DNA Profiling and Forensic Genetics PDF

DNA Profiling and Forensic Genetics Assoc. Prof. Mahmut Çerkez Ergören Near East University Department of Medical Genetics Faculty of Medicine  Analysis of Microsatellites Microsatellites are widely used for DNA profiling in forensic identifica...

DNA Profiling and Forensic Genetics Assoc. Prof. Mahmut Çerkez Ergören Near East University Department of Medical Genetics Faculty of Medicine  Analysis of Microsatellites Microsatellites are widely used for DNA profiling in forensic identification. Microsatellites are used for mapping within the genome, specifically in locations genetic linkage analysis/marker assisted selection to locate a gene or a mutation responsible for a given trait or disease. As a special case of mapping, they can be used for studies of gene duplication or deletion.  Origin of tandem repeats by unequal pairing and crossing over Length variation can be generated at tandem repeat loci by unequal pairing and crossing over. This Figure illustrates the general principle behind the generation of DNA variation at tandem repeat loci such as minisatellites and microsatellites.  Human Identity Testing 1. Forensic cases -- matching suspect with evidence 2. Paternity testing -- identifying father 3. Historical investigations 4. Missing persons investigations 5. Mass disasters -- putting pieces back together 6. Military DNA “dog tag” 7. Convicted felon DNA databases  Methods of identification Used Since? Identification Method Accuracy? 1800 Measurement of height 1 in 4 (Quételet’s method) Comparison of Pubic 1 in 800 hair Late 1800’s Early 1900’s Comparison of Scalp 1 in 4500 hair Late 1800’s early 1900’s Anthropometry 1 in 268 million (Bertillon’s method) Forensic odontology 1 in 2.5 billion Teeth bite marks Evidence in Early Egypt – Dactylography ? documented forensic use (Fingerprints) 1800’s -1900’s Late 1900’s DNA Fingerprinting 1 in 2 x 1022 Late 1900’s early 2000’s Facial recognition ?  Brief History of Forensic DNA Typing Ø 1980 - Ray White describes first polymorphic RFLP marker Ø 1985 - Alec Jeffreys discovers multi-locus VNTR probes Ø 1985 - first paper on PCR Ø 1988 - FBI starts DNA casework Ø 1991 - first STR paper Ø 1995 - FSS starts UK DNA database Ø 1998 - FBI launches CODIS database  DNA fingerprinting Principles – Restriction Fragment Length Polymorphism (RFLP) – Multiplex PCR Techniques – Southern blot – PCR – Capillary Electrophoresis Population genetics Prof. Sir Alec Jeffreys Molecular geneticist Inventor of DNA fingerprinting Department of Genetics University of Leicester-UK The first DNA fingerprint co 9:05am, Monday 10 Sep 1984 ac r the r ild he tob ch mo fat  Steps in Sample Processing Sample Obtained from Crime Scene or Paternity Biology Investigation DNA DNA PCR Amplification Extraction Quantitation of Multiple STR markers Technology Separation and Detection of Sample Genotype PCR Products Determination (STR Alleles) Genetics Comparison of Sample Generation of Case Genotype to Other Report with Probability Sample Results of Random Match If match occurs, comparison of DNA profile to population databases Using microsatellites for identification 1. PCR-RFLP method (Restriction Fragment Lenght Polymorphism) Oldest/Cheapest test Requires large amounts of non-degraded DNA Utilizes the longer sequences of VNTR’s  Type II Restriction enzyme or restriction endonuclease – A group of enzymes, produced by bacteria, that cleave molecules of DNA internally at specific base sequence EcoRI restriction enzyme ↓ 5’ GAATTC 3’ 5’ G AATTC 3’ 3’ CTTAAG 5’ 3’ CTTAA G 5’ ↑ Amplification 1) Microsatellites can be amplified for identification by the polymerase chain reaction (PCR) process: using the unique sequences of flanking regions as primers; a)DNA is repeatedly denatured at a high temperature to separate the double strand, b) cooled to allow annealing of primers c)the extension of nucleotide sequences through the microsatellite. 2) This process results in production of enough DNA to be visible on agarose or polyacrylamide gels. Primers that flank microsatellite loci are simple and quick to use, but the development of correctly functioning primers is open a tedious and costly process  Design of microsatellite primers If searching for microsatellite markers in specific regions of a genome, for example within a particular exon of a gene, primers can be designed manually. This involves searching the genomic DNA sequence for microsatellite repeats, which can be done by eye or by using automated tools such as repeat masker. Once the potentially useful microsatellites are determined, the flanking sequences can be used to design oligonucleotide primers which will amplify the specific microsatellite repeat in a PCR reaction.  