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Genetica forense curso 2012


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Genetica forense curso 2012

  1. 1. Bioq. Raúl Horacio LuceroJefe de Area Biología MolecularInstituto de Medicina Regional
  2. 2.  Exámenes generales:  Diagnóstico de especie. datos fisonómicos, sexo,  Datación de los restos. peso, talla, cabello, color  Exámenes generales. de ojos y piel, marcas.  Elementos extrínsecos. Registro de voz.  Caracteres patológicos, Trazado caligráfico. naturales o traumáticos. Huellas dactilares.  Identidad radiográfica y Huellas genéticas. dental.  Métodos bioquímicos.
  3. 3.  Grupos sanguíneos: ABO,  Minisatélites: sondeos de RH, MNS, Duffy, Lewis. locus múltiple y locus Proteínas plasmáticas: único. haptoglobina, α1-  Microsatélites: métodos de antitripsina, transferrina. PCR. Enzimas eritrocitarias:  Variantes de secuencia: fosfatasa ácida HLA-DQα, Polymarker, eritrocitaria, adenilato genoma mitocondrial. kinasa, PGM, ADA. HLA: Antígenos de histocompatibilidad.
  4. 4.  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.
  5. 5. Sitio de adhesión de Sitio de adhesión delos primers los primers Alelo 1 Alelo 2 Unidad de Repetición
  6. 6.
  7. 7.  Herramienta indispensable para la investigación forense. Gran número de STRs disponibles. Alta sensibilidad. Evaluación manual o automatizada.
  8. 8. Analizando el Caso.Material Indubitadode la Víctima.Material SubunguealTomado de la Víctima Tejido Dactilar de la Víctima. Match Match MatchSangre delSospechoso1. Sangre del Sospechoso 2
  9. 9. 2006: DNA is an important part of the criminal justice system Justice for All Act ($1B 2006 over 5 years) Identifiler 5-dye kit and ABI 3100 UK National Database 2002 2004 Y-STRs launched (April 10, CODIS loci 1995) defined PowerPlex® 16 (16Gill et al. (1985) Forensic loci in single amp)application of DNA fingerprints‘. 1998 2000Nature 318:577-9FSS Quadruplex STR typing with CE is fairly routine 1994 1996 First commercial First STRs fluorescent STR mtDNA developed multiplexes 1990 1992 Capillary electrophoresis of STRs first described 1985 PCR developed DQA1 & PM (dot blot) Multiplex STRs RFLP
  10. 10. 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 2005Contains more than 2.8 million DNA profiles
  11. 11.  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
  12. 12. An accordion-like DNA sequence that occurs between genesTCCCAAGCTCTTCCTCTTCCCTAGATCAATACAGACAGAAGACAGGTGGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATATCATTGAAAGACAAAACAGAGATGGATGATAGATACATGCTTACAGATGCACAC = 12 GATA repeats (“12” is all that is reported) 7 repeats The number of consecutive repeat units 8 repeats can vary between people 9 repeats 10 repeats 11 repeats The FBI has selected 13 12 repeats core STR loci that must be 13 repeats run in all DNA tests in order to provide a Target region common currency with (short tandem repeat) DNA profiles
  13. 13. The polymerase chain reaction (PCR) is used to amplify STR regions and label the amplicons with fluorescent dyes using locus-specific primers 8 repeats Locus 1 10 repeats 8 repeats Locus 2 9 repeatsScanned Gel Image Capillary Electropherogram
  14. 14. YCAII ~45% Requires size based DNA separation to resolve different alleles from one anotherHigh stutter  Dinucleotide (CA)(CA)(CA)(CA)  Trinucleotide (GCC)(GCC)(GCC)  Tetranucleotide (AATG)(AATG)(AATG)  Pentanucleotide (AGAAA)(AGAAA)Low stutter  Hexanucleotide (AGTACA)(AGTACA) DYS448 Short tandem repeat (STR) = microsatellite = <2% simple sequence repeat (SSR)
  15. 15. Category Example Repeat 13 CODIS Loci StructureSimple repeats – contain (GATA)(GATA)(GATA) TPOX, CSF1PO,units of identical length and D5S818, D13S317,sequence D16S539Simple repeats with (GATA)(GAT-)(GATA) TH01, D18S51, D7S820non-consensus alleles(e.g., TH01 9.3)Compound repeats – (GATA)(GATA)(GACA) VWA, FGA, D3S1358,comprise two or more D8S1179adjacent simple repeatsComplex repeats – (GATA)(GACA)(CA)(CATA) D21S11contain several repeatblocks of variable unit length These categories were first described by Urquhart et al. (1994) Int. J. Legal Med. 107:13-20
  16. 16.  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.
