1.

DNA in Forensic

by Dr. Tiana Milanda

Introduction

In the late 1800s, efforts to fight crime were given by photography. Alphonse Bertillon was the first to approach a crime scene with the systematic methods. He'd capture images at various distances and take both ground level and overhead shots. Photographs were and are still being used to record evidence around an crime scene that then allow forensic scientists and criminal investigators to have an reference guide for that particular crime scene. Limitations : criminals found may ways to alter their appearance.

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Introduction

In 1823, John Evangelist Purkinje published his thesis discussing 9 fingerprint patterns, but he made no mention of the value of fingerprints for personal identification. In 1892, Juan Vucetich made the first criminal fingerprint identification, because no two fingerprints have ever been found alike They need a huge collections of prints and clerks were responsible for examining each set of possible prints to find matches the process became less tedious and more realiable with a computer. Limitations : fingerprints can be wiped away and gloves can be worn to keep from leaving fingerprints behind.

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Introduction

In 1985, Alec Jeffrey developed the method of DNA fingerprinting. In the course of his research on variability in human DNA, Alec Jeffreys developed a method of forensic DNA typing. This method, termed DNA Fingerprinting

Human DNA

In human cells, DNA are packing in nuclear chromosome and cytoplasmic mitochondria chromosome

male

Nuclear chromosomes

Human cells : somatic cells and gametes Each somatic cells have two sets of 23 linear chromosomes (diploid), 23 maternal chromosomes and 23 paternal chromosomes homologous pairs

female

LOCUS AND ALLELE

Allele: Refers to the type of DNA.

Locus or Loci: Refers to the location on the chromosome

2 alleles per locus

male

Nuclear chromosomes
Chromosomes 1-22 autosomes, chromosomes 23 sex chromosomes, consisting of X and Y chromosome Autosomes contain genes that affects other body features unrelated to sex Sex chromosome (X and Y) contain genes that influence sex traits and the development of reproductive organs

female

Autosomes : coding and non coding sequences

Nuclear (and mitochondrial) chromosomes consist of two types of nucleotides 1. Those that make up the genes, called coding sequences 2. Those whose function is largerly unknown, called non coding regions The nucleotide in coding and non coding portion are alike, they differ solely in whether or not they contribute to one or more of the individuals trait (phenoptype)

Nuclear : Non coding sequence

3 5 Intergenic DNA Gene 1 Gene 2 5

3 5

Gene 1
Introns

Gene 2

5 3

Nucleus : Eukaryotic genomes full of repeated DNA

Repeat Unit # Repeats ???

Satellite DNA Minisatellite DNA or Variable Number of Tandem Repeats (VNTR) Microsatellite DNA or Short Tandem Repeats

100 1000 bp

7 100 bp

7 - 80

2 6 bp

5 - >40

VNTR

Rectangle blocks represent repeated DNA sequences at a particular VNTR location Repeats are tandem clustered together and oriented in same direction Repeats can be removed or added leading to alleles with different numbers of repeats.

History of DNA Testing (Soon)

DNA fingerprint : - RFLP Multilocus (fingerprint) - RFLP single locus - PCR strips DNA profiling : - PCR STR Mitochondrial DNA testing Y chromosome testing Another testing

Types of STRs
Dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, hexanucleotide.
Simple repeats: identical length and sequence.
Compound repeats: two or more adjacent simple repeats. Complex repeats: repeat blocks varying in unit length and sequence

Short Tandem Repeats

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..

1 1 1

2 2 2

3 3 3

4 4 4

5 5 5

Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT.. Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..

On average, occur every 10,000 nucleotides.

LOCUS AND ALLELE

Allele: Refers to the type of DNA.

Locus or Loci: Refers to the location on the chromosome

2 alleles per locus

Person 1..GCCAGCTAGCTAGCTAGCTTTCAT..

