Bioq. Raúl Horacio LuceroJefe de Area Biología MolecularInstituto de Medicina Regional

Exámenes generales: datos fisonómicos, sexo, peso, talla, cabello, color de ojos y piel, marcas.

Registro de voz. Trazado caligráfico. Huellas dactilares. Huellas genéticas.

Diagnóstico de especie. Datación de los restos. Exámenes generales. Elementos extrínsecos. Caracteres patológicos,

naturales o traumáticos.

Identidad radiográfica y dental.

Métodos bioquímicos.

Grupos sanguíneos: ABO, RH, MNS, Duffy, Lewis.

Proteínas plasmáticas: haptoglobina, 1-antitripsina, transferrina.

Enzimas eritrocitarias: fosfatasa ácida eritrocitaria, adenilato kinasa, PGM, ADA.

HLA: Antígenos de histocompatibilidad.

Minisatélites: sondeos de locus múltiple y locus único.

Microsatélites: métodos de PCR.

Variantes de secuencia: HLA-DQPolymarker, genoma mitocondrial.

Existen regiones codificantes -que contienen la información para la síntesis proteica-, y regiones no codificantes.

Dentro de las no codificantes existen regiones polimórficas.

Existen alrededor de 3 x 106 sitios polimóficos en nuestro genoma.

Alelo 1

Alelo 2

Sitio de adhesión de los primers

Sitio de adhesión delos primers

Unidad de Repetición

Herramienta indispensable para la investigación forense.

Gran número de STRs disponibles.

Alta sensibilidad. Evaluación manual

o automatizada.

Analizando el Caso.

Material Indubitado de la Víctima.

Material SubunguealTomado de la Víctima

Tejido Dactilar de la Víctima.

Sangre del Sospechoso1.

Sangre del Sospechoso 2

Match Match Match

1985

1990

1994 1996

1998 2000

2002

1992 Capillary electrophoresis of STRs first described

First STRs developed

FSS Quadruplex

First commercial fluorescent STR

multiplexes

CODIS loci defined

STR typing with CE is fairly routine

Identifiler 5-dye kit and ABI 3100

PCR developed

UK National Database launched (April 10,

1995) PowerPlex® 16 (16 loci in single amp)

2006: DNA is an important part of the

criminal justice system

2004

2006

Y-STRs

www.dna.govJustice for All Act ($1B

over 5 years)

RFLPDQA1 & PM

(dot blot) Multiplex STRs

mtDNA

Gill et al. (1985) Forensic application of DNA 'fingerprints‘. Nature 318:577-9

Combined DNA Index System (CODIS)

Launched in October 1998 and now links all 50 statesUsed for linking serial crimes and unsolved cases with repeat

offendersConvicted offender and forensic case samples along with a missing

persons indexRequires 13 core STR markers>27,000 investigations aided nationwide as of Sept 2005

Contains more than 2.8 million DNA profiles

http://www.fbi.gov/hq/lab/codis/index1.htm

Small product sizes are generally compatible with degraded DNA and PCR enables recovery of information from small amounts of material

Multiplex amplification with fluorescence detection enables high power of discrimination in a single test

Commercially available in an easy to use kit format

Uniform set of core STR loci provide capability for national and international sharing of criminal DNA profiles

TCCCAAGCTCTTCCTCTTCCCTAGATCAATACAGACAGAAGACAGGTGGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATATCATTGAAAGACAAAACAGAGATGGATGATAGATACATGCTTACAGATGCACAC

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

Target region (short tandem repeat)

7 repeats

8 repeats9 repeats

10 repeats

11 repeats12 repeats

13 repeats

The number of consecutive repeat units can vary between people

An accordion-like DNA sequence that occurs between genes

The FBI has selected 13 core STR loci that must be run in all DNA tests in order to provide a common currency with DNA profiles

Scanned Gel Image Capillary Electropherogram

The polymerase chain reaction (PCR) is used to amplify STR regions and label the amplicons with fluorescent

dyes using locus-specific primers

8 repeats

10 repeatsLocus 1

8 repeats

9 repeatsLocus 2

Dinucleotide Trinucleotide Tetranucleoti

de Pentanucleoti

de Hexanucleoti

de

(CA)(CA)(CA)(CA)(GCC)(GCC)(GCC)(AATG)(AATG)

(AATG)(AGAAA)(AGAAA)(AGTACA)(AGTACA)

