DNA FINGERPRINTING AND FORENSIC

ENDRIKA WIDYASTUTI

INTRODUCTION OF BIOTECHNOLOGY

2014

INSUFFICIENT EVIDENCE ? Mr. Chan ingin pergi ke Hongkong untuk tinggal bersama anaknya. Sebelum pergi ke Hongkong, Mr. Chan harus menunjukkan dokumen tes laboratorium pada Pihak Imigrasi bahwa Mr. Chan memiliki hubungan kekerabatan dengan anaknya. Akhirnya Mr. Chan menjalankan tes golongan darah. Akan tetapi golongan darahnya tidak sama dengan anaknya, sehingga berkas pengurusan visa Mr. Chan ditunda oleh pihak imigrasi. Mr. Chan memberitahu pihak imigrasi bahwa hubungan kekerabatan tidak hanya dibuktikan melalui golongan darah saja.

• Mengapa Golongan Darah tidak selalu mencerminkan ada/tidaknya hubungan kekerabatan? • Apakah ada tes lain yang dapat digunakan untuk membuktikan hubungan kekerabatan antara Mr. Chan dan Anaknya?

Blood group typing is much less sensitive. May use DNA fingerprint to provide biological evidence as a proof in a paternity test.

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IDENTIFICATION: CLASSIC AND DNA

...OF THE LIVING AND THE DEAD • investigation of any death • the living – inability to identify themselves, immigration, inheritance

MORPHOLOGICAL CHARACTERISTICS • only by MATCHING the parameters • specific feature possesed by the individual • height, weight and general physique • hair color and length, moustache, skin pigmentation, eye color • clothing, jewellery, ornaments • facial appearance, age

FINGERPRINTS • position of minute defects on the ridges • no less than 16 points of similarity • chances for identical fingerprint: 1:64 billion

IDENTITY FROM TEETH • identification of the dead, bite marks, estimation of age • commonly comparison of antemortem dental chartings with the direct examination of the teeth • mass disasters • no previous records – age, sex and ethnic origin

IDENTIFICATION OF THE ORIGIN OF TISSUE OR SAMPLES • blood, semen, saliva, stains on bodies, weapons, clothing • matching remains • resolution of paternity, maternity and inheritance disputes

THE INDIVIDUALITY OF CELLS • blood grups and Rh factor – possibility to exclude 93% of nonmatching samples

• replaced with DNA analysis of human tissue and fluid

TATOOS AND BODY PIERCING • there has to be comparison • photographs and drawings if visual identification is not possible

IDENTIFICATION BY DNA PROFILING • nearly 100% in exclusion and in identification • 10% are active genes • 90 % are “silent” – zone of 200 to 14 000 repeats of identical sequences of bases are constant and transmitted from parents – ESSENTIAL for DNA analysis! • “bar code”

• risk of contamination • blood, hair with roots; samples of spleen; buccal smears; vaginal, anal and penile swabs • paternity/maternity testing • every bar in the “bar code” must have come from either the father or mother, half from each

IDENTITY OF DECOMPOSED OR SKELETALIZED REMAINS • are the remains actually bones? • are the remains human? • one or more bodies? • what sex are the bones? – pelvis and skull • age, height, race? • can a personal identity be discovered? • YouTube - Crime 360: Human Bone Identification

FACIAL RECONSTRUCTION FROM SKULLS • measuring the average soft-tissue thickness at many points on the skull and rebuilding this layer with plasticine on unknown skull • computerized reconstruction • individuality of faces? • what about eyes and lips?

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ACTIVITY 8.1 1.Decide whether the following statements are TRUE or FALSE. FALSE

TRUE

TRUE

a) There are 46 pairs of chromosomes in each nucleus of a human cell. Chromosomes are made of DNA and proteins. b) DNA determines the body characteristics of an organism. c) DNA may be extracted from red blood cells found in a blood sample.

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ACTIVITY 8.1 TRUE

e)

The Hair and Teeth of the same person are composed of same DNA molecules.

FALSE

f)

Identical twins have different base sequences of DNA .

g)

In the DNA structure, adenine (A) will only link with cytosine (C) and guanine (G) will only link with thymine (T).

FALSE

Define DNA FINGERPRINTING Preparation of DNA FINGERPRINTING Putting DNA to the TEST Familial Relationships and DNA PROFILE CASE STUDY

KEY CONCEPT DNA fingerprints identify people at the molecular level.

Define DNA FINGERPRINTING Chemical structure of DNA always same, but the order of basepairs are differs DNA fingerprints are based on parts of an individual’s DNA that can by used for identification.

DNA fingerprinting is used for identification. • DNA fingerprinting depends on the probability of a match. (mother) (child 1) (child 2) (father) – Many people have the same number of repeats in a certain region of DNA. – The probability that two people share identical numbers of repeats in several locations is very small.

– Individual probabilities are multiplied to find the overall probability of two DNA fingerprints randomly matching. 1 1 1 1 1 chance in 5.4 million people 500 x 90 x 120 = 5,400,000 =

– Several regions of DNA are used to make DNA fingerprints.

• DNA fingerprinting is used in several ways. – evidence in criminal cases – paternity tests – immigration requests – studying biodiversity

Define DNA FINGERPRINTING Preparation of DNA FINGERPRINTING Putting DNA to the TEST Familial Relationships and DNA PROFILE CASE STUDY

PROCEDURE • Collected DNA • DNA isolation • DNA Analysis

DNA COLLECTION & COMPARISON •

DNA samples can be from: Saliva blood hair strands skin finger or toe nails, tooth with root material

DNA COLLECTION & COMPARISON • 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

MAKING DNA FINGERPRINTS • DNA fingerprinting is a laboratory procedure that requires six steps: • 1: Isolation of DNA.