  17. 17.  Compatible primers are the key to successful multiplex PCR  STR kits are commercially available  15 or more STR loci can be simultaneously amplified Challenges to Multiplexing primer design to find compatible primers (no program exists) reaction optimization is highly empirical often taking monthsAdvantages of Multiplex PCR –Increases information obtained per unit time (increases power of discrimination) –Reduces labor to obtain results –Reduces template required (smaller sample consumed)
  18. 18.  Most are rape cases (>2 out of 3) Looking for match between evidence and suspect Must compare victim’s DNA profile Challenges•Mixtures must be resolved•DNA is often degraded•Inhibitors to PCR are often present
  19. 19.  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
  20. 20. Steps in DNA Sample Processing Sample Obtained fromCrime Scene or Paternity Investigation Biology DNA DNA DNA DNA PCR Amplification PCR Amplification Extraction Extraction Quantitation Quantitation of Multiple STR markers of Multiple STR markers Technology Separation and Detection of Sample Genotype PCR Products Determination (STR Alleles) Genetics Comparison of Sample Comparison of Sample Generation of Case Generation of Case Genotype to Other Genotype to Other Report with Probability Report with Probability Sample Results Sample Results of Random Match of Random Match If match occurs, comparison If match occurs, comparison of DNA profile to population of DNA profile to population databases databases
  21. 21. Sources of Biological Evidence• Blood• Semen• Saliva• Urine• Hair• Teeth• Bone• Tissue
  22. 22. AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constantHomozygote = both alleles are the same lengthHeterozygote = alleles differ and can be resolved from one another
  23. 23. 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
  24. 24. AmpFlSTR® Profiler Plus™ Kit available from PE Biosystems (Foster City, CA) 100 bp 200 bp 300 bp 400 bp Size Separation D3 vWA FGA 5-FAM (blue) Color Separation A D8 D21 D18 JOE (green) D5 D13 D7 NED (yellow) ROX (red) GS500-internal lane standard9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction
  25. 25. ABI Prism 310 Genetic Analyzer capillarySyringe with polymer solution Injection electrode Outlet Autosampler tray buffer Inlet buffer
  26. 26. Close-up of ABI Prism 310 Sample Loading Area Electrode Capillary Sample Vials Autosampler Tray See Technology section for more information on CE
  27. 27. Human Identity Testing with Multiplex STRs AmpFlSTR® SGM Plus™ kit DNA Size (base pairs) D3 amelogenin D8 TH01 D19 VWA D21 D16 D18Two different individuals D2 FGA probability of a random match: ~1 in 3 trillion Results obtained in less than 5 hours with a spot of blood the size of a pinhead amelogenin D3 D19 D8 VWA D16 D21 D18 D2 FGA TH01 Simultaneous Analysis of 10 STRs and Gender ID
  28. 28. STR genotyping is performed by comparison of sample data to allelic ladders Microvariant allele
  29. 29. 45 40 TH01 Marker 35 30 25 Caucasians (N=427)Frequency 20 Blacks (N=414) 15 Hispanics (N=414) 10 5 *Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34 0 6 7 8 9 9.3 10 Number of repeats
  30. 30. 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMELD13S317 D16S539 D18S51 D21S11 AMEL
  31. 31.  Applications  forensic investigations (98% of violent crime by men)  genealogical purposes  evolutionar y 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
  32. 32. Y Chromosome Structure SRY ~60 Mb total DNA sequence (only chromosome 22 is smaller) p AMEL ~2.5 Mb on tips recombine with X (pseudoautosomal regions) q 35-36 Mb euchromatin 9.5 Mb sequenced (27%) heterochromatin Genetic variation at Genetic variation at multiple points along the Y multiple points along the Y chromosome is combined chromosome is combined to form a Y haplotype for a to form a Y haplotype for aNucleic Acids Res. 28(2), e8 (2000) sample sample
  33. 33.  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)
  34. 34. Map of Y Chromosome STR Markers p q Nucleic Acids Res. 28(2), e8 (2000)
  35. 35. Profiler Plus™100 bp 200 bp 300 bp 400 bp D3 vWA FGA A D8 D21 D18 D5 D13 D7
  36. 36. PowerPlex™ 16100 bp 200 bp 300 bp 400 bp D3 TH01 D21 D18 Penta E D5 D13 D7 D16 CSF Penta D A vWA D8 TPOX FGA
  37. 37. Located in cell nucleus Autosomes 2 copies Located in per cell mitochondria (multiple copies in cell cytoplasm) mtDNA 1 2 3 4 5 6 7 8 9 10 11 12 16,569 bp Mitochondrial 13 14 15 16 17 18 19 20 21 22 X Y DNA Sex- Nuclear DNA chromosomes 3.2 billion bp 100s of copies per cellButler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 2.3, ©Elsevier Science/Academic Press
  38. 38.  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
  39. 39. Lineage Markers CODIS STR Loci Autosomal Y-Chromosome Mitochondrial (passed on in part, (passed on complete, but (passed on complete, from all ancestors) only by sons) but only by daughters)Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.1, ©Elsevier Science/Academic Press
  40. 40. Advantages Disadvantages Extend possible reference  Lower power of discrimination due samples beyond a single to no genetic shuffling with generation (benefits missing recombination persons cases and genetic genealogy)  Family members have indistinguishable haplotypes unless Family members have mutations have occurred indistinguishable haplotypes unless mutations have occurred
  41. 41. Historical Investigation of Jefferson-Hemings DNA Thomas Jefferson II Field Jefferson Peter Jefferson Genetic Genealogy Companies President Thomas Jefferson ? Eston Hemings Thomas Woodson Same Y Haplotype Jefferson Y Haplotype Different Y Haplotype JeffersonY Haplotype SOURCE: Foster et al. (1998) Nature 396:27-28 Figure 9.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
  42. 42.  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.