DNA is cut into different size VNTR’s by the use of restriction enzymes Fragments are placed on a gel plate  Gel Electrophoresis System Loading well anode cathode - + Gel - Buffer DNA bands Voltage + Gel lanes Side view Top view Separation of DNA sequence length amplified products Larger - fragments Smaller fragments + Fragments are separated by electrophoresis The DNA is then transferred from the gel plate and made visible bsapp.com  RFLP EcoRI EcoRI Longer restriction fragment length Detection of restriction fragment length by Southern blot EcoRI EcoRI Shorter restriction fragment length Note: Restrictions enzymes may be located within the VNTR, thereby giving multiple bands  DNA Extraction and Quantitation Extraction Chemicals Type 1: DNA Blood Stain Extraction Type 2: Different Chemicals Chemicals Vaginal Swab Epithelial Sperm DNA DNA (Female) (Male) 2. Automatic microsatellite (Short Tandem Repeats; STRs) Analysis: Fluorescen t dye AATG AATG AATG label 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another Primer positions define PCR product size Capillary Electrophoresis Fluorescently labeled DNA fragments separated by size migrate by the laser detection region on the capillary electrophoresis instrument Fluorescent dyes with excitation and emission traits result in detection of DNA fragments STR Analysis Genotyping is performed by comparing to STR allelic ladder STR allelic ladder represents all possible STR designations for a given DNA site Alleles represent different lengths of STRs on a chromosome Sizing assured by internal sizing standard STR genotyping is performed by comparison of sample data to allelic ladders Microvariant allele  Heterozygous versus Homozygous in SINGLE SOURCE samples Locus 1 Heterozygous Locus 2 Heterozygous Locus 3 Homozygous At each locus there are either one or two peaks. Two peaks at a locus site are called heterozygous while one peak is called homozygous.  Why STRs are Preferred Genetic Markers Rapid processing is attainable Abundant throughout the genome Highly variable within various populations Small size range allows multiplex development Discrete alleles allow digital record of data Allelic ladders simplify interpretation PCR allows use of small amounts of DNA material Small product size compatible with degraded DNA  FBI’s CODIS DNA Database Combined DNA Index System: http://www.fbi.gov/hq/lab/codis/index1.htm Used for linking serial crimes and unsolved cases with repeat offenders Launched October 1998 Links all 50 states Requires >4 RFLP markers and/or 13 core STR markers As of September 2003 – Total number of profiles: 1,472,150 – Total Forensic profiles: 64,523 – Total Convicted Offender Profiles: 1,407,627 – 9,842 Investigations Aided through September 2003 Position of Forensic STR Markers on Human Chromosomes TPOX 13 CODIS Core STR Loci D3S1358 TH01 D8S1179 D5S818 VWA D2S1338 FGA D7S820 CSF1PO AMEL Sex-typing Penta E D19S433 D13S317 D16S539 D18S51 D21S11 AMEL Penta D  Why mtDNA SNPs? Well characterized and studied (population, evolutionary, medical and forensic studies) Uniparental maternal inheritance missing persons- mat. lineage ref smpls Relatively small size (16kb) and high copy number – good on low quantity/quality samples (hair, bone, teeth- ancient/degraded)-(Think Peterson case) Implicated in maternally inherited diseases : diabetes, deafness, hypertrophic cardiomyopathy and myopathy Analysis by DNA sequencing- more complex than STR analysis mtDNA - many mitotypes are only found 1X. Some use counting method for statistics. Commonly found mitotypes are as frequent as 1 in 10.  Why Y? Applications – Forensic investigations (98% of violent crime by men) – Biodefense- Male terrorist profiling – Genealogical and Evolutionary studies Advantages to Human Identity Testing – Male component isolated without differential extraction – Paternal lineages – Some cases with no spermatazoa- use Y STRs – Assess number of male donors/contributors – Same analysis as autosomal STRs Challenges – Y STR kits not as abundant- now 12plexes available in 2003 – Some Y Haplogroups are common – Population specific haplotying needed for new markers Other Applications of DNA Analysis PATERNITY FATHER CHILD 1 CHILD 2 CHILD 3 MOTHER  Other Applications of DNA Analysis IDENTIFICATION OF MASS DISASTER VICTIMS Ø World Trade Center, Tsunami, Hurricane Katrina Ø Comparison of biological samples from the scene of disaster (bone, teeth, hair) to personal effects from a missing person (razor, toothbrush)  Wildlife forensics Esther Signer Dr Josef Mengele  T C G A fem so ur n Mfd49 wi fe Immigration dispute April 1985 mother boy in dispute undisputed children DNA testlerinin okunması bsapp.com bsapp.com bsapp.com  Overview of Steps Involved in DNA Typing TH01 TPOX CSF D7 D3 D16 D5 D13 Penta D AMEL VWA D21 D8 D18 FGA Penta E Blood Stain PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis DNA Quantitation using Slot Blot Genotyping by Comparison to Allelic Ladder