contribution of the national institute of standards and technology, 2010 final figures fundamentals...

Contribution of the National Institute of Standards and Technology, 2010

Law Enforcement Scientific Analysis Legal Proceedings

Police Agencies(local, state, federal)

Forensic Laboratory

Investigators/Detectives

CSI

DNA UnitDNA Analysts

Court System

Prosecution

Defense

Judge

Trial

Legal framework and precedent

Evidence submitted

Scientific report(s)

completed

References submitted

Validated scientific testsLaws and police training

Conviction or

exoneration

Other Forensic

Disciplines

Research (introduces new methods)

Evidence returned


Sample Obtained from Crime Scene or

Paternity Investigation

DNAExtraction

DNAExtraction

DNAQuantitation

DNAQuantitation

PCR Amplificationof Multiple STR markers

PCR Amplificationof Multiple STR markers

Biology

Separation and Detection of PCR Products

(STR Alleles)

Technology

Sample Genotype Determination

Genetics

Comparison of Sample Genotype to Other

Sample Results

Comparison of Sample Genotype to Other

Sample Results

If match occurs, comparison of DNA profile to population databases

If match occurs, comparison of DNA profile to population databases

Generation of Case Report with Probability

of Random Match

Generation of Case Report with Probability

of Random Match


DNA Profile ComparisonQ K

Court

Database Search

Exclusion (no match)

Inclusion (match)

May match another (K’)

Evidence (Question) sample “Q”

Profile put on database

Plea

Report(with statistical weight)

Q = K

Q ≠ K

Crime committedBiological material transferred

Collection

Extraction

Quantitation

STR Markers

DataInterpretation

Sample Storage

Amplification

Separation/Detection

Reference (Known) sample “K”

Profile put on database

Steps Involved

Suspect developed

Collection

Extraction

Quantitation

STR Markers

DataInterpretation

Sample Storage

Amplification

Statistical Interpretation

Characterization

Separation/Detection

Bio

log

yT

ech

no

log

yG

en

etic

sS

ero

log

y

Steps Involved

QUALITY

ASSURANCE

QUALITY

ASSURANCE

May match another (K’)


A = TG C

T = A

A = T

C G

T

CCA

GG

TA

G C

T = A

T = A

C G

A = T

A = TG C

5’

3’

5’

3’

5’ 3’

3’ 5’denatured

strands

hybridizedstrands

Hydrogen bonds

C G C

G

G C

Phosphate-sugar backbone


The Human Genomeh

ttp

://w

ww

.ncb

i.nlm

.nih

.go

v/g

en

om

e/g

uid

e/

1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 X Y

Sex-chromosomes

Autosomes

3.2 billion bp

Nuclear DNA - Located in cell nucleus

2 copies per cell

mtDNA

16,569 bp

mtDNA

Located in mitochondria

(multiple copies in cell cytoplasm)

100s of copies per cell

Onl

y si

ngle

cop

y of

ea

ch a

utos

ome

show

n


p(short arm)

centromere

telomere

q(long arm)

telomere

Band 5

Band 3

Chromosome 12

12p312p3

12q512q5


2 repeats

3 repeats

--------AGACTAGACATT-------

--------AGATTAGGCATT-------

---------(AATG)(AATG)(AATG)----------

---------(AATG)(AATG)----------

(B) Length polymorphism

(A) Sequence polymorphism


63

4 5

Homologous pair of chromosomes

Homologous pair of chromosomes

Locus A

Locus B

Allele 1 Allele 2

Allele 2Allele 1


1 2 3 4 5 6 7 8 9 1110 12 13 14 15 16 17 18 19 20 21 22

X mtDNA

Y1 2 3 4 5 6 7 8 9 1110 12 13 14 15 16 17 18 19 20 21 22

Maternal Contribution

Paternal Contribution

1 2 3 4 5 6 7 8 9 1110 12 13 14 15 16 17 18 19 20 21 22X Y

mtDNAMale Child’s Full Genome

Nuclear DNAMitochondrial DNA

AutosomesSex

chromosomes

X

or

Sex chromosome

Sex chromosome

Zyg

ote

(d

iplo

id)

Sp

erm

(h

aplo

id)

Eg

g

(hap

loid

)