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

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

High stutter

Low stutter

YCAII

DYS448

~45%

<2%

Category Example Repeat Structure

13 CODIS Loci

Simple repeats – contain units of identical length and sequence

(GATA)(GATA)(GATA) TPOX, CSF1PO, D5S818, D13S317, D16S539

Simple repeats with non-consensus alleles

(e.g., TH01 9.3)

(GATA)(GAT-)(GATA) TH01, D18S51, D7S820

Compound repeats – comprise two or more adjacent simple repeats

(GATA)(GATA)(GACA) VWA, FGA, D3S1358, D8S1179

Complex repeats – contain several repeat blocks of variable unit length

(GATA)(GACA)(CA)(CATA) D21S11

These categories were first described by Urquhart et al. (1994) Int. J. Legal Med. 107:13-20

The efforts of the Human Genome Project have increased knowledge regarding the human genome, and hence there are many more STR loci available now than there were 10 years ago when the 13 CODIS core loci were selected.

More than 20,000 tetranucleotide STR loci have been characterized in the human genome (Collins et al. An exhaustive DNA micro-satellite map of the human genome using high performance computing. Genomics 2003;82:10-19)

There may be more than a million STR loci present depending on how they are counted (Ellegren H. Microsatellites: simple sequences with complex evolution. Nature Rev Genet 2004;5:435-445).

STR sequences account for approximately 3% of the total human genome (Lander et al. Initial sequencing and analysis of

the human genome. Nature 2001;409:860-921).

Butler, J.M. (2006) Genetics and genomics of core STR loci used in human identity testing. J. Forensic Sci., in press.

Compatible primers are the key to successful multiplex PCR

STR kits are commercially available

15 or more STR loci can be simultaneously amplified

Advantages of Multiplex PCR–Increases information obtained per unit time (increases power of discrimination)–Reduces labor to obtain results–Reduces template required (smaller sample consumed)

Challenges to Multiplexingprimer design to find compatible primers (no program exists)reaction optimization is highly empirical often taking months

Most are rape cases (>2 out of 3) Looking for match between

evidence and suspect Must compare victim’s DNA profile

•Mixtures must be resolved

•DNA is often degraded

•Inhibitors to PCR are often present

Challenges

Forensic cases -- matching suspect with evidence

Paternity testing -- identifying father Historical investigations Missing persons investigations Mass disasters -- putting pieces back

together Military DNA “dog tag” Convicted felon DNA databases

Sample Obtained from Crime Scene or

Paternity Investigation Biology

DNAExtraction

DNAExtraction

DNAQuantitation

DNAQuantitation

PCR Amplificationof Multiple STR

markers

PCR Amplificationof Multiple STR

markers

Technology

Separation and Detection of PCR Products(STR Alleles)

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

Steps in DNA Sample Processing

Sources of Biological Evidence

• Blood• Semen• Saliva• Urine• Hair• Teeth• Bone• Tissue

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

7 repeats

8 repeats

AATG

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another

Multiplex PCR Over 10 Markers Can Be

Copied at Once Sensitivities to levels

less than 1 ng of DNA Ability to Handle

Mixtures and Degraded Samples

Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

D3 FGAvWA 5-FAM (blue)

D13D5 D7 NED (yellow)

A D8 D21 D18 JOE (green)

GS500-internal lane standard

ROX (red)

AmpFlSTR® Profiler Plus™Kit available from PE Biosystems (Foster City, CA)

9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction

100 bp 400 bp300 bp200 bpSize Separation

Colo

r Sep

arati

on

ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer solution

Autosampler trayOutlet buffer

Injection electrode

Inlet buffer

Close-up of ABI Prism 310 Sample Loading Area

Autosampler Tray

Sample Vials

Electrode

Capillary

See Technology section for more information on CE

amelogenin

D19

D3

D8

TH01

VWA D21FGA

D16D18 D2

amelogeninD19

D3D8 TH01

VWA D21

FGA

D16D18 D2

Two

diffe

rent

indi

vidu

als

DNA Size (base pairs)

Results obtained in less than 5 hours with a spot of blood the size of a pinhead

probability of a random match: ~1 in 3 trillion

Human Identity Testing with Multiplex STRs

Simultaneous Analysis of 10 STRs and Gender ID

AmpFlSTR® SGM Plus™ kit

STR genotyping is performed by comparison of sample data to allelic ladders

Microvariant allele

0

5

10

15

20

25

30

35

40

45

6 7 8 9 9.3 10

Caucasians (N=427)Blacks (N=414)Hispanics (N=414)