  43. 43. 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 picograms 1 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 167diploid cells 1 ng genomic DNA (1000 pg)/6pg/cell = ~333 copies of each locus (2 per 167 diploid genomes)
  44. 44. We generally shoot for 0.5-2 ng DNA Size (bp) -A DNA Size (bp)Relative Fluorescence (RFUs) +A 100 pg 10 ng template (overloaded) template D3S1358 5 pg 2 ng template (suggested level) template
  45. 45.  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?
  46. 46.  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
  47. 47. During the exponential expansion of the PCR the amount of product produced is proportional to the amount of template. Here Exponential PCR we show the total amount of product following 32 cycles. 1.00E+10 9.00E+09 8.00E+09 7.00E+09ng product 6.00E+09 5.00E+09 2ng template 4.00E+09 3.00E+09 1ng template 2.00E+09 0.5ng template 1.00E+09 0.00E+00 0 5 10 15 20 25 30 35 # Cycles
  48. 48.  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  Plateau stage exponential baseline
  49. 49.  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
  50. 50. Even if same amount of template, different tubes will reach different PCR plateaus 25 Equal template in all tubes 20 PCR product 15 10 5 0 0 10 20 30 40 Cycle Karen Carleton Hubbard Center for Genome Studies and Department of Zoology
  51. 51. Different wells reach plateau at different cycle numbers. When you lookchanges what you see. 16 14 12 PCR product 10 8 6 4 2 0 0 10 20 30 40 Cycle Karen Carleton Hubbard Center for Genome Studies and Department of Zoology
  52. 52.  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 Carleton Hubbard Center for Genome Studies and Department of Zoology
  53. 53.  Ec is a function of: • Hybridization efficiency • Quantity of reactants/target DNA • Temperature
  54. 54.  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
  55. 55.  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.
  56. 56. Development of a standard curve 5.0 ng 1.3 ng 0.31 ng 0.0 ng 0.078 ng (reagent blank)Ct
  57. 57. Cycle # nanograms Concentration = 10^(-0.297*CT+ 4.528)
  58. 58.  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
  59. 59.  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 Intercalation
  60. 60.  Consist of ssDNA with an internal complementary sequence that keeps reporter and quencher dyes close → No fluorescence Reporter Molecular beacon Quencher Following denaturation, beacon anneals to template, separating both dyes and yielding fluorescence proportional to PCR product concentration
  61. 61.  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 < TextAbove Tm loop structure reforms and probe leaves template
  62. 62. 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 R Q Taq Taq
  63. 63.  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.
  64. 64. Lugar del Hecho = Quirófano !
  65. 65.  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.
  66. 66.  Soportes adsorbentes. Conservación a temperatura ambiente. Largo tiempo de conservación. Permite generar un banco de muestras.
  67. 67. Otras evidencias
  68. 68.  Sangre de sospechoso/ s. Evidencias: hisopados, prendas, pelos, uñas, etc. Sangre de la víctima.
  69. 69.  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.
  70. 70. 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 paralela El material genético de un indicio se mezcla con ADN de otro origen en el momento de los hechos INEVITABLE, VALORABLE y UTIL  Contaminación posterior Debido al depósito de material genético de diversos orígenes en el indicio con posterioridad al momento de los hechos EVITABLE
  71. 71. TOMA DE MUESTRAS DE REFERENCIA1. Personas vivas • Siempre con consentimiento informado • Debe existir un documento firmado con la autorización expresa para realizar el análisis En PERSONAS TRANSFUNDIDAS evitar la toma de sangre, podría detectarse • Punción venosa (5 ml con EDTA) SANGRE el ADN procedente de la sangre transfundida al menos en un corto periodo de tiempo después a la transfusión • 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
  72. 72. TOMA DE MUESTRAS DE REFERENCIA2. Cadáveres en buen estado de conservación 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.
  73. 73. TOMA DE MUESTRAS DE REFERENCIA4. Cadáveres carbonizados • Cuando la carbonización no es total es posible analizar MÚSCULO ESQUELÉTICO de zonas profundas. • Cuando la carbonización es total recomendable contactar con el laboratorio5. 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…)
  74. 74. FINMás información sobre análisis de ADN:• Laboratorio:• Sociedad Latinoamericana de Genética Forense:• Tesis