AA

A

A

a

a Aa

aA

aa

Freq (A) = p

Freq (a) = q (p + q)2 = p2 + 2pq + q2

Punnett square

p2 qp

pq q2

p

q

p q

Fat

her

gam

etes

(sp

erm

)Mother gametes (egg)

p + q = 1

Resulting genotype combinations and frequencies

AA

Aa

p2

2pq

aa

q2


AA aa

Aa

1.0

Frequency of a allele (q)

Frequency of A allele (p)

1.0 0.8 0.6 0.4 0.2 0.0

0.0 0.2 0.4 0.6 0.8

Fre

quen

cy o

f ge

noty

pe in

pop

ulat

ion

0.2

0.4

0.6

0.8 p2

2pq

q2


1 2

3 4 56 7 8

9 10 11

20,22 23.2,25

20,25 20,25

20,22

20,22

22,25

20,22

22,25 20,22 20,25

14 15 16

22,2422,

23.222,

23.2

12 13

22,23.2

22,22

17

(a)

(b) (c)

22,2522,23.2

20,2520,23.220

22

23.2 25

Fat

her

’s a

llele

s

Mother’s alleles

#3

#5#4#1

#2

22,2520,22

20,2520,2020

22

20 25

Fat

her

’s a

llele

s

Mother’s alleles

#13

#14

#12

#7

#4


Speed of Analysis (Technology)

Power of Discrimination

(Genetics)

Low

High

Slow Fast

Markers Used (Biology)

Markers Used (Biology)

RFLPSingle Locus Probes

RFLPMulti-Locus Probes

ABO blood groups

Multiplex STRs

DQsingle STR

D1S80mtDNA

PolyMarker


A B AB O

A A or OA,B,AB,

or OA,B, or

ABA or O

BA,B,AB,

or OB or O

A,B, or AB

B or O

ABA,B, or

ABA,B, or

ABA,B, or

ABA or B

O A or O B or O A or B OFat

her

’s B

loo

d T

ype

Mother’s Blood Type

Child’s Blood Type


Multi-Locus Probe

Probe 1 Probe 2 Probe 3

D1S7 D2S44 D4S139

Single-Locus Probe

Probe 33.6


probe

Restriction site

Restriction site

VNTR

Small allele

Large allele

Small allele

Large allele

Sizing ladder

13 repeats

7 repeats

Bands seen on autoradiogram of probed membrane

probe


TGCAGG CCTAACGACGTCC GGATTGC

TGCACTGCA GTAACGACGTG ACGTCATTGC

TGCAG ANTCTAACGACGTCTNA GATTGC

HaeIII

HinfI

PstI


GTCCAGTCG PCR product (denatured)Biotin

Strepavidin

HRP

TMB (colorless)

Colored precipitate

CAGGTCAGC

Nylon membrane

Immobilized SSO probe5’

3’

hybridization

match no match

View from above nylon membrane

Allele 1 Allele 2


Nominal allele specific dots

Control dot

Subtype allele specific dots

4.24.3

1.21.3 4

1 2 3 4 CAll but1.3

1.3 4.11.1 DQA1

1.21.3 4

1 2 3 4 CAll but1.3

1.31.1 AMPLITYPETM

DQ-Alpha

S A B

LDLR

A B

GYPA

A B

HBGG

A B

D7S8

A B

GC

C C

“S” dot

(A)

(B)

1.2/3

AB AB BB AB BC


AllelicLadder

AllelicLadder

Pos

itive

Con

trol

Sam

ple

1

Sam

ple

2

14

18

24

34

31

1617

1920212223

41

252627282930

14

18

24

34

31

1617

1920212223

41

252627282930


Minisatellite Marker (D1S80)

GAGGACCACCAGGAAGGAGGACCACCAGGAAG

Repeat region

Flanking regions

16 bp repeat unit

STR Marker (TH01)

TCATTCAT

Repeat region

Flanking regions

4 bp repeat unit


AllelicLadders

Sam

ple

1

Sam

ple

2

5

8

67

91011

CSF1PO

TPOX

TH01

89

101112

AllelicLadders

6

14


3’-TAAATGATTCC-5’

ATT

ATTTACTAA

ATTTACT ATTTAC

ATTTATTTA

AT

ATTTACTA

ATTTACTAAGATTTACTAAGG

A

DNA template5’ 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 noting peak color in electropherogram (possessing single base resolution)


ORGANIC FTA PaperCHELEX

Blood stain

PUNCH

WASH Multiple Times with extraction buffer

PERFORM PCR

PCR Reagent

s

SDS, DTT, EDTA and

proteinase K

INCUBATE (56 oC)

