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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