1 2 3 4 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT.. 1 2 3 4 5 6 1 2 3 4 5 6 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTTTCAT.. 1 2 3 4 5 6 7 1 2 3 4 5 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT.. 1 2 3 4 5

Person 2..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..

Person 3..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..

Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another

Loci Numenclature
3 5 Intergenic DNA Gene 1 Gene 2 5

3 5

Gene 1
Introns

Gene 2

5 3

Loci Nomenclature

Introns: STR name based on gene.

TH01 - located in the 1st intron (01) of gene for tyrosine hydroxylase

Intergenic DNA: based on chromosome

D5S818
D = DNA 5 = chromosome 5 S = single copy in genome 818 = 818th locus

History of DNA Testing

DNA fingerprint : - RFLP Multilocus (fingerprint) - RFLP single locus - PCR strips DNA profiling : - PCR STR Mitochondrial DNA testing Y chromosome testing Another testing

male

Nuclear chromosomes
Human males have an X and a Y chromosomes (XY) and females have a pair of X chromosomes (XX) A set genes found on the Y chromosomes, called SRY (sex-determining region) determine an embryo into a male

female

Y Chromosome Structure
SRY ~60 Mb total DNA sequence (only

AMEL

chromosome 22 is smaller)
~2.5 Mb on tips recombine with X

(pseudoautosomal regions)

35-36 Mb euchromatin 9.5 Mb sequenced (27%)

heterochromatin

Nucleic Acids Res. 28(2), e8 (2000)

Genetic variation at multiple points along the Y chromosome is combined to form a Y haplotype for a sample

History of DNA Testing (Soon)

DNA fingerprint : - RFLP Multilocus (fingerprint) - RFLP single locus - PCR strips DNA profiling : - PCR STR Mitochondrial DNA testing Y chromosome testing Another testing

Mitochondrial DNA
Mitochondria - The powerhouse of the cell. The number of mitochondria varies greatly with the type of cell and stage of its development (ranging 200-1,000)

Mitochondria

Mitochondria have their own DNA

Mitochondrial DNA

Double Helix

Nuclear Chromosomes

Mitochondrial DNA

Each mitochondrion contains two or three circular DNA

Mitochondrial DNA is only 16,569 letters long

[compared to 3 billion in nuclear DNA]

Coding and non coding sequences

In a mitochondrial chromosome, the coding and non coding areas are entire separate. Non coding portion being located in a region reffered as the control region (also called displacement loop or D-loop) The control region contains 1,100 bp and is devided into 2 distinct sections, hypervarible 1 (HV1) and hypervarible 2 (HV2)

History of DNA Testing (Soon)

DNA fingerprint : - RFLP Multilocus (fingerprint) - RFLP single locus - PCR strips DNA profiling : - PCR STR Mitochondrial DNA testing Y chromosome testing Another testing

Summary
Two aspects of DNA : The uniform nature of DNA in a single individual every cell in a body shares the same DNA The genetic variability between individual The chemical structure of DNA is the same, but the order of the base pairs differs

The unique signature found in each persons genetic makeup DNA fingerprint

DNA Fingerprint
The basis of genetic testing is that no two humans DNA is alike (except identical twins).
Polymorphisms are positions where the nucleotide sequence is not the same in every member of the population.

Preparation of a DNA fingerprint

Specimen collection Extracting DNA for analysis DNA Testing

Specimen Collection

DNA samples can be from: Saliva, blood, hair strands, skin, finger or toe nails, and/or a tooth with root material Dirty laundry, a cigarette butt, a coffee cup, tiny blood stains, a smear of dried semen, etc. DNA is collected at crime scenes in a variety of ways using tools such as: Smear slides, scalpels, tweezers, scissors, sterile cloth squares, blood collection kits (for sample collection of suspects or living victims)