Phenol,chloroform,

isoamyl alcohol

QUANTITATE DNA

Apply blood to paper and

allow stain to dry

Blood stain

VORTEX

(NO DNA QUANTITATION TYPICALLY PERFORMED

WITH UNIFORM SAMPLES)

Water

INCUBATE (ambient)

5% Chelex

INCUBATE (100 oC)

REMOVE supernatant

INCUBATE (56 oC)

QUANTITATE DNA

PERFORM PCR

PERFORM PCR

Centrifuge

Centrifuge

Centrifuge

Centrifuge

REMOVE supernatantTRANSFER aqueous (upper) phase to new tube

CONCENTRATE sample (Centricon/Microcon-100 or

ethanol precipitation)

Centrifuge

TE buffer


Perpetrator’s sperm mixed with victim’s

epithelial cells

Centrifuge

REMOVE supernatant

SDS, EDTA and proteinase K

(cell lysis buffer)

Remove a portion of the mixed stain

SDS, EDTA and proteinase K + DTT

Incubate at 37 oC

sperm pellet

DTT lyses sperm heads

“Male Fraction” “Female Fraction”sperm pellet

Differential Extraction


Too much DNA amplified

(a) (b)

Too little DNA amplified

(c)

Within optimal range


20 ng

10 ng

5 ng

2.5 ng

1.25 ng

0.63 ng20 ng

10 ng

5 ng

2.5 ng

1.25 ng

0.63 ng

Calibration standards

Calibration standards

Unknown Samples

≈2.5 ng


Polymerization and Strand Displacement

RQ

Forward primer

Reverse primer

3’

5’ 3’

5’3’5’

5’

5’

Forward primer

Reverse primer

3’

5’ 3’

5’5’

5’

Q

R

3’ Probe Cleavage (release of reporter dye)

Forward primer

Reverse primer

3’

5’ 3’

5’5’

5’

QR

Completion of Polymerization

TaqMan probe

Fluorescence occurs when reporter dye and quencher dye are no longer in close proximity


Cycle Number

Nor

mal

ized

Flu

ores

cenc

e

threshold

CT

Exponential product growth

Linear product growth

Plateau

ΔRn

Negative control

a b c d e

Standard curve

CT

Log[DNA]

a

b

c

d

e

Nc = No (1 + E)c

If efficiency is close to 100% (E = 1), then the product copy number (Nc) doubles the target copy number (No) with each cycle (c).


94 oC

60 oC

72 oC

TimeTemperature

Single Cycle

Typically 25-35 cycles performed during PCR

94 oC 94 oC 94 oC

60 oC60 oC

72 oC72 oC

The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a “hot-start” PCR


Separate strands

(denature)

Add primers (anneal)

Make copies (extend primers)

Repeat Cycle, Copying DNA Exponentially

Starting DNA

Template

5’

5’

3’

3’

5’

5’

5’3’ 3’

3’3’5’

Forward primer

Reverse primer


(a)

(b)


(a) Simultaneous amplification of three locations on a DNA template

Locus A Locus CLocus B

(b) Resolution of PCR products with size-based separation method

A

CB

small large


STR repeat region

GATA GATAGATAGATA

PCR product size generated

DNA template containing STR marker

Reverse PCR primer

Forward PCR primer

Fluorescent dye

(a)

(b)

Flanking regions

4 repeat units

5 repeat units

6 repeat units


1 2 3 4 5 65’-TTTCCC TCAT TCAT TCAT TCAT TCAT TCAT TCACCATGGA-3’3’-AAAGGG AGTA AGTA AGTA AGTA AGTA AGTA AGTGGTACCT-5’

6 5 4 3 2 1


Combine and re-amplify

Allelic Ladder

Individual Samples

6 8

7 9

8

9 10 6 7 8 9 10


AmpFlSTR Identifiler kit (Applied Biosystems)

6-FAM (Blue)

VIC (Green)

NED (Yellow)

PET (Red)

D8S1179 D21S11 D7S820 CSF1PO

D3S1358 TH01 D13S317 D16S539 D2S1338

D19S433 D18S51TPOXVWA

AMEL D5S818 FGA

GS500 LIZ size standardLIZ (Orange)

D3S1358 TH01 D21S11 D18S51 Penta E

D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D

AMEL VWA D8S1179 TPOX FGA

PowerPlex 16 kit (Promega Corporation)

