INTRODUCTION OF BIOTECHNOLOGYINTRODUCTION OF BIOTECHNOLOGY

DNAFINGERPRINTING

AND FORENSIC

20142014

ENDRIKA WIDYASTUTI

INSUFFICIENT EVIDENCE ?Mr. Chan ingin pergi ke Hongkonguntuk tinggal bersama anaknya.Sebelum pergi ke Hongkong, Mr.Chan harus menunjukkandokumen tes laboratorium padaPihak Imigrasi bahwa Mr. Chanmemiliki hubungan kekerabatandengan anaknya.Akhirnya Mr. Chan menjalankan tesgolongan darah. Akan tetapigolongan darahnya tidak samadengan anaknya, sehingga berkaspengurusan visa Mr. Chan ditundaoleh pihak imigrasi. Mr. Chanmemberitahu pihak imigrasi bahwahubungan kekerabatan tidakhanya dibuktikan melalui golongandarah saja.

• Mengapa Golongan Darahtidak selalu mencerminkanada/tidaknya hubungankekerabatan?

• Apakah ada tes lain yangdapat digunakan untukmembuktikan hubungankekerabatan antara Mr. Chandan Anaknya?

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Blood group typing is much lesssensitive.

May use DNA fingerprint to providebiological evidence as a proof in apaternity test.

Blood group typing is much lesssensitive.

May use DNA fingerprint to providebiological evidence as a proof in apaternity test.

IDENTIFICATION:CLASSIC AND DNA

...OF THE LIVING AND THE DEAD

• investigation of anydeath

• the living – inabilityto identifythemselves,immigration,inheritance

MORPHOLOGICAL CHARACTERISTICS

• only by MATCHING theparameters

• specific featurepossesed by theindividual

• height, weight andgeneral physique

• hair color and length,moustache, skinpigmentation, eye color

• clothing, jewellery,ornaments

• facial appearance, age

FINGERPRINTS

• position of minutedefects on theridges

• no less than 16points of similarity

• chances foridentical fingerprint:1:64 billion

IDENTITY FROM TEETH

• identification of thedead, bite marks,estimation of age

• commonlycomparison ofantemortem dentalchartings with thedirect examination ofthe teeth

• mass disasters• no previous records –

age, sex and ethnicorigin

IDENTIFICATION OF THEORIGIN OF TISSUE OR SAMPLES

• blood, semen,saliva, stains onbodies, weapons,clothing

• matching remains

• resolution ofpaternity, maternityand inheritancedisputes

THE INDIVIDUALITY OF CELLS

• blood grups and Rhfactor – possibility toexclude 93% of non-matching samples

• replaced with DNAanalysis of humantissue and fluid

TATOOS AND BODY PIERCING

• there has to becomparison

• photographs anddrawings if visualidentification is notpossible

IDENTIFICATION BY DNA PROFILING

• nearly 100% in exclusionand in identification

• 10% are active genes• 90 % are “silent” – zone

of 200 to 14 000 repeatsof identical sequencesof bases are constantand transmitted fromparents – ESSENTIAL forDNA analysis!

• “bar code”

• risk of contamination• blood, hair with roots;

samples of spleen;buccal smears;

vaginal, anal andpenile swabs

• paternity/maternitytesting

• every bar in the “barcode” must have comefrom either the father ormother, half from each

IDENTITY OF DECOMPOSED ORSKELETALIZED REMAINS

• are the remains actuallybones?

• 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 BoneIdentification

FACIAL RECONSTRUCTION FROMSKULLS

• measuring the averagesoft-tissue thickness atmany points on the skulland rebuilding this layerwith plasticine onunknown skull

• computerizedreconstruction

• individuality of faces?• what about eyes and

lips?

ACTIVITY 8.1

a) There are 46 pairs of chromosomes ineach nucleus of a human cell.Chromosomes are made of DNA andproteins.

b) DNA determines the bodycharacteristics of an organism.

c) DNA may be extracted from redblood cells found in a blood sample.

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1.Decide whether the followingstatements are TRUE or FALSE.

FALSE

TRUE

TRUE

ACTIVITY 8.1

e) The Hair and Teeth of the same person arecomposed of same DNA molecules.

f) Identical twins have different base sequences ofDNA .

g) In the DNA structure, adenine (A) will only link withcytosine (C) and guanine (G) will only link withthymine (T).

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TRUE

FALSE

FALSE

Define DNA FINGERPRINTING

Preparation of DNAFINGERPRINTING

Putting DNA to the TEST

Familial Relationships and DNAPROFILE

CASE STUDY

KEY CONCEPTDNA fingerprints identify people at the molecularlevel.

Define DNAFINGERPRINTING

Chemical structure ofDNA always same, butthe order of basepairsare differs

DNA fingerprints arebased on parts of anindividual’s DNA thatcan by used foridentification.

DNA fingerprinting is used for identification.• DNA fingerprinting depends on the probability of a

match.– Many people have the

same number ofrepeats in a certainregion of DNA.

– The probability that twopeople share identicalnumbers of repeats inseveral locations isvery small.

(mother) (child 1) (child 2) (father)

– Individual probabilities are multiplied to find theoverall probability of two DNA fingerprints randomlymatching.

– Several regions of DNA areused to make DNA fingerprints.

