Forensic• Environmental• Food Testing• Ecology
• Target users • Researchers • Practitioners
80
R&D: Point-of Care system
Advantages• Avoiding chain of custody and storage
problems – Sampling on the site– One person vs. officer/transport/receptionist/lab
assistant; less risk for manipulation and mix-up• Speed and cost
– Flexible platform, microscale analysis– Very fast feedback
• Decision making possible onsite
– Economical price per assay
Monozygotic identical twins
• Probability of monozygotic twins is app. 3 out of 1,000
• Next Generation Sequencing• Rare mutations (SNP’s)
• Forensic Science International: Genetics, Volume 9, March 2014, Pages 42–46
Identical twins
• Methylation of DNA affected by environmental factors –> twins have different methylation profile
• Cheaper than mutation profiling
• Problems can arise if :– Methylation changes after
sampling– Identical twins living in
similar environment
Analytical Biochemistry, Volume 476, 1 May 2015, Pages 36–39
FacialreconstructionbasedonDNAinformation
Musghot DNApaper fromMarch 2014
Demonstrationoftoday'saccuracyisshowninthe
photo=>
Inpractice5-10yearsmoredatarequired;large
populationsincludedinthecomputermodel
2016: Full facial effects of two different loci in PAX3, rs7559271 and rs974448
A/A A/G G/G
C/C, C/T, TT
T/T predominant