ILS600 CXR size standardCXR (Red)

FL (Blue)

JOE (Green)

TMR (Yellow)

PCR product size (bp)


D3S1358 TH01 D21S11D18S5

1

Penta E

D5S818

D13S317D7S820

D16S539Penta D

CSF1PO

Amelogenin(sex-typing)

VWA D8S1179

TPOXFGA

blue panel

Overlay of all 4 colors

(including internal size standard)

yellow panel

green panel

red panel

ILS600 DNA sizing standard

200 bp 300 bp100 bp 400 bp 500 bp325 350 375 425 450 475225 250 275

120 140 160 180


D3S1358(8 alleles)

VWA(14 alleles)

D16S539(9 alleles)

D2S1338(14 alleles)

Blue panel

Green panel

Yellow panel

Orange panel

D21S11(24 alleles)

D8S1179(12 alleles)

D18S51(23 alleles)

TH01(10 alleles)

FGA low(19 alleles)

FGA high(9 alleles)

250 bp*139bp 200 bp160 bp 300 bp 340 bp 350 bp150 bp LIZ-labeled GS500 DNA sizing

standard100 bp

Red panel

D19S433(15 alleles)

D5S818(10 alleles)

TPOX(8 alleles)

D13S317(8 alleles)

D7S820(10 alleles)

CSF1PO(10 alleles)

AMEL(2 alleles)


Fluorescent dye at 5’end

Non-nucleotide linkers (mobility modifiers)

Primer sequence

PCR amplification generates a labeled PCR product containing the mobility modifiers

5’-end

3’-end

For each linker unit added, there is an apparent

migration shift of ≈2.5 bp


D7S820

CSF1PO

6 15

6 15

NED-labeled (yellow)

JOE-labeled (green)

D7S820 CSF1PO6 15 6 15

6FAM-labeled (blue) 6FAM-labeled (blue)

(a) COfiler kit allele relative size ranges

(b) Identifiler kit allele relative size ranges

256.01 bp 292.62 bp

279.65 bp 317.67 bp

255.15 bp 291.58 bp 304.69 bp 341.84 bp

Size overlap

10 non-nucleotide linkers

= ≈ +25 bp shift


CSF1PO forward primer

CSF1PO reverse primer

(AGAT)6-15

(a) PowerPlex 1.1 Kit

91 bp 128 bp

TMR-labeled

PCR product sizes = 291-327 bp

CSF1PO forward primer

CSF1PO reverse primer

(b) PowerPlex 16 Kit

13 bp238 bpJOE-labeled

(AGAT)6-15

PCR product sizes = 221-357 bp +30 bp shift in size+30 bp shift in size


X

Y

6 bp deletion

Female: X, X

Male: X, Y

1:1 Mixture: 3X + 1Y

X = 212 bpY = 218 bp

X = 106 bpY = 112 bp

AmpFlSTR kits and PowerPlex 16

PowerPlex 1.1


-

Voltage

Gel

Loading well

+anode cathode

Side view Top view

Gel lanes

DNA bands

Buffer

+

-

Gel stand


Laser

InletBuffer

Capillary filled with polymer solution

5-20 kV- +

OutletBuffer

Sample tray

Detection window

(cathode) (anode)

Data Acquisition

Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession


Mixture of dye-labeled PCR products from multiplex PCR

reaction

CCD Panel (with virtual filters)

Argon ion

LASER (488 nm)

ColorSeparationFluorescenc

e

ABI Prism spectrograph

SizeSeparation

Processing with GeneScan/Genotyper software

Sample Interpretation

Sample Injection

Sample Separation

Sample Detection

Sample Preparatio

n

Capillary(filled with

polymer solution)


(a)

Larger DNA molecules interact more frequently with the gel and are thus retarded in their migration through the gel

Gel

(b)

Ogston Sieving Reptation

Small DNA molecules

Long DNA molecules

Gel


hex hem1

2

3

So

S’1S1energy

(a)

Excitation Emission

Wavelength (nm)

1 3

ex max em max

Fluorescence

(b)

Stokes shift


Fluorescent dNTPs are incorporated into both strands of PCR product

Ethidium bromide

DNA labeled with intercalating dye

Unlabeled DNA

SYBR Green

Intercalator inserts between base pairs on double-stranded DNA

One strand of PCR product is labeled with fluorescent dyeFluorescent dye

labeled primer

(a)

(b)

(c)


FAM(blue)

