DNA FORENSIK MK. Kimia Forensik Oleh : Dr. I Nengah Wirajana Kimia FMIPA UNUD 2011
Oct 22, 2014
DNA FORENSIKMK. Kimia Forensik
Oleh :Dr. I Nengah Wirajana
Kimia FMIPA UNUD2011
Forensic Science
• Process of gathering (pengumpulan) and examining (pengujian)
evidence of a crime• First practiced forensic medicine in 1958• Italy• Application of medical knowledge to legal
questions
Brief History of Forensic DNA Typing
• 1980 - Ray White describes first polymorphic RFLP marker
• 1985 - Alec Jeffreys discovers multilocus VNTR (variable number tandem repeat) probes (penanda/kuar)
• 1985 - first paper on PCR• 1988 - FBI starts DNA casework• 1991 - first STR paper• 1995 - FSS starts UK DNA database• 1998 - FBI launches CODIS database
Pengidentitas yang unik (Unique Identifiers)
• Fingerprints– When a persons hand
touched a surface, it left a barely visible mark
– Each person’s fingerprint is a unique identifier of that person, no two people’s are alike
• DNA – Genetic material that is
found in DNA is also a unique identifier
– Not until1985 when DNA became part of forensic science
DNA (deoxyribonucleic acid)
• Nucleic acid that carries the genetic information • Double helix• 2 long chains of nucleotides twisted and joined by
hydrogen bonds• Can be found in white blood cells• Polymorphic: vary in shape from person to person
Penggunaan Identifikasi DNA secara Umum
– Investigations of criminal cases involving victims (korban-korban)• Assault (sergapan)• Kidnapping (penculikan)• Robbery (perampokan)• Rape (perkosaan)• Murder (pembunuhan)
– Catastrophe victims (korban-korban bencana)– Paternity/family relationships (hub. garis keturunan ayah/keluarga)– Identify endangered and protected species (identifikasi spesies yang
berbahaya dan dilindungi)– Detect bacteria/organisms that may pollute the air, water, food, and soil
(mendeteksi bakteri/organisme lain yg mungkin mencemari udara, air, makanan, dan tanah)
– Match organ donors with recipients (mencocokan organ donor dg penerima)– Determine pedigree for seed/livestock breeds (menentukan silsilah benih /
keturunan ternak)– Authenticate consumables such as caviar and wine (membuktikan dapat
dimakan/tidak, seperti telur sejenis ikan [terubuk] dan anggur
DNA Use in Forensic Cases
• Most are rape (perkosaan) 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 (tantangan)
Human Identity Testing
• 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
Sources of Biological Evidence
• Blood• Semen• Saliva• Urine• Hair• Teeth• Bone• Tissue
Progression of DNA Typing Markers
• RFLP (Restriction Fragment-Length Polymorphisms)– multilocus VNTR probes– single locus VNTR probes (32P and chemiluminescence)
• PCR– DQ-alpha (reverse dot blot)– PolyMarker (6 plex PCR; dots for SNPs)– D1S80 (AMP-FLPs)
– singleplex STRs with silver staining– multiplex STRs with fluorescent dyes
Changing Technologies Paradigm Shift: Restriction Fragment-Length Polymorphisms
to Short Tandem Repeats
• Five RFLP probes provide almost exclusive identity (~ 1 in 109 individuals)
• RFLP requires a minimum of 25 ng of relatively undegraded DNA (1000 - 20,000 basepairs)
• Short Tandem Repeats (STRs) only require ~ 1 ng DNA that can be partially degraded
• Discrimination power: 5 RFLP probes equals ~ 12 STR loci
Contoh data berupa
elektroforegram
(hasil elektroforesis)
sampel DNA pada
RFLP
Which Suspect,
A or B, cannot
be excluded from
potential perpetrators (pelaku)
of this assault?
