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Forensic DNA profiling: mitochondrial DNA and STR analyses

Daniele Podini [email protected]

Outline •  History of Forensic DNA Analysis •  STRs •  mtDNA

–  Structure and funcCon – mtDNA for human idenCficaCon – AnalyCcal process

•  ExtracCon •  AmplificaCon •  Sequencing •  Results and InterpretaCon

•  QuesCons

Where did it all start?

•  Mendel showed that the inheritance of certain traits follows particular laws which were later named after him.

•  The significance of Mendel's work was not recognized until the turn of the 20th century.

•  The independent rediscovery of these laws formed the foundation of the modern science of genetics.

James Watson & Francis Crick

Using x-ray diffusion data they proposed the double helix or spiral staircase structure of the DNA molecule in 1953

Base-Pairing •  In double-stranded DNA the

bases are paired up –  As with Ts and –  Gs with Cs

•  The bases are held together by hydrogen bonds –  These bonds are weak

(∼1/10 the strength of a C-C bond)

The Human Genome

Nuclear DNA

3 billion bp

High Power Of DiscriminaCon

Mitochondrial DNA

16.5 Kbp

High Copy Number

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Early 1980s: Restriction Fragment Length Polymorphism (RFLP)

•  Genetic variation in the distance between restriction enzyme sites

•  Template DNA digested by enzymes, electrophoresed, detected via Southern blotting

•  Power of discrimination in the range of 106-108 for a six probe analysis

Sir Alec Jeffreys

Mechanisms

for RFLPs

Mid-1980s: The Colin Pitchfork Case

  Two young women raped and murdered in Narborough, England

  5,000 local men are asked to provide blood/saliva samples

  1st exoneraCon and convicCon on forensic DNA evidence

THEN THERE WAS PCR

Polymerase Chain Reaction PCR

•  Polymerase Chain Reaction = molecular Xeroxing

•  “Amplify” the desired DNA fragment(s)

•  Increased sensitivity •  1988 FBI starts DNA section

Dr. Kary Mullis Eccentric Genius

1985

h\p://www.youtube.com/watch?v=L51UvB5za7c

h\p://www.karymullis.com/pcr.shtml

  Repeat unit 2-‐7 b.p. in length   Repeated 5-‐40 Cmes

  Length appropriate for PCR ~ 400 nt

  Highly polymorphic

  Spread throughout the genome

Short Tandem Repeats1991

10 repeats

GATA

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STRBase

h\p://www.cstl.nist.gov/biotech/strbase/

Types of STR Repeat Units

•  DinucleoCde •  TrinucleoCde •  TetranucleoCde •  PentanucleoCde •  HexanucleoCde

(CA)(CA)(CA)(CA)

(GCC)(GCC)(GCC) (AATG)(AATG)(AATG) (AGAAA)(AGAAA)

(AGTACA)(AGTACA)

Requires size based DNA separa1on to resolve different alleles from one another

Short tandem repeat (STR) = microsatellite = simple sequence repeat (SSR)

Short Tandem Repeat (STR) Markers

TCCCAAGCTCTTCCTCTTCCCTAGATCAATACAGACAGAAGACAGGTGGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATATCATTGAAAGACAAAACAGAGATGGATGATAGATACATGCTTACAGATGCACAC

= 12 GATA repeats (“12” is all that is reported)

Target region (short tandem repeat)

7 repeats

8 repeats

9 repeats

10 repeats

11 repeats

12 repeats

13 repeats

The number of consecu>ve repeat units can vary between people

An accordion-‐like DNA sequence that occurs between genes

5

D5S818

10 repeats

8 repeats

Short Tandem Repeats

GATA

LOCUS

ALLELE

Profile 8/10

Mitochondrial DNA

The Mitochondrion Cytoplasmic organelle

Double membrane

Outer membrane – porin proteins for the transportaCon of materials.

Inner membrane – highly folded (increased surface area) and highly impermeable.

