Easy Breezy Species IDfor the Forensic LabUsing a method that works for

even the most challenging samples

Terry Melton, PhDPresident and Laboratory Director

Mitotyping TechnologiesState College, PA

www.mitotyping.com

12S mtDNA species testing• Prior to 2004, 8% of our casework

hairs failed to amplify with human specific primers*

• 2 possible explanations:– Human hair with no detectable

(degraded) mtDNA– Hair is of non-human origin

May be important and useful information for investigation…

*Journal of Forensic Sciences: 691 Casework Hairs, 50:73-80

For example:

• (Actual case): 26 year old homicide of young boy…• Evidence was collected at the time of the crime, but

mitochondrial DNA analysis was not available until the late 1990s.

• Several crime scene hairs gave partial to full human mtDNA profiles that will be useful in examining crime theories and testing suspects.

• Two crime scene hairs gave no results…• Availability of species testing will keep investigators from

worrying if human hairs were just too degraded.• With 12S testing, species testing revealed one hair was

from a raccoon and one hair was from a pig.

Believe it or not:• Microscopic examination (although we

recommend it on every hair submission) is becoming less common prior to submission of hairs

• Even if carried out, not all examiners are successful in recognizing a non-human hair

HUMAN DEERHUMAN CAT HUMANDOG

Highly conserved regionstraditionally used for species testing

• 16S rRNA Mitochondrial DNA• 12S rRNA Mitochondrial DNA• Cytochrome b Mitochondrial DNA

• Original protocols used restriction endonucleases, later sequencing

Forensic application requires:

• Ease of use

• Specificity (at least to rule out human source)

• Applicability to degraded samples

• In our lab, most likely to be forensically relevant when larger animals (vertebrates->bones) and mammals (hair) are involved…

D-Loop = Forensic Analysis12s = Species Determination

From: Kitano T et al, International Journal of Legal MedicineDOI 10:1007/s00414-006-0133-7 (June 2007: Two universalprimer sets for species identification among vertebrates)

Primer selection attributed to Kocher et al. 1989, PNAS 86:6196

Protocol applied to extraction product

• PCR using primers that amplify ~100bp from the 12S ribosomal gene

• PCR product gel

• Sequence PCR product using same primers

• Import sequence into NCBI BLASTn tool and search for homologous sequences (www.ncbi.nlm.nih.gov/BLAST)

Q + -

Samples used in validationCommon Name

of Known Sample

GenBank Species Closest Match

12S rRNA

Product Size Trimmed of

Primers (bp)

Percent Homology of Lab Product to GenBank

Blast Search 12S rRNA

Human Homo sapiens 104 100%

Mountain lion Felix concolor 105 100%

Mouse Mus musculus 105 100%

Kangaroo rat Dipodomys ordii 102 100%

Squirrel Spermophilus lateralis 103 100%

Mule deer Odocoileus hemionus 104 100%

Chipmunk Tamias sibiricus 103 96% (closest match)

Dog Canis familiaris 103 100%

Testing strategy for hairsAmplify with regular human primer set ~250bp to capture

intact DNA

If no human-specific product

Amplify with human mini-primer set ~80-100bp to capture degraded DNA

If no human-specific product

Amplify with 12S primers to determine species

Case results using 12S species testing

Non-human species obtained from

casework hairs:– Dog– Cat– Goat– Rat– Pig– Raccoon

Case background

• Beauty supply retail store contacts Mitotyping regarding their wigs and hair extensions

• Received tip that their supplier is selling them non-human hair and claiming that it is 100% human

• Wanted us to determine whether their hair products are human or non-human

12S testing results

• A result was obtained using the 12S primers on the hair DNA extract

• The 12S sequence was searched in GenBank using BLASTn

• The resulting species was…….

“Bos grunniens”

???????

Bos grunniens = YAK

Learning something new

• Yak hair has all the properties of human hair

• Yak hair is often used in wigs, hair weaves, and hair extensions (especially Santa Claus costumes)

• 100% human hair is often diluted with yak hair to lower the cost

Will this method work on fish, birds, and other species?