TH01 Marker

*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

Number of repeats

Freq

uenc

y

13 CODIS Core STR Loci with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

Applications forensic investigations (98% of violent crime by men) genealogical purposes evolutionary studies

Advantages to Human Identity Testing male component isolated without differential

extraction paternal lineages

Needs population studies to evaluate diversity of

haplotypes robust assay for accurate characterization of Y

markers

Nucleic Acids Res. 28(2), e8 (2000)

Y Chromosome Structure

~2.5 Mb on tips recombine with X (pseudoautosomal regions)

~60 Mb total DNA sequence (only chromosome 22 is smaller)p

q

heterochromatin

35-36 Mb euchromatin 9.5 Mb sequenced (27%)

AMEL

SRY

Genetic variation at multiple points along the Y chromosome is combined to form a Y haplotype for a sample

Genetic variation at multiple points along the Y chromosome is combined to form a Y haplotype for a sample

STRs (microsatellites) DYS19, DYS385, etc. mostly tetranucleotide repeats

Bi-allelic markers (unique event polymorphisms--UEP) SNPs (single nucleotide polymorphisms) Y Alu polymorphism (YAP) or other

insertions/deletions (“indels”) Minisatellite

MSY1 (DYF155S1) composed of 48-114 copies of a 25 bp repeat unit with 5 sequence variant repeat types

typed by MVR-PCR (minisatellite variant repeat)

Nucleic Acids Res. 28(2), e8 (2000)

Map of Y Chromosome STR Markers

p

q

100 bp 400 bp300 bp200 bp

D13

D3

A

FGAvWA

D8 D21 D18

D5 D7

Profiler Plus™

100 bp 400 bp300 bp200 bp

D13

D3 D21TH01

D5 D7

PowerPlex™ 16

D16

D18 Penta E

A vWA D8 TPOX

CSF Penta D

FGA

http:

//w

ww

.ncb

i.nlm

.nih

.gov

/gen

ome/

guid

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

mtDNA

16,569 bp

Autosomes

Mitochondrial DNA

Nuclear DNA

3.2 billion bp

Located in cell nucleus

Located in mitochondria

(multiple copies in cell cytoplasm)

2 copies per cell

100s of copies per cell

Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 2.3, ©Elsevier Science/Academic Press

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

Due to capabilities for male-specific amplification, Y-chromosome STRs (Y-STRs) can be useful in extreme female-male mixtures (e.g., when differential extraction is not possible such as fingernail scrapings)

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

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

Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.1, ©Elsevier Science/Academic Press

CODIS STR Loci

Advantages

Extend possible reference samples beyond a single generation (benefits missing persons cases and genetic genealogy)

Family members have indistinguishable haplotypes unless mutations have occurred

Disadvantages

Lower power of discrimination due to no genetic shuffling with recombination

Family members have indistinguishable haplotypes unless mutations have occurred

Thomas Jefferson II

Field Jefferson Peter Jefferson

President Thomas Jefferson

Eston Hemings Thomas Woodson

Different Y Haplotype

Same Y Haplotype

Jefferson Y Haplotype

Jefferson Y Haplotype

?

Figure 9.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Historical Investigation of Jefferson-Hemings DNA

Genetic Genealogy Companies

SOURCE: Foster et al. (1998) Nature 396:27-28

All sources of DNA are extracted when biological evidence from a crime scene is processed to isolate the DNA present.

Thus, non-human DNA such as bacterial, fungal, plant, or animal material may also be present in the total DNA recovered from the sample along with the relevant human DNA of interest.

For this reason, the DNA Advisory Board (DAB) Standard 9.3 requires human-specific DNA quantitation so that appropriate levels of human DNA can be included in the subsequent PCR amplification.

Multiplex STR typing works best with a fairly narrow range of human DNA – typically 0.5 to 2.0 ng of input DNA works best with commercial STR kits.