Inner Matrix – several copies of mtDNA

FuncCon of the mitochondrion: ProducCon of ATP Apoptosis – programmed cell death ElongaCon of fa\y acids OxidaCon of epinephrine (adrenaline) DegradaCon of tryptophan Heme synthesis Heat producCon

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mtDNA Genome 16,569 bp

h\p://www.mitomap.org/

Coding Region 13 polypepCdes 2 rRNAs 22 tRNAs

All necessary for OXPHOS

Highly compact (few intergenic spaces)

LocaCon and Copy Number of mtDNA

•  Found within the mitochondria in the cellular cytoplasm.

•  On average 4-‐5 copies of mtDNA molecules per mitochondria (range of 1-‐15 mtDNA copies).

•  Number of mitochondria vary by cell type (e.g., muscles have more…).

•  Generally, hundreds of mitochondria per cell.

Control region (D-‐loop)

1/16,569

cyt b

ND5 ND6

ND4

ND4L

ND3

COIII ATP6

ATP8 COII

12S rRNA

16S rRNA

ND1

ND2

COI

OH

9-‐bp dele1on

OL

F

V

L1

I Q M

W

A N

C Y

S1

D K

G

R

H S2

L2

E

P

T

HV1 HV2

16024 16365 73 340

16024 576

“16,569” bp

1

22 tRNAs

2 rRNAs

13 genes

Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press

Forensic Focus Control Region (16024-‐576) Original Reference Sequence

•  Human mtDNA was first sequenced in 1981 in Frederick Sanger’s lab located in Cambridge, England.

•  Authors for this paper (Nature 1981, 290:457-‐465) were listed in alphabeCcal order so Stan Anderson was the first author.

•  This sequence has come to be referred to as the “Anderson” sequence (GenBank accession: M63933).

•  This first sequence is someCmes called the Cambridge Reference Sequence (CRS).

•  This revised Cambridge Reference Sequence (rCRS) is now the accepted standard for comparison.

Maternal Inheritance of mtDNA

•  FerClizing sperm contributes only nuclear DNA.

•  Cellular components including the mitochondria in the cytoplasm come from the mother’s ovum.

•  Any sperm mitochondria that may enter a ferClized egg are selecCvely destroyed due to a ubiquiCn tag added during spermatogenesis.

•  Barring mutaCon, a mother passes her mtDNA type on to her children.

Summary – mtDNA CharacterisCcs

•  High copy number of mtDNA.

•  Circular molecule

•  Maternal inheritance of mtDNA.

•  Lack of recombinaCon.

•  High mutaCon rate compared to single copy nucDNA.

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Methods for Measuring mtDNA VariaCon

•  Low-‐resoluCon RFLP (1980s)

•  High-‐resoluCon RFLP (1990s)

•  Sequence analysis of HV1 and HV2 within control region (1991-‐present)

•  Sequence analysis of complete mtDNA genome (2000-‐present)

Role of mtDNA Compared to Autosomal STRs

•  Autosomal STRs provide a higher power of discriminaQon and are the preferred method whenever possible

•  Due to high copy number, mitochondrial DNA (mtDNA) may be the only source of surviving DNA in highly degraded specimens or low quanCty samples such as hair shass

•  A mtDNA result is be\er than no result at all…

Candidates for mtDNA TesCng

•  Shed hairs lacking root bulb or a\ached Cssue

•  Fragments of hair shass.

•  Aged bones or teeth that have been subjected to long periods of exposure.

•  Crime scene stains or swabs that were unsuccessful for nuclear DNA tesCng.

•  Tissues (muscle, organ, skin) that were unsuccessful for nuclear DNA tesCng.

Terry Melton – InternaConal Symposium on the ApplicaCon of DNA Technologies in AnalyCcal Sciences

Mitochondrial DNA as a Means of IdenCficaCon

Why go to mtDNA?