• Scenario: Object in a restaurant-prepared salad…(an actual case):

• Is it a human fingertip, or calamari?• We have to question if 12S will work for

every situation.• Fortunately (!) the sample was human

skin…

Other species?

• Examination of the first 1000 closest sequences to a variety of GenBank Blastn Search sequences shows an immense variety of possible species (including non-mammalian species)

• As always, match possibilities are limited by the contents of the database

• Nucleotide database search yielded the following:– Total sequences for search “12S ribosomal” = 34,809– Total sequences for search “12S + vertebrata” = 23,717– Total sequences for search “12S + mammalia” = 7,619

• Most mammals are in GenBank, and the most common North American mammals certainly are

Consequences of using 12S

• Availability of 12S testing on hairs has reduced failure rate of hair testing from 7.2% to 4.5% (e.g, 95.5% of hairs give a profile).

• The assay is suitable for the most degraded samples, with an amplicon size equal to that of the standard “mini-primer” sets used for ancient DNA.

• Until hair microscopy (a forensic skill that we recommend be enhanced with training and experience) improves and increases, this assay will be a helpful adjunct for mitochondrial DNA analysis.

MtDNA Typing of Canid Species

Canine Hairs as Evidence

• 2002: David Westerfield on trial for murder of 7 year old Danielle VanDam.

• Dog hairs from suspect’s motor home, quilt, and dryer lint matched Danielle’s dog.

• Frequency of the mtDNA type in question was about 9%

• First trial in the US to admit canine mtDNA• Westerfield was convicted on all counts

High potential utility

• www.denverda.org lists 31 significant cases where non-human DNA played a role in conviction

• 18 of these cases involved dog DNA, including hair, blood, feces, and saliva

• Prior limitation was lack of a validated mitochondrial DNA database to provide a statistical context

Laying the groundwork:

Canine D-loop (non-coding region)

• Two hypervariable regions connected by a region consisting of 10bp tandem repeats

“HV1”:15431-15782

“HV2”: 15739-16092Repeat region:16130-16430 “HV3”:16451-00014

Protocols are unchanged from human mtDNA analysis

• Hair extraction method uses glass grinder, 2 hour proteinase K incubation, and phenol-chloroform-isoamyl alcohol (PCIA)

PCR and sequencing

• Amplification and sequencing of three fragments results in 1,004 bp of sequence data and a profile of ~953 bp

• Protocols differ from human mtDNA only with respect to the primers used

• Resulting sequence is compared to a “dog reference sequence” from Kim et al. 1998 that is also in GenBank

Material Modification Experiments

• Development of a “positive control animal”

• Species specificity:– HV1 primers amplify cat and human but

amplicon size is different from dog– HV2 primers amplify cat but amplicon size is

different– HV3 primers do not amplify cat or human– Results indicate that all PCR products should

be sequenced and compared with Genbank

Material Modification Experiments

• Reproducibility: a total of 18 dogs and wolves were sampled

• Probative evidence types and sensitivity: guard hairs and undercoat hairs all performed well at different sizes

• Mixtures: hairs with saliva from a second dog were sufficiently cleaned to get the hair type only

First Project

• For a private client: can one distinguish dogs and wolves via control region sequencing?

• Non-forensic, investigational, allowed us to combine implementation of new methods and material modification experiments

Blind Testing

• Four bindles of hair were received• We were told only that they were collected from

dogs or wolves• Hair appearance was not informative at least at

the macroscopic level• Hairs were profiled using standard hair methods

and primers designed for dogs and other canines from published literature

• Results were compared with the published literature on mtDNA variation in dogs and wolves

Results

• Full mitochondrial DNA sequences were obtained from all four samples

• Two of the samples had identical profiles

• The other two were different

• Profiles were searched in GenBank

Sample Closest Match % Similarity to Closest

Match

% Coverage with

Closest Match

LikelySpecies

W1 C. lupus 100%(662/662)

100%(662/662)

gray wolf

W2 C. lupus 99% (660/661)

99%(661/663)

gray wolf

W3 C. lupus 100%(662/662)

100%(662/662)

gray wolf

W4 C. familiaris 100%(662/662)

100%(662/662)

domestic dog

All results were correct; W4 is product of domestic dog mother and wolf father

Providing a forensic context for a canine mtDNA match

• Requires a database to estimate the frequency of a particular type

• Database must be searchable with user-friendly interface

• Current model for this software and database is “MitoSearch”, provided by the FBI for human mitochondrial DNA cases.

