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