JOE(green)

TAMRA(yellow)

ROX(red)


520 540 560 580 600 620 640

WAVELENGTH (nm)

100

80

60

40

20

0

310 Filter Set F with color contributions

5-FAM JOE NED ROX

Laser excitation(488 nm, 514.5 nm)

Laser excitation(488 nm, 514.5 nm)

Normalized Fluorescent Intensity


Scan number

Relative Fluorescence Units

DNA size in base pairs

Relative Fluorescence UnitsRegion shown below

(a)

(b)


Capillary

Heat plate

Detection

window

electrode

Autosampler

Gel block

Syringe (with

polymer)

Outlet buffer

reservoir

Inlet buffer reservoir

Sample tray

Samples


Mechanical pump(with polymer)

Capillaryarray Oven

Detection

window

electrodes

Autosampler

Lower gel

block

Polymer bottle Outlet

buffer reservoir

Inlet buffer reservoir

Sample tray

Fan


Capillaries

Electrodes for Injection


Data Collection

Peak Identification

Data Review by Analyst/Examiner

Color Separation

Peak Sizing

Comparison to Allelic Ladder

Confirmation of Results by Second Analyst/Examiner

Genotype Assignment to

Alleles

GeneScan software

Genotyper software

Internal size standard

Matrix file (spectral

calibration)

Allelic ladder sample

GeneMapperIDsoftware

Expert Systems (e.g., FSS-i3, TrueAllele)

Peak Editing to Remove Artifact

Calls

User-defined thresholds


50 RFUs

150 RFUs

Analytical Threshold

Interpretation Threshold

Baseline Noise

Peak reliable, but only used for exclusions

Peak reliable, can be used for inclusions

Peak not considered

reliable

Val

ues

show

n fo

r ex

ampl

e pu

rpos

es o

nly

(sho

uld

be

base

d em

piric

ally

on

a la

b’s

inte

rnal

val

idat

ion)


DNA fragment peaks in sample

DNA Size

Data Point

147.32 bp147.32 bp

165.05 bp

165.05 bp

100

139

150160

200

250

DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard

3550

75 100139 160 200 250

300 340350

400 450 490500150

(a)

(b)

Time (minutes)


Allelic ladder PCR-amplified sample

Internal size standard Internal size standard

Co

lor-

sep

arat

ed

an

d s

ize

d

alle

le p

eaks

fo

r e

ach

lo

cu

s

10 11 12 13 14 15

Data from CE instrument (prior to color separation

and peak sizing)

Genotyping performed by comparing allelic

ladder to sample results

Color separation and peak

sizing

Color separation and peak

sizingLocus 1

Genotype = 12, 14All ladder

alleles sized using internal size standard

All sample alleles sized

using internal size standard

Genotyping allele bins (+/-0.5 bp around ladder allele)

Alleles (# repeats)


Dye blob

STR alleles

stutter

Pull-up (bleed-through)

spike

Blue channel

Green channel

Yellow channel

Red channel

D3S1358

Stutter products

6.0% 7.8%

Incomplete adenylation

D8S1179

-A

+A

-A

+A

Biological (PCR) artifacts


(a) (b) (c)

Allelic ladder Allelic ladder Allelic ladder


28.128.1

1 = S25-L25 = 244.34 - 244.46 = -0.12 bp

2 = SOL - L28 = 257.51-256.64 = +0.87 bp

c = |1 -2| = |-0.12-0.87| = 0.99 bp


(a) (b)

12

31 2 3

Type 1 Type 2


*

*8

86

6 8

Allele 6 amplicon has ‘dropped out’

Imbalance in allele peak heights

Heterozygous alleles are well balanced

No mutation

Mutation at 3’-end of primer binding site (allele dropout)

Mutation in middle of primer

binding site

(a)

(b)

(c)


DNA Profile(with specific alleles)

Rarity estimate of DNA profile(e.g., RMP or LR)

Genetic formulas

Population allele frequencies


Decide on Number of Samples and Ethnic/Racial Grouping

Gather Samples

Analyze Samples at Desired Genetic Loci

Summarize DNA types

Ethnic/ Racial Group 1

Ethnic/ Racial Group 2

Determine Allele Frequencies for Each Locus

Perform Statistical Tests on Data

Hardy-Weinberg equilibrium for allele independenceLinkage equilibrium for locus independence