1 1 1 1500 90 120 5,400,000 1 chance in 5.4 million peoplex x = =

• DNA fingerprinting is used in several ways.

– evidence in criminalcases

– paternity tests– immigration requests– studying biodiversity

Define DNA FINGERPRINTING

Preparation of DNAFINGERPRINTING

Putting DNA to the TEST

Familial Relationships and DNAPROFILE

CASE STUDY

PROCEDURE• Collected DNA• DNA isolation• DNA Analysis

DNA COLLECTION &COMPARISON

• DNA samples can be from:Salivabloodhair strandsskinfinger or toe nails,tooth with root material

DNA COLLECTION &COMPARISON

• How is blood collected?• Blood on Clothing?

• Investigators submit whole pieces ofclothing or they may use a sterilecloth square and a small amount ofdistilled 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 tomove to the lab?

• Investigators scrape the blood sampleinto a sterile container for furtheranalysis

MAKING DNA FINGERPRINTS

• DNA fingerprinting is a laboratory procedure thatrequires 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 sheetproduces 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

PCR

STR

RFLPRESTRICTION FRAGMENT LENGTH POLYMORPHISM

• Analyzes variable lengthsof DNA fragments

• One of the originalapplications of DNAanalysis

• Not used as muchanymore because itrequires a large quantityof DNA sample andsamples degraded by theenvironment do not workwell with RFLP

RestrictionFragmentLengthPolymorphismRFLP

Allele 1

Allele 2

GAATTCGTTAAC

GAATTCGTTAAC

CTGCAGGAGCTC

CGGCAGGCGCTC

PstI EcoRI

1 2 3

3Fragment 1+2DifferentBase PairsNo restriction site

+

M A-1 A-2

Electrophoresis ofrestriction fragments

M: MarkerA-1: Allele 1 FragmentsA-2: Allele 2 Fragments

THIS METHOD ANALYSESTHE LENGTH OF THESTRANDS OF DIFFERENTDNA MOLECULES. THEDNA IS BROKEN INTOSMALL PIECES BYRESTRICTION ENZYMES.THEN ALL THOSE SMALLPIECES BECOMESEPARATED BY GELELECTROPHORESIS.RFLP, UNFORTUNATELY, ISNOT USED AS MUCHANYMORE.

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PCRPOLYMERASE CHAIN REACTION

• Used to makemillions of exactcopies of DNA froma biological sample

• Allows very smallsamples to beanalyzed, such as asample of a few skincells

• Must be very carefulaboutcontamination inthis process

USED FOR DNA FINGERPRINTING AND PATERNITY TESTING. PCR IS USEDTO ENLARGE A FEW PIECES OF DNA WHICH WOULD CREATE THOUSANDS

TO MILLIONS OF COPIES OF THAT ONE SAMPLE OF DNA.

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WHICH IS SHORT TANDEMREPEAT, IS THE MOST WIDELY

USED DURING CRIMINALINVESTIGATIONS. STR

ANALYZES HOW MANY TIMESBASE PAIRS REPEATTHEMSELVES ON A

PARTICULAR LOCATION ONA STRAND OF DNA. THIS

METHOD IS A PCR-BASEDTOOL BECAUSE IT USES SOME

COMPONENTS OF THE PCRMETHOD.

40STRSHORT TANDEM REPORT

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DNA COMPARISON &ANALYSIS

• A DNA profile iscreated….how??

Markers are found bydesigning small piecesof DNA (probes) thatwill seek out and bindto complementaryDNA sequences. Thiscreates a distinctpattern. Again, onemarker is not usuallyunique, but with fouror five regions thematch is likely

DNA COMPARISON &ANALYSIS

The DNA profiles are compared with samples fromsuspects to find possible matches.

DNA COMPARISON &ANALYSIS

If there are no suspects, anational database calledCODIS may be used to findpotential 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 ofStained Gel

Determinerestriction fragmentsizes

• Create standard curveusing DNA marker

• Measure distancetraveled by restrictionfragments

• Determine size of DNAfragments

Identify the relatedsamples

Define DNA FINGERPRINTING

Preparation of DNAFINGERPRINTING

Putting DNA to the TEST

Familial Relationships and DNAPROFILE

CASE STUDY

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Activity 8.5DNA Fingerprint Analysis

Case 1

Mr. Chan’s family consists of mom, dad and four kids. The parentshave one daughter and one son together, another daughter is from themother’s previous marriage, and the other son is adopted. Here arethe 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 8.5Answers:• 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 2A blood sample from a crimescene was collected. DNAsamples of the victim and thepotential suspects (June,Scarlet and John) were alsocollected for DNA analysis.The DNA profile is shown.Now, you should be able toidentify the potential murderer.

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Activity 8.5

Answers:• All of the DNA fragments of Scarlet can be

found in the crime scene sample makingher 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 evidencesuch as eyewitness accounts?

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Activity 8.5Answers:

1. This pattern may arise if the two women are related orif this pattern were common in population.

2. Someone’s DNA is found at a crime scene does notmean that they committed the crime because of thefollowing reasons:(i)The DNA sample may be contaminated by theenvironment.(ii) The sample may be a mixture of more than oneperson’s DNA.(iii) The DNA evidence may be degraded or brokendown.