•  Disadvantages – mtDNA is not a posiCve form of idenCficaCon

(You have many maternal relaCves!!)

– Easily contaminated with modern DNA

ContaminaCon

•  Modern DNA can easily be introduced and overwhelm target DNA from the sample. – Due to the sensiCvity of the reacCon

•  Appropriate controls must be implemented to assure that the mtDNA sequence being reported is authenCc.

•  Laboratories need to be designed to lessen the chances of contaminaCon.

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Why go to mtDNA?

•  Disadvantages – mtDNA is not a posiCve form of idenCficaCon

(You have many maternal relaCves!!)

– Easily contaminated with modern DNA

– Time-‐consuming and costly

Nuclear DNA Analysis

•  24-‐36 hours •  ~$100 per sample •  Use commercially available kits for processing

Sample CollecCon Laboratory

Profile generaCon

MtDNA Analysis

•  1-‐6 weeks post-‐submission to the laboratory

•  ~$1,000 per sample •  Custom designed primers

Sample CollecCon Laboratory

Profile generaCon

Why go to mtDNA?

•  Advantages – Maternally inherited

•  The pool of potenCal references is greatly increased.

Limited references available for nDNA Maternal inheritance

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Why go to mtDNA?


•  The pool of potenCal references is greatly increased. – Numerous copies of the mitochondrial DNA genome in each cell.

Why go to mtDNA?


•  The pool of potenCal references is greatly increased. – Numerous copies of the mitochondrial DNA genome in each cell.

– Small genome size and mulCple copies increase chances of recovering DNA from degraded samples.

Extract mtDNA from evidence (Q) sample

PCR Amplify HV1 and HV2 Regions

Sequence HV1 and HV2 Amplicons

(both strands)

Confirm sequence with forward and reverse strands

Note differences from Anderson (reference) sequence

Compare with database to determine haplotype frequency

Compare Q and K sequences

QuesQon Sample

Reference Sample

Extract mtDNA from reference (K) sample



(both strands)



Performed separately and preferably

aCer evidence is completed

Process for EvaluaCon of mtDNA Samples


Pre-‐ and post-‐PCR SeparaCon

Pre-‐PCR

ExtracCons & AmplificaCon set-‐ups

Post-‐PCR

Thermalcyclers & Sequencing

AmplificaCon ReacCons

Amplified Product

Pre-‐ and post-‐PCR SeparaCon

Pre-‐PCR

ExtracCons & AmplificaCon set-‐ups

Post-‐PCR

Thermalcyclers & Sequencing

AmplificaCon ReacCons

Amplified Product

Degraded Skeletal Remains

What to choose and how to generate a full mtDNA profile.

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Degraded Skeletal Remains

•  Sample SelecCon •  ExtracCon Methods

•  AmplificaCon Strategies

•  Sequencing Strategies

Degraded Specimens

•  In general terms all skeletal remains are degraded.

•  Some are more degraded then others due to environmental stressors.

•  Prudent sample selecCon will increase the rate of success.

Environment

•  Recovery sites vary –  Extreme condiCons

•  Salt-‐water marshes •  Glaciers

–  High/Low temperatures

–  Repeated freezing and thawing

–  High/Low pH –  High water levels –  Salt or brackish water

Environment

•  Remains may be –  On the surface –  Buried in soil or other substrates –  Highly fragmented –  Subjected to burning or high heat –  Exposed to fuel or other chemicals

–  Disturbed or moved by humans or animals

–  Animal destrucCon (feeding)

Sample SelecCon

•  Unknown skeletal remains – Remains are examined and samples selected by anthropologists or medical examiners

SelecCng samples for analysis

•  What are the best skeletal elements to use for analysis?

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Bone Structure

•  Bones with dense corCcal structure tend to have a greater success rate. –  Compact bone may inherently afford greater protecCon for it’s deeper layers.