1. Molecular Weight of a DNA Basepair = 618g/mol A =: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol

G = 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol

2. Molecular Weight of DNA = 1.85 x1012 g/mol There are 3 billion base pairs in a haploid cell ~3 x 109 bp

(~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol

3. Quantity of DNA in a Haploid Cell = 3 picograms1 mole = 6.02 x 1023 molecules (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules) = 3.08 x 10-12 g = 3.08 picograms (pg)

A diploid human cell contains ~6 pg genomic DNA

4. One ng of DNA contains the DNA from 167 diploid cells

1 ng genomic DNA (1000 pg)/6pg/cell = ~333 copies of each locus (2 per 167 diploid genomes)

D3S1358

-A

+A

10 ng template (overloaded)

2 ng template (suggested level)

DNA Size (bp)

Rela

tive

Fluo

resc

ence

(RFU

s)

100 pg template

5 pg template

DNA Size (bp)

We generally shoot for 0.5-2 ng

What is rtPCR or qPCR? How does it work? How does it compare to traditional

methods of Human DNA quantitation?

What techniques are available? What systems are available?

RtPCR is a very recently developed technique Developed by Higuchi in 1993 Used a modified thermal cycler with a UV detector and a

CCD camera Ethidium bromide was used as intercalating reporter As

[dsDNA] increased fluorescence increased

First paper on qPCR: Higuchi, R.; Fockler, C.; Dollinger, G.; Watson, R. “Kinetic

PCR analysis: real-time monitoring of DNA amplification reactions” Biotechnology (N Y). 1993 Sep;11(9):1026-30

Warning: RT-PCR also means reverse transcriptase PCR which is used when working with RNA

Exponential PCR

0.00E+00

1.00E+09

2.00E+09

3.00E+09

4.00E+09

5.00E+09

6.00E+09

7.00E+09

8.00E+09

9.00E+09

1.00E+10

0 5 10 15 20 25 30 35

# Cycles

ng

pro

du

ct

During the exponential expansion of the PCR the amount of product produced is proportional to the amount of template. Here we show the total amount of product following 32 cycles.

2ng template

1ng template

0.5ng template

To use PCR as a quantitative technique, the reaction must be clearly defined

In fact there are several stages to a PCR reaction Baseline stage Exponential stage Plateau stage

baseline

exponential

plateau

PCR product can not double forever Limited by Amount of primer Taq polymerase activity Reannealing of product strands

Reach plateau No more increase in product

End point detection Run for fixed # cycles and then quantify on

agarose gels

0

5

10

15

20

25

0 10 20 30 40Cycle

PC

R p

rod

uct

Equal template in all tubes

Even if same amount of template, different tubes will reach different PCR plateaus

Karen CarletonHubbard Center for Genome Studies and Department of Zoology

0

2

4

6

8

10

12

14

16

0 10 20 30 40Cycle

PC

R p

rod

uct

Different wells reach plateau at different cycle numbers. When you look changes what you see.

Karen CarletonHubbard Center for Genome Studies and Department of Zoology

Use data when still in exponential phase PCR product proportional to initial

template Need to look at PCR product each cycle

Use fluorescent detection, where fluorescence is proportional to PCR product

Use real time PCR machine which records fluorescence for each well at each cycle

Karen CarletonHubbard Center for Genome Studies and Department of Zoology

Ec is a function of:

• Hybridization efficiency

• Quantity of reactants/target DNA

• Temperature

http://www.med.sc.edu:85/pcr/realtime-home.htm

Quantitation of DNA is a based on the number of cycles required to reach a threshold intensity, Ct.

The greater the amount of starting DNA, the sooner this threshold value is reached.

Ct

http://www.med.sc.edu:85/pcr/realtime-home.htm

The log of DNA template concentration vs Ct is plotted using a series of stds yielding a calibration curve

The unknown is then run and the number of cycles required to reach threshold, Ct is compared to the calibration curve.

0.0 ng

5.0 ng

1.3 ng

0.31 ng

0.078 ng

Ct

Development of a standard curve

(reagent blank)

Concentration = 10^(-0.297*CT+ 4.528)

nanograms

Cycl

e #

Fluorescent intercalating dye - SYBR Green Fluorescence increases with

concentration of dsDNA

Taqman probes Fluorescence increases as quenched

probe is digested

Molecular beacons Fluorescence increases as quenched

probe hybridizes to template

Easy Fluorescence only with dsDNA Use with existing PCR primers

Generic, Detects all double stranded

products, including primer dimers

However, can be very specific with proper primer design

Singleplexed Multiple probes cannot be used

dsDNA Intercalationhttp://www.probes.com/handbook/figures/1557.html

Consist of ssDNA with an internal complementary sequence that keeps reporter and quencher dyes close → No fluorescence

Following denaturation, beacon anneals to template, separating both dyes and yielding fluorescence proportional to PCR product concentration

Reporter

QuencherMolecular beacon

Improved specificity and multiplexing Non-specific amplification will not produce a signal Can multiplex several probes (quantify nuclear, Y, int std.)