Usually >100 per group

Use Database(s) to Estimate an Observed DNA Profile Frequency

Often anonymous samples from a blood bank

See Table 11.1

Examination of genetic distance between populations


Paternal Allele

Maternal Allele

Genotype

Locus 1

DNA Profile

Paternal Allele

Maternal Allele

Genotype

Locus 2

Paternal Allele

Maternal Allele

Genotype

Locus 3

HWE

Linkage Equilibrium (product rule)


National LevelNDIS

(FBI Laboratory)

SDIS(Richmond, Virginia)

SDIS(Tallahassee, Florida)

LDIS(Tampa)

LDIS(Orlando)

LDIS(Broward County)

LDIS(Roanoke)

LDIS(Norfolk)

LDIS(Fairfax)

State Level

Local Level


Convicted Offender Index

Offenders (N) Crime Samples (C)

Forensic Index Arrestee Index

Arrestees (A)

1

2

3

‘Offender Hit’

‘Forensic Hit’


Degraded DNA sample

D5S818D13S317

D7S820D16S539

CSF1PO Penta D

Agarose yield gel results

Smear of degraded DNA fragments

High relative molecular mass DNA in a tight band

(a)

(b)

Good quality DNA

Degraded DNA


Full Profile (Good Quality)

Partial Profile (Poor Quality)

(a)

(b)

DNA size (bp) relative to an internal size standard (not shown)R

ela

tiv

e fl

uo

resc

enc

e u

nit

s (

RF

Us)


STR repeat regionminiSTR primer

miniSTR primer

Conventional PCR primer

Conventional PCR primer

(a)

(b)

Conventional STR test (COfiler kit)

MiniSTR assay (using Butler et al. 2003 primers)

150 bp smaller


(a) Single SourceD3S1358 TH01 D13S317 D16S539 D2S1338

16,16 9,9.3 8,12 9,9 17,19

(b) Mixed Source

D3S1358 TH01 D13S317 D16S539 D2S1338


Type A Type B Type C


>2 alleles at a

locus, except tri-allelics?

Single Source DNA Sample

NO

Mixed DNA Sample

YES

Differentiate a

Major/Minor Component?

Determine STR profile and compute RMP

YES

Is the sample a mixture?

TYPE B

NO

YES

Stochastic Effects ? Possible

Low Level DNA) ?

YES

Assume # Contributor

s?

TYPE C

TYPE ANO

A biostatistical analysis must be performed

Probability of Exclusion [CPE]

“RMNE”

Likelihood Ratio [LR]

YES

NO

Are # of contributors

defined?

A biostatistical analysis should not be performed

Determine component profile(s) and compute RMP for

major


Identify the Presence of a Mixture

Consider All Possible Genotype Combinations

Estimate the Relative Ratio of the Individuals Contributing to the Mixture

Identify the Number of Potential Contributors

Designate Allele Peaks

Compare Reference Samples

Step #1

Step #2

Step #3

Step #4

Step #5

Step #6


100 pg

50 pg

10 pg

Allele dropout

Severe imbalance

Good heterozygote peak balance


Two possible allelesTwo possible alleles

Human Alu Repeat(~300 bp)

AluI

400 bp

100 bp

“long” (+) allele

“short” (-) allele


Autosomal (passed on in part, from all ancestors)

Y-Chromosome(passed on complete,

but only by sons)

Mitochondrial (passed on complete, but only by daughters)

Lineage Markers


Female-Male Mixture Performance with Autosomal vs. Y-Chromosome DNA Markers

Female Victim DNA Profile

Male Perpetrator DNA Profile

DNA Profile from Crime Scene

Autosomal STR Profile

Y-Chromosome STR Profile

No signal observed


?uncle 3rd cousin

(paternal)


Control region (D-loop)

1/16,569

cyt b

HV1 HV2

16024 16365 73 340

16024 576

mtGenome‘16,569’ bp

1

Heavy (H) strand

Light (L) strand

Coding region

HV3

438 574


1 2

3 54

1211109

6 7 8

181715 16

13 14

MtDNA Haplotype Groups:1

2,3,6,8,11,13,15,164,9,10

57

1214,17,18

MtDNA Haplotype Groups:1

2,3,6,8,11,13,15,164,9,10

57

1214,17,18

A B

B C

C C

D B

B

B

B

B B

E

F

G

G

G


Compare with database to determine haplotype frequency

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 Q and K sequences

Performed separately and preferably after

evidence is completed

Extract mtDNA from reference

(K) 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


Sample Q16093C16129A

Sample K16093C16129A

ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT 16090 16100 16110 16120 16130 16140

rCRS

ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT Q

K ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT

(a) mtDNA Sequences Aligned with rCRS (positions 16071-16140)

(b) Reporting Format with Differences from rCRS


Thomas Jefferson II

Field Jefferson Peter Jefferson

President Thomas Jefferson

Eston Hemings

Same Y Haplotype

Jefferson Y Haplotype

Jefferson Y Haplotype

?