–  Trabecular bone and elements composed of thin corCcal bone have a greater surface area

•  Cranial fragments vary in success –  Formed of a layer of trabecular bone sandwiched between two layers of corCcal bone

–  Temporal and occipital tend to have denser corCcal bone

Bones SubmiUed for Analysis

DenCCon •  Dental remains provide a parCcular challenge –  The enamel gives a greater protecCon to the denCn from which the DNA is extracted.

–  Anecdotally shown to provide copious quanCCes of DNA from even medieval era remains.

–  Require a lot of handling.

Cleaning the Sample

•  The exterior of the bone fragment needs to be cleaned of any possible contaminants: – Dirt – Plant material – Extraneous DNA – Dried Tissue

Cleaning

•  An easy way to clean the surface is using a sanding bit in a Dremel tool.

How far to clean?

•  Everything on the surface needs to come off, along with the spongy bone.

•  But, you’ll hit a point where there is no solid bone les.

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Other Cleaning Methods

•  Bleaching – Bones can be subjected to a bleach sonicaCon to remove external contaminants.

– A fresh water sonicaCon should follow to get rid of the bleach or DNA can be lost.

•  “DNA Off” or other DNA removal products

Too much?

•  Aggressive cleaning can remove or otherwise damage available DNA.

ExtracCon Methods

•  Numerous extracCon methods available. •  Involve different methods of –

– pulverizing the samples

–  removing the DNA from the samples

•  Different starCng quanCCes of bone can also be used.

PulverizaCon Methods •  Freezer Mill

– Uses liquid nitrogen and a magnet to pulverize the bone into a very fine powder.

– Disadvantage: •  Requires storage and handling of liquid nitrogen. •  Grinders and sample vials are reused – potenCal contaminaCon.

PulverizaCon Method

•  Waring Blender Cup – Also grinds bone to a relaCvely fine powder – Disadvantage: Cups are reused, so there is a possibility of contaminaCon.

“Freeing” the DNA

•  AFDIL now uses a complete demineralizaCon protocol – Demineralizes the bone matrix.

•  Other chemical/physical treatments are commercially available to more easily acquire the DNA. –  Silica gel –  Charge Switch™ –  DNA IQ™

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ExtracCon of Skeletal Remains

•  The powdered bone is extracted with –  Proteinase K and extracCon buffer (Demin)

–  Overnight at 56°C •  DNA is removed from the extracCon buffer with –  a series of washes with Phenol/ Chloroform/ Isoamyl alcohol

–  PurificaCon of product with filters.

PCR -‐ Primer Design Origin 16024 576

HV1 F15971/R16410

HV2 F15/R389

Primer Design Origin 16024 576

HV1 F15971/R16410

HV2 F15/R389

PS1 F15989/R16251

PS2 F16190/R16410

PS3 F15/R285

PS4 F155/R389

PS5 F16381/R16569

Primer Design Origin 16024 576

HV1 F15971/R16410

HV2 F15/R389

PS1 F15989/R16251

PS2 F16190/R16410

PS3 F15/R285

PS4 F155/R389

mps1a mps2a

mps1b mps2b

mps3a mps4a

mps3b mps4b

mps5a

mVR1 mVR2

PS5 F16381/R16569

Science of DNA Sequencing

dideoxyNTP (ddNTP)

•  Fred Sanger – developed the dideoxy method of sequencing in the 1970s… sCll used today.

AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGAC

-‐OH

dNTP G

-‐OH PPP

ddNTP G

-‐H PPP

* ddNTP

T

-‐H PPP

*

Template

Primer

A blend of dNTPs and ddNTPs

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-‐OH

dNTP G

-‐OH PPP

ddNTP G

-‐H PPP

* ddNTP

T

-‐H PPP

*

IncorporaCon

AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGACG

-‐H

dNTP G

-‐OH PPP

ddNTP T

-‐H PPP

*

TerminaCon of the reacCon!

