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

TrainingCSI’s

Cheshireexcavationsite

• Noupstandingremainsleft

• Associatedwith12-13thcenturyAbbeyandChapel

• Saxon,Romanandprehistoricelements

• Highproportionofthefarmlandistodayusedforpasturage

• Soilmostlystagnogley

DNA analysis of medieval population

Differencesinpreservationundersoil

2 feet

- Lots of variation in preservation of skeletons within just few feet distance (same layer)

Soilacids

• Fulvic acid• Humic acid;complexmixtureofacids

Location

- EightkilometerssouthofChester- Mostlyhomogenouslandscape

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

5

DNAisolation

• First:collectthecellularmaterial

• Lysis ofcells– Bonemill– Mortarandpestle– Liquidnitrogen

• -196C(−321 F)• Nobiologicalactivitywhentemperatureislow

– Enzymes:proteinaseK

DNAbindingcolumns

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

Amplify/copyDNA

Current DNA profilingMultiplex microsatellite analysis:

1) Sex of the victim/suspect2) Identity, unless identical twins or DNA old or in bad condition (fragmented)

Role of genotyping in Forensic Science

Analysisofold,fragmentedorancientDNA

• GenomicDNAcanbevirtuallyfullydegradeinjustover10years.Deadend?Solution:mtDNA

• ManylaboratoriesanalyzeDNAupto10,000yearsold

• Bloodresidueshavebeenfoundfromtoolswhichhavebeendatedtobe100,000yearsold

GenomicDNAvs.mtDNA

• BecauseofthenatureofthemtDNA,itcannever,regardlessofcondition,giveasgoodmatchasthegenomicDNA.– mtDNA vs CODIS/SGM+/DNA17systems– Romanovfamily– RemainsofNikolasCopernicus

KinshipanalysisusingmtDNA

TVdocumentary:PrimeTimeSuspect:JackTheRipper(2011)

Robin Napper Russell Edwards

“Theshawl”

Over2mlongSilk200yearsold

JacktheRipperVictims

Doubleswab/wetswabtechniqueusedinthe2011documentary

• Removesmaterialfromthesurfacefordrugand/orDNAprofiling

• Workswellwithhardmaterials

• Contaminationrisk

NewlowcopynumberDNAlab

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

SoevenifwegetmtDNA profile,thenwhat?

Catherine Eddowes

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.

ç ç

2001- >

30ofthe31mainsuspects

“Heywethinkyourgreatgreat grandfatherisa

murdered,canwehaveyourDNAsampletoverifyit?”

Goodluck…

CatherineEddowesAaronKosminsky

12345678910111213

1234567891011

Lane7=ReferenceLane8=Victim

Lanes1-6and9-13correspondtomainsuspect

Thisisapartialhaplotype,containingmultiplemtDNA “markers”.Forexample,lane7and8

TheGreatSemenhuntSingle cell => single genomic DNA = from one person only => no mixed profiles

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

15minutesbeforeBBCBreakfastshowIsawthebackcoverofthe

bookfortheveryfirsttime

TVandradio:BBCLondon

ITVITNCNNABCCBS

Al-JazeeraFoxNewsChicagoTVSpanishTVFinnishTVPolishTV

RadioIsraelRadioJapanRadioChina

RadioAustraliaRadioToronto

etc.Over100interviews,andovertwoyearslater…stillgoingon

What followed

• Phones tapped • Laptops hacked• Landline cut• Stalkers• Blackmail• Etc.

a

R

R&D: Water profiling project

a

R

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

Twins: 1999 crime - 2004 arrest – 2014?

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