• 2: Cutting, sizing, and sorting. • Special enzymes called restriction enzymes are used to cut the DNA at specific places

• 3: Transfer of DNA to nylon. The distribution of DNA pieces • is transferred to a nylon sheet • by placing the sheet on the gel • and soaking them overnight. • 4-5: Probing. Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA fingerprint.

• 4-6: DNA fingerprint. • The final DNA fingerprint is built by using several probes (5-10 or more) simultaneously.

DNA ANALYSIS

RLFP

STR

PCR

RFLP

RESTRICTION FRAGMENT LENGTH POLYMORPHISM • Analyzes variable lengths of DNA fragments

• One of the original applications of DNA analysis • Not used as much anymore because it requires a large quantity of DNA sample and samples degraded by the environment do not work well with RFLP

Restriction Fragment Length Polymorphism RFLP

Allele 1 1

Allele 2

PstI

EcoRI

CTGCAG GAGCTC

GAATTC GTTAAC 2

3

CGGCAG GCGCTC

Different Base Pairs No restriction site

GAATTC GTTAAC

3

Fragment 1+2

M

Electrophoresis of restriction fragments

M: Marker A-1: Allele 1 Fragments A-2: Allele 2 Fragments

+

A-1

A-2

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THIS METHOD ANALYSES THE LENGTH OF THE STRANDS OF DIFFERENT DNA MOLECULES. THE DNA IS BROKEN INTO SMALL PIECES BY RESTRICTION ENZYMES. THEN ALL THOSE SMALL PIECES BECOME SEPARATED BY GEL ELECTROPHORESIS. RFLP, UNFORTUNATELY, IS NOT USED AS MUCH ANYMORE.

PCR

POLYMERASE CHAIN REACTION • Used to make millions of exact copies of DNA from a biological sample • Allows very small samples to be analyzed, such as a sample of a few skin cells • Must be very careful about contamination in this process

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USED FOR DNA FINGERPRINTING AND PATERNITY TESTING. PCR IS USED TO ENLARGE A FEW PIECES OF DNA WHICH WOULD CREATE THOUSANDS TO MILLIONS OF COPIES OF THAT ONE SAMPLE OF DNA.

STR 40

SHORT TANDEM REPORT WHICH IS SHORT TANDEM REPEAT, IS THE MOST WIDELY USED DURING CRIMINAL INVESTIGATIONS. STR ANALYZES HOW MANY TIMES BASE PAIRS REPEAT THEMSELVES ON A PARTICULAR LOCATION ON A STRAND OF DNA. THIS METHOD IS A PCR-BASED TOOL BECAUSE IT USES SOME COMPONENTS OF THE PCR METHOD.

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DNA COMPARISON & ANALYSIS • A DNA profile is created….how?? Markers are found by designing small pieces of DNA (probes) that will seek out and bind to complementary DNA sequences. This creates a distinct pattern. Again, one marker is not usually unique, but with four or five regions the match is likely

DNA COMPARISON & ANALYSIS The DNA profiles are compared with samples from suspects to find possible matches.

DNA COMPARISON & ANALYSIS If there are no suspects, a national database called CODIS may be used to find potential suspects. • More on CODIS: • Stands for Combined DNA Index System • National Network that helps identify leads for crimes with no suspects • Three tiers: Local (LDIS), State (SDIS), National (NDIS) • Uses 13 DNA regions that vary from person to person • Looks for matches at more than one location on a genome for more accurate results

Analysis of Stained Gel Determine restriction fragment sizes • Create standard curve using DNA marker

• Measure distance traveled by restriction fragments • Determine size of DNA fragments Identify the related samples

Define DNA FINGERPRINTING Preparation of DNA FINGERPRINTING Putting DNA to the TEST Familial Relationships and DNA PROFILE CASE STUDY

Activity 8.5 DNA Fingerprint Analysis Case 1 Mr. Chan’s family consists of mom, dad and four kids. The parents have one daughter and one son together, another daughter is from the mother’s previous marriage, and the other son is adopted. Here are the DNA analysis results:

1. Which child is adopted? Why?

2. Which child is from the mother’s previous marriage? Why? 3. Who are the own children of Mr and Mrs Chan?

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Activity 8.5 Answers: • Child 4 is adopted. • Child 2 is the child from the mother’s previous marriage. • Child 1 and Child 3 are own children of Mr and Mrs Chan.

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Activity 8.5 Case 2 A blood sample from a crime scene was collected. DNA samples of the victim and the potential suspects (June, Scarlet and John) were also collected for DNA analysis. The DNA profile is shown. Now, you should be able to identify the potential murderer. 50

Activity 8.5 Answers: • All of the DNA fragments of Scarlet can be found in the crime scene sample making her the most likely suspect.

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Activity 8.5 Extended discussions: 1. Both June and Scarlet have the same DNA fragments (“8” and “12”), why? 2. Why DNA evidence must be combined with the traditional forms of evidence such as eyewitness accounts?

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Activity 8.5 1.

2.

Answers: This pattern may arise if the two women are related or if this pattern were common in population. Someone’s DNA is found at a crime scene does not mean that they committed the crime because of the following reasons: (i) The DNA sample may be contaminated by the environment. (ii) The sample may be a mixture of more than one person’s DNA. (iii) The DNA evidence may be degraded or broken down. 53