Specimen Collection

The ideal specimen is 1 mL or more of fresh, whole blood trated with anti-clotting agent EDTA, but DNA has been successfully analyzed from samples that were a decade old How is blood collected? Blood on Clothing : investigators submit whole pieces of clothing or they may use a sterile cloth square and a small amount of distilled water Dried blood on furniture : investigators send the whole object to the lab Dried blood on a wall, tub or some other object too big or difficult to move to the lab :investigators scrape the blood sample into a sterile container for further analysis

Specimen collection

Evidence should not be stored in plastic bags, because they can retain damaging mositure evidence bag Every crime scene is full of sources of contamination. To protect the evidence, workers at crime scene must : - wear disposable gloves - use disposable or cleaned instruments - avoid talking, sneezing and coughing - avoid touching any item that might be contain DNA - air dry the evidence thoroughly before packaging. - Especially, do not mix known samples (from victim or suspect) with unknown samples. The enemy of evidence : sunlight, high temperature, bacteria, etc.

Putting DNA to the test

The evidence collected from the crime scene is compared to evidence from a known source DNA samples are collected- in the case of parentage testing, from the mother, child and putative (possible) father(s) They are usually blood, but a buccal (cheek cell) swab is acceptable

Extracting DNA for analysis

It is possible to extracting DNA from a childs parentage before birth

Two different methods can be used to test a developing fetus : Amniocentensis Chrionic villus sampling

Amniocentensis

Amniocentensis is performed when the fetus is around 16 weeks of age

Chorionic villus sampling

A suction tube is used to remove a chrionic villi (fetal tissue that helps from the placenta) Advantage : - enough cells are obtained - this procedure can be done around 8-10 weeks of pregnancy. Disadvantage : - this procedure carrier a higher risk for disturbing the fetus and causing a miscarriage

Extracting DNA for analysis

Physical methods: the cell are disrupted by mechanical force, such as disruption cell walls by grinding Chemical methods : cell lysis is brought about by exposured to chemical agent that affect the integrity of the cell barrier, involves: - agent attacking the cell wall - agent disrupting the cell membrane in extraction buffer Combination of physical and

chemical methods

Separation of nucleic acids

Separation of nucleic acids from other cellular components. Extraction/Precipitation method Adsorption Chromatography method (see : Biotechnology)

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Extracting DNA for analysis

Sir Alex Jefferies et al had recently developed techniques for extracting DNA and preparing profiles using old stains and developed a method for separating sperm from vaginal cells (detergent can break open vaginal cells first and the sperm cells) The first reported use of this DNA extracting method in criminal case involved the sexual assaults and murder of a schoolgirl in the UK.
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History of DNA Testing

DNA fingerprint : - RFLP Multilocus (fingerprint) - RFLP single locus - PCR strips DNA profiling : - PCR STR Mitochondrial DNA testing Y chromosome testing Another testing

Some uses of DNA Fingerprint/Profiling

Forensic work on crime scenes Parentage testing Victim identification in mass disasters Sex Identification Non human DNA analysis

Forensic work on crime scenes

The Narborough Village Murder

DNA evidence was compared to DNA samples from 5,500 suspects blood by RFLP multilocus the DNA did not match. A man had given a sample in the name of friend an exact match Limitation : unless there is a known sample to be used as a comparison, identity cannot be established
Defendants blood Blood from defendants clothes Victims blood

The Forest Hill Rapist

In 1988, Victor Lopez was tried for the sexual assault of three women. Oddly, all three women had described their assailant as a black man. Lopez was not black. The accuseds blood was analyzed and compared with sperm left at the sceene the DNA was a match Lopez was found guilty of the attacks. DNA evidence is especially valuable when used to expose faulty eye-witness testimony

Simpson/Goldman murders

In 1994, Nicole Brown Simpson and Ronald Goldman were murdered and Nicoles ex-husband, O.J. Simpson was a suspect. The DNA collected at the crime scene matched that of O.J. Simpson. Defense lawyers attacked the procedures used in collecting,labelling and testing the evidence As a result, the DNA evidence was not effective, O.J. Simpson was found not guilty