Fragment = TCGACG


-‐OH

dNTP G

-‐OH PPP

ddNTP G

-‐H PPP

* ddNTP

T

-‐H PPP

*

IncorporaCon

AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGACG

-‐OH

ddNTP G

-‐H PPP

* ddNTP

T

-‐H PPP

*

IncorporaCon

AGCTGCATGCAATT-‐-‐-‐-‐-‐ TCGACGT

-‐H

ddNTP G

-‐H PPP

*

TerminaCon of the reacCon!

Fragments = TCGACG TCGACGT

1 bp difference!

Sanger Sequencing 3’-TAAATGATTCC-5’

ATT

ATTTACTAA

ATTTACT ATTTAC

ATTT ATTTA

AT

ATTTACTA

ATTTACTAAG ATTTACTAAGG

A DNA template 5’ 3’

Primer anneals

Extension produces a series of ddNTP terminated products each one base different in length

Each ddNTP is labeled with a different color fluorescent dye

Sequence is read by no1ng peak color in electropherogram (possessing single base resolu1on)


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InterpreQng and ReporQng mtDNA Results

Data Review and EdiCng

Trimming of data (primer sequences too)

Data Review and EdiCng

rCRS

Differences from rCRS are noted by the sosware

ReporCng Differences from rCRS

rCRS

489 T-‐C 493 A-‐G

Point MutaCons are listed as differences

from the rCRS

DeleCons

•  DeleCons – report the posiCon and bases deleted…

rCRS

523 A-‐del 524 C-‐del

InterpretaConal Issues -‐ Heteroplasmy

•  Heteroplasmy – the presence of more than one mtDNA type in an individual (Melton 2004).

•  Once thought to be rare, heteroplasmy exists (at some level) in all Cssues (Melton 2004).

•  Especially important in hair analysis (semi-‐clonal).

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InterpretaConal Issues -‐ Heteroplasmy

•  Two types: Length (most common) and Point Heteroplasmy.

AAACCCCCCCCCTCCCCCCGCTTC

AAACCCCCCC : : :TCCCCCGCTTC

Sequence 1

rCRS

303 310 315

Sequence 2 AAACCCCCCCCCCTCCCCCCGCTTC

Sequence 1 has 9 Cs before 310T Sequence 2 has 10 Cs before 310T

“Out of phase!”

HV2 Length Heteroplasmy

AAACCCCCCCCCTCCCCCCGCTTC Sequence 1 Sequence 2 AAACCCCCCCCCCTCCCCCCGCTTC

“Out of phase!”

Dr. CinCa Friedman, São Paulo, Brasil

Double coverage is important to determine sequences surrounding HV1, HV2, HV3 C-‐stretches.

Point Heteroplasmy

16093 (C/T)

16086 16101


“Hotspot” for heteroplasmy “Y” Pyrimidine

OriginaCon of Heteroplasmy

Chinnery et al. (2000) Trends in GeneCcs

Ovum – 100K mitochondria

Very li\le mito growth unCl implantaCon

Females – produce ~7 million ova during fetal development only a few hundred become mature oocytes

Heteroplasmic VariaCon

Sekiguchi et al. (2003) 16291

Buccal Swabs

Heteroplasmy DetecCon

•  DetecCon of heteroplasmy – sequencing can detect only to ~10% level.

•  Other methods (e.g. Denaturing Gradient Gel Electrophoresis) are much more sensiCve.

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Famous Case Involving Heteroplasmy

IdenQficaQon of the Romanov Remains (the Last Russian Czar)

Tsarina Alexandra

Tsar Nicholas II

Xenia Cheremeteff-Sfiri

Prince Philip Duke of Edinburgh

Georgij Romanov

Mitotype 16111T 16357C 263G 315.1C

Mitotype 16126C 16169T 16294T 16296T 73G 263G 315.1C

16169T/C

16169T/C Louise of

Hesse-Cassel

SOURCES: Gill et al. (1994) Nature Gene1cs, 6, 130‑135.; Ivanov et al. (1996) Nature Gene1cs, 12, 417‑420; Stone, R. (2004) Science, 303, 753.