• “MitoSearch” is used in every human case where there is a failure to exclude (match)

Sampling Statistics

Case where type previously observed:

n

ppp

)1)((96.1

Case where type never observed:

np 11

Where p=frequencyn=size of databaseα=significance level (0.01

or 0.05)

95% Confidence Interval

95% Upper Bound

FidoSearchTM

• Mitotyping Technologies is developing a software package to search the canine profiles already available in GenBank

• There are 2800 canine mtDNA profiles in GenBank, representing about 2400 domestic dogs (C. familiaris) as well as wolves, dingos, coyotes, extinct species (specimens from museums)

• We have downloaded the 2800 sequences and are creating a user-friendly interface that will be resident on our website (www.mitotyping.com)

Dog Diversity

• Dogs are not nearly as diverse with respect to their mitochondrial DNA as humans

• There are only 10 major haplogroups, and only 4 of those are found in Europe

• Therefore, the ability to provide a powerful forensic statistic is reduced…“Only 78% of dogs can be excluded as the donor of this

particular mtDNA profile”…for example

Diversity of canine mtDNA

• Savolainen et al:– 1576 dogs and 40 wolves: 582 bp for CR– 169 dogs and 8 wolves: whole genome

• Dog mtDNA haplotypes are distributed across the wolf diversity, in 6 phylogenetic clades

• Dogs across the Old World share a common homogeneous mtDNA genepool, but diversity follows a gradient from high values in southeastern Asia to low in Europe

• 100% of dogs have a haplotype belonging to the main phylogenetic groups

Genetic Diversity

• East Asia: 735 dogs have 121 haplotypes• West(Europe/SW Asia/Africa/India): 585

dogs have 68 haplotypes• Europe: 336 dogs have 39 haplotypes

Comparatively: in any human population, the proportion of haplotypes to individuals is 2/3, meaning that out of 900 individuals, there would be 600 different haplotypes

Conclusions:

• Dogs originated in Southeastern Asia from multiple wolf founders

• Using mtDNA mutation rate to estimate time to most recent common ancestor give a date range of 5,400-16,300 years ago

• At that time, there existed at least 51 different mtDNA lineages leading to today’s dogs (most likely hundreds)

• Domestication of wolves most likely resulted from close contact with increasingly sedentary hunter-gatherers or early rice farmers…

• Question: was the wolf a food source as opposed to hunting helper/guard/or companion?

“Well, well—another blonde hair…conducting a little more “research” with that Jane Goodall tramp?”

Animal DNA Forensics

• Animal-on-animal crimes• Animal cruelty cases• Animal on human crimes (bear attack)• Suspect/victim pets leave transfer

evidence such as urine, feces, and hair• CITES: importing endangered species

illegally, protecting endangered populations, museum specimens

• Hunting violations, poaching

Not just mtDNA, also nuDNA

• Most famous case? 1997…• Snowball the cat, from PEI, Canada, has her

own Wikipedia entry• White hairs were recovered from a jacket found

in the grave of murdered Shirley Duguay• STR analysis showed Snowball, the cat of

Duguay’s estranged husband, was the donor of the hairs

• First known usage of animal DNA in a crime• Article published in Nature indicated significance

Endangered Species and Forensics

DNA registry for whales

DNA test confirms tiger meat for sale at Chinese farm

NY museum to bank endangered species DNA

Frankfurt zoo stores frozen stem cells from Aye-Aye

DNA shows what species of sharks were illegally finned

My lab from the lab, Sam, says:

HONORARY

YOU’RE ALL