Randolph Jefferson


TsarinaAlexandra

Tsar Nicholas II

Xenia Cheremeteff-Sfiri

Xenia Cheremeteff-Sfiri

Prince PhilipDuke of Edinburgh

Prince PhilipDuke of Edinburgh

GeorgijRomanov

GeorgijRomanov

Mitotype16111T16357C263G

315.1C

Mitotype16126C16169T16294T16296T

73G263G

315.1C

16169T/C

16169T/C Louise of Hesse-Cassel

Louise of Hesse-Cassel


11,14 8,12

12,14 11,128,14 12,14 8,11

Obligate paternal allele 14 14 11 14 11

C,DA,B

B,C

mother father

child

(b) Example

(a) Mendelian Inheritance


child

Mother (known parent)

Alleged father

?Rules of Inheritance

1) Child has two alleles for each autosomal marker (one from mother and one from biological father)

2) Child will have mother’s mitochondrial DNA haplotype (barring mutation)

3) Child, if a son, will have father’s Y-chromosome haplotype (barring mutation)

Random man

Missing child

Alleged mother

Alleged father

?

Parentage (Paternity) Testing

Reverse Parentage Testing (Missing Persons Investigation)

(a)

(b)


DNA profile from mass disaster victim

DNA profile from direct reference

(toothbrush believed to have belonged to the victim)

(a) Direct comparison

(b) Kinship analysis

D5S818 D13S317D7S820

D16S539CSF1PO

Penta D

?

son

wife

victim D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D

10,10 9,109,138,98,1411,13 son

wife10,12 8,108,98,128,1211,13

10,10 9,1211,139,911,1412,13

?,10 9,??,139,??,1411,? or ?,13

victim (father)

actual profile

Predicted victim profile

mass disaster victim profile


14,18

(a) (b)

15,18

15,17 14,18

13,17

15,17


Core set of markers(e.g., CODIS 13 STRs)

Past and Present

Future

(a)

(b)

(c)

(d)


Loci Described

Use in Casework

Court Presentation/ Acceptance

Internal Validation

ResearchGovernment

Funded or Private

Development

Typically by Commercial Manufacturer

ForensicApplicationForensic Labs

Assay Constructed

Population Study

Information GatheredReleased to Community

Kit Developed

Kit Tested


Lower amounts of DNA being

tested

Challengingkinship search

questionsStandard

STR Typing (DNA Profile)

Core Competency

Sufficient DNA quantity (ng)

Direct Matching (or Parentage)

Solution: Replicate Testing

Solution: Additional Markers (Y-chromosome, more STRs) and

Multiple Reference Samples

Touch DNA Attempts (poor quality, mixtures, low-level

stochastic effects)

Familial Searching Attempts (fishing for brothers or other relatives)


8,128,13

12,138,9

9,129,13

Q(query)

K(database)

(b)

9,139,12

8,138,128

9

12 13

Fat

her’s

alle

les

Mother’s alleles

(a)


Set up two hypotheses (H0 and H1)

Collect data and calculate the test

statistic (S)

Look up the critical value (C) and define

the region of rejection for the test statistic

Is S ≤ C?

yes

Accept H0 (Reject H1)

Accept H1 (Reject H0)

no

Select appropriate statistical model

Specify the level of significance and its

critical value (C)


Truth about the population

Decision based on sample examined

Correct decision

Type II error

Type I error Correct decision

H0 True H0 False

Accept H0

Reject H0 (Accept H1)

Correct decision

Wrongfully acquitted

Wrongfully accused

Correct decision

(b) Example

Not Guilty

Guilty

Courtroom VerdictSaint Sinner

Defendant

(a) Hypothesis Testing Decisions

contribution of the national institute of standards and technology, 2010 final figures fundamentals...

Documents

t c g t c c

t g c t

pq aa q2q2 slide

pq q2q2 slide

comparison of dna profile

allele p

aa aa aa freq

forensic dna typing