Can be tricky to design Loop portion – binds to DNA template Stem portion – must be complementary to other

stem Probe must denature from template below 72º so Taq

polymerase does not chew it up during extension step

Tanneal< Tm < Text

Above Tm loop structure reforms and probe leaves template

Probe also binds to PCR product during extension but is always quenched 5’-3’ exonuclease activity of Taq

polymerase digests probe and frees reporter dye from quencher

Free dye accumulates with PCR product

TaqR Q

Taq

Constituye el punto crítico para la resolución de una causa judicial.

Cada Servicio de Investigación Forense deberá establecer claros protocolos para la manipulación, transporte y conservación de las evidencias.

Cualquier manipulación inadecuada permitirá a la defensa invalidar los resultados del análisis.

Lugar del Hecho = Quirófano !

Fue históricamente la fuente de ADN más común y eficiente.

Los criterios de conservación han cambiado, simplificándose: pequeñas cantidades a temperatura ambiente, en papeles de filtro, bastan para un estudio completo si el Laboratorio cuenta con equipamiento de última generación.

Soportes adsorbentes.

Conservación a temperatura ambiente.

Largo tiempo de conservación.

Permite generar un banco de muestras.

Otras evidencias

Sangre de sospechoso/ s. Evidencias: hisopados,

prendas, pelos, uñas, etc. Sangre de la víctima.

Material cadavérico fresco: congelado -20C. Material cadavérico descompuesto: en mezcla de

sales (hasta 2 meses), congelado si el periodo es mayor.Material Cadavérico esqueletizado: conservar a temperatura ambiente, en sobres limpios, luego de lavados.

Se debe evitar en todos los casos el empleo de fijadores con formol.

2. Protección de la muestra

2.1 Contaminación por material biológico humano

Contaminación anterior o previa

Se debe a la aparición de la material biológico en el lugar donde luego aparecerán los indicios.

INEVITABLE Contaminación coetánea o paralelaEl material genético de un indicio se mezcla con ADN de otro origen en el momento de los hechos

INEVITABLE, VALORABLE y UTIL

Contaminación posteriorDebido al depósito de material genético de diversos orígenes en el indicio con posterioridad al momento de los hechos

EVITABLE

TOMA DE MUESTRAS DE REFERENCIA

1. Personas vivas

• Siempre con consentimiento informado• Debe existir un documento firmado con la autorización expresa para

realizar el análisis

SANGRE • Punción venosa (5 ml con EDTA)

• Punción dactilar (gotas depositadas en papel secante y se dejan secar a TA)

CEL EPITELIALES BUCALES • 2 Hisopos de ambos carrillos (importante dejarlos secar antes de enfundar para evitar la proliferación bacteriana)

PELOS CON BULBO • 10-15 pelos con bulbo

En PERSONAS TRANSFUNDIDAS evitar la toma de sangre, podría detectarse el ADN procedente de la sangre transfundida al menos en un corto periodo de tiempo después a la transfusión

2. Cadáveres en buen estado de conservación

TOMA DE MUESTRAS DE REFERENCIA

SANGRE post-mortem 1 ml (anticoagulante tipo EDTA)

MUSCULO ESQUELETICO Aprox 1 gr. Se almacena en un recipiente de plástico y tapón de rosca.

PIEZAS DENTALES 2 (molares). Dejar en reserva con el fin de evitar la exhumación.

3. Cadáveres en avanzado estado de putrefacción o esqueletizados

HUESO LARGO Fémur, húmero…

PIEZAS DENTALES 2 (molares). No dañados externamente ni sometidos a endodoncias.

4. Cadáveres carbonizados

TOMA DE MUESTRAS DE REFERENCIA

• Cuando la carbonización no es total es posible analizar MÚSCULO ESQUELÉTICO de zonas profundas.

• Cuando la carbonización es total recomendable contactar con el laboratorio

5. Otras muestras de referencia de personas fallecidas

• En hospitales (muestras de sangre, biopsias en parafina, preparaciones histológicas…

No utilizar tejidos fijados en formol.• Ámbito familiar (peines, maquinillas de afeitar, saliva en sellos o sobres…)

FINMás información sobre análisis de ADN:

• Laboratorio: www.adn.ac• Sociedad Latinoamericana de

Genética Forense: www.slagf.org• Tesis doctoral:www.secretpaternity.com