Humans error and sources of contamination

The great threats to DNA evidence is : Human error the chain of custody requires that the collection of evidence must be systematically recorded and access to the evidence must be controlled. The process of the samples adhere to high standard. Damage during the analysis itself defined standard of laboratory practice and procedure can help guard againts errors during DNA forensics analysis Band shifting occurs during electrophoresis, when the DNA fragments in one lane migrates more rapidly than identical fragments in second line minor inconsistencies in the gel or a surplus DNA in one of the lanes

Parentage Testing

Parentage Testing

Because DNA is shared to some degree by members of the same family, relationships can be conclusively determined by comparing samples between two individuals It is possible to verify a childs parentage before birth One sort of DNA analysis in establishing familial relationships focuses on mitocondrial DNA (mtDNA) mtDNA is inhereted from the mother only (in the cytoplasm of the egg) relationships can be traced through the unbroken maternal line, because it change only slightly through time as a result of random mutation.

Parentage Testing

MtDNA evidence was essential in reuniting families torn apart by military government of Argentina Family ties established by DNA have also been used to identify the remains of historic figures, ex. Jesse James.

Victim identification in mass disasters

Victim Identification : September 11

Nearly 3000 people died as a result of the terrorist attacks on September 11th, 2001 Dangerous conditions at ground zero made it very difficult for forensic scientists to determine the identity of victims based on their remains. Most remains were small bone fragments and teeth Collection points were set up around New York City where family members could make missing person reports as well as donate items that may contain DNA of the victims.

M-FISys
Mass Fatality Identification System Software system created in the wake of September 11th attacks First software to combine STR, mtDNA, and SNP in an integrated matter. Out of the 3000 people who died, only about 1700 were identified. Can be modified to be used in other disasters or in natural disasters Altered, and used to identify over 800 individuals in three months following the South Asian Tsunami

Sex Identification

Sex Identification
Y chromosome-specific loci

Some genetic markers are found only on the Y chromosome. Presence of male only amplified band identifies the subject as male. Some of these Y markers can be used to ID the individual male in mixed samples.

Y Chromosome Structure
SRY ~60 Mb total DNA sequence (only

AMEL

chromosome 22 is smaller)
~2.5 Mb on tips recombine with X

(pseudoautosomal regions)

35-36 Mb euchromatin 9.5 Mb sequenced (27%)

heterochromatin

Nucleic Acids Res. 28(2), e8 (2000)

Genetic variation at multiple points along the Y chromosome is combined to form a Y haplotype for a sample

Sex Identification
Amelogenin gene

Amelogenin gene is found on each sex chromosome, but is longer on Y. Male gives 2 bands, female gives just one band.

Non Human DNA Analysis

Non human DNA analysis

Many legal questions have been answered by the genetic profiles of plants and animals. For example, the two type of ginseng (American ginseng and Asian ginseng) look almost identical, but they have very different reputations. Asian ginseng purportedly boosts energy, whereas American ginseng is prized for its ability to calm nerves using DNA sequencing to help make distinction between two varieties. Another example : - Distinct the varieties derived crossbreeding are inherently less valuable as wine grapes. - DNA profiling is regularly used by wildlife management authorities.

Paternity of Plants
Ancestry of Cabinet Sauvignon grapes Wine purists believe hybrid grapes are inferior Scientists can examine the DNA cabinet sauvignon plants Found two other varieties : Cabernet Franc & Sauvignon Blanc This challenged the idea that the varieties of these plants that come from cross-breeding are inherently less valuable as wine grapes.

Animal Profiling

Evidence has been used to form genetic profiles of animals Example Is a case in Pennsylvania DNA fingerprinting was used to prove that a hunter had illegally killed a bear. Authorities collected blood samples from the bear den and then compared it with samples from the check station Because of this the DNA Analysis investigators were able to find the hunter guilty.

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