D.N.A. Box 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd EdiCon © 2005 Elsevier Academic Press

AFDIL – ConfirmaCon of FSS

TSAR

GEORGIJ

Dr. Thomas Parsons

Extract mtDNA from evidence (Q) sample



(both strands)



Compare with database to determine haplotype frequency

Compare Q and K sequences

QuesQon Sample

Reference Sample

Extract mtDNA from reference (K) sample



(both strands)



Performed separately and preferably

aCer evidence is completed

Process for EvaluaCon of mtDNA Samples


InterpretaCon of mtDNA Results

•  Once the sequence has been generated (Q and K), and the differences from the rCRS are noted, what next?

SWGDAM Guidelines for Mitochondrial DNA (mtDNA) NucleoCde Sequence InterpretaCon

(1) Exclusion

(2) Inconclusive

(3) Cannot Exclude (Failure to Exclude)


•  Exclusion – if there are two or more nucleoCde differences between the quesConed and known samples, the samples can be excluded as originaCng from the same person or maternal lineage.

Q TATTGCACAG K TATTGTACGG

Exclusion

Sample Q Sample K

6 T-‐C 9 G-‐A 263 A-‐G 315.1 C

263 A-‐G 315.1 C

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•  Inconclusive – if there is one nucleoCde difference between the quesConed and the known samples, the result will be inconclusive.

Q TATTGCACGG K TATTGTACGG

Sample Q Sample K

6 T-‐C 263 A-‐G 315.1 C

263 A-‐G 315.1 C

Inconclusive


•  Cannot Exclude – if the sequences from quesConed and known samples under comparison have a common base at each posiCon or a common length variant in the HV2 C-‐stretch, the samples cannot be excluded as originaCng from the same person or the same maternal lineage.

Q TATTGTACGG K TATTGTACGG

Sample Q Sample K

152 T-‐C 263 A-‐G 315.1 C

152 T-‐C 263 A-‐G 315.1 C

Cannot Exclude

ReporCng StaCsCcs

•  When “cannot exclude” is the interpretaCon, then a staCsCcal esCmate is needed in order to weigh the significance of the observed match

•  CounCng method is most common approach used and involves counCng the number of Cmes that a parCcular mtDNA haplotype (sequence) is seen in a database

•  The larger the number of unrelated individuals in the database, the be\er the staCsCcs will be for a random match frequency esCmate.

Control region (D-‐loop)

1/16,569

cyt b

ND5 ND6

ND4

ND4L

ND3

COIII ATP6

ATP8 COII

12S rRNA

16S rRNA

ND1

ND2

COI

OH

9-‐bp dele1on

OL

F

V

L1

I Q M

W

A N

C Y

S1

D K

G

R

H S2

L2

E

P

T

HV1 HV2

16024 16365 73 340

16024 576

“16,569” bp

1

22 tRNAs

2 rRNAs

13 genes


Forensic Focus

Uses of Haplogroup Typing

www.payvand.com

h\p://www.rcfp.org/

Anthropological Studies

Forensic Identification

Mass Graves/ Commingled Remains

h\p://www.dccam.org

KEY:

EU = European

AS = Asian

AF = African

NA = Native American L3

M

10398G, 10400T

13263G

5178A

7600A 4833G

C

D

E G

AS/NA

AS/NA

AS AS

N

8272-8280 del

A10398, C10400

C7028

1719A 663G

12406A

X

H

F

A B

EU

EU/AS/NA

AS

AS/NA AS/NA

10398G, 1719A

I

EU

G3594 10398G, 7028T

3594T, 10398G, 7028T

L1/L2

AF

AF/EU/AS

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Acknowledgments

Many thanks to •  Organizers •  Mike Coble NIST (Former chief research secCon AFDIL)

QUESTIONS? [email protected]

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