Short Tandem Repeats (STR)
Teknik Baru untuk Identifikasi Personal
Disunting dari berbagai sumber
Short Tandem Repeats (STR)• STR regions are nucleotides along the backbone of a
chromosome• 13 markers used in forensic science• Classified into groups depending on the size of the repeat
regions– Mini satellites– Micro satellites
• The chance that any two peoples DNA fingerprint for a particular set of regions is exceptionally small– 1/10th of a single % of DNA, about 3 million bp, differ from one person
to the next*(kemungkinan bahwa ada dua sidik jari DNA manusia untuk daerah
posisi utama yg sama amat kecil sekali )
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
The Process
• Isolation• Quantifying• Polymerase Chain Reaction (PCR)• Short Tandem Repeat-Polymerase Chain
Reaction (STR-PCR)• Interpretation• Database
Isolation
• Scientist extract DNA from the nucleus of cells in tissue
• Quality of tissue DNA samples degrades as body decomposes
• 1-4 hours
Quantifying
• Tests are run to determine the amount of DNA recovered
• Targeted amount 1 nanogram (billionth of a gram)
• If inadequate quantity, isolation must be repeated
• 1-2 hours
Polymerase Chain Reaction (PCR)
• One of the most popular and widely used techniques in molecular biology
• Reproduces millions of exact copies of specific fragments of DNA
• Enables even highly degraded samples to be analyzed
• 3 hours
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
• Based on polymerase enzyme• Break apart double helix, two single strands• Rebuild two strands into two complete helixes• DNA deposited into polymerases and nucleotides• Repeated rapidly, doubling amount of DNA
STR-PCR
• Mainly same process• Focus solely on STR regions• Since these repeat regions are usually
bounded by specific restriction enzyme sites, it is possible to cut out the segment of the chromosome
Capillary Electrophoresis (CE)• Early 1990’s• Automated analytical technique • Generally used for separating ions, which move at different
speeds when the voltage is applied depending on their size and charge. The solutes are seen as peaks as they pass through the detector
• Area of each peak is proportional to their concentration • Produces a chart mapping a person’s exact genetic makeup • This is the information used to compare suspects to a crime • http://chemi.muni.cz/~analytika/ce/ce-animation.gif
Capillary Electrophoresis
Interpretation
• A DNA scientist reviews the DNA profile produced through the capillary electrophoresis to determine if there is a match
• STR markers are examined (2-5 bp)• 1-3 are not enough to determine is the sample came
from the suspect• 4-5, beyond a reasonable doubt• 5 very rare
National DNA Databank: CODIS
• The COmbined DNA Index System• Blends computer and DNA technologies into a tool
for fighting crime• 2 indexes
– Convicted Offender Index• DNA profiles of individuals convicted of criminal crimes
– Forensic Index• DNA profiles developed from crime scene evidence
• All profiles stored in CODIS are generated using STR analysis
FBI’s CODIS DNA Database
Combined DNA Index System• 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• Current backlog of >600,000 samples
The Future
• March 2004: President Bush proposed $1 billion in funding over the next 5 years– Reduce DNA testing backlog (mengurangi pekerjaan uji DNA
yg tertunda)
– Build crime lab capacity– Stimulate research and development– Support training– Protect the innocent (TIDAK BERSALAH)– Identify missing persons
Short Tandem Repeats (STRs)
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
170 bp170 bp195 bp195 bp
Different primer sets produce different PCR product sizes for the same STR allele
TCAT repeat unitTCAT repeat unit
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
An Example Forensic STR Multiplex Kit
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
Available Kits for STR Analysis
• Kits make it easy for labs to just add DNA samples to a pre-made mix
• 13 CODIS core loci– Profiler Plus and COfiler (PE Applied Biosystems)– PowerPlex 1.1 and 2.1 (Promega Corporation)
• Increased power of discrimination– CTT (1994): 1 in 410– SGM Plus™ (1999): 1 in 3 trillion– PowerPlex ™ 16 (2000): 1 in 2 x 1017
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
STR Allele Frequencies
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
Terima kasih
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