Lacunae of DNA Fingerprinting

8/14/2019 Lacunae of DNA Fingerprinting

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Introduction

DNA fingerprinting isthe newest techniquefor identification inmedicolegal cases


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Introuction: Pro-view

Marine Jacot, UNESCO:

Police and judges in Western countries allagree that the arrival of genetic testing in

their daily lives is a far more revolutionary

development than the introduction of

fingerprinting at the end of the 19thcentury.


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Introduction: Contrapunto

The National Academy of Sciences:

This new technology burst on the scene so

rapidly that there are essentially no

standards and no regulations


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THE LACUNAE

There are four major classes of fallacies:

LEGAL FALLACIESTECHNICAL FALLACIES

STATISTICAL FALLACIES

FALLACIES IN METHODOLOGY


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A. THE LEGAL FALLACIES

1. THE FRYE CRITERIA

2. PROSECUTORSFALLACY

3. THE DEFENDANTS

FALLACY


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A.1) THE FRYE CRITERIA

The introduction of the DNA fingerprinting in

the US legal system was based on the Fryecriterion, which states:

a new scientific technique "must be

sufficiently established to have gained

general acceptance in the particular field

to which it belongs" before presentation

to the jury.


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A.2) THE PROSECUTORS FALLACY

This is in relation to the presentation

of the case by the prosecutor. A DNAsequence which has an occurrence of

one in one million, can be presented

to the court as:

There is only a one in a million

chances that the defendant is

innocent.


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ELIMINATING THE PROSECUTORS

FALLACY

However, if it was presented in the

following manner, this would cast lessdetrimental effect on the defendant;

The chance of obtaining this DNA

Profile from the forensic sample if itoriginated from a person other that

the defendant is one in a million.


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B. THE TECHNICAL FALLACIES

1. METHYLATED DNA

2. SHORTCOMINGS OF RFLP3. SHORTCOMINGS OF PCR

4. SHORTCOMINGS OF SOUTHERNBLOT

5. MT-DNA IN MATERNALLYRELATED

6. STANDARDISATION

7. SIMILARITY AMONG RELATIONS


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B.1) METHYLATED DNA

The cytidylate residues in a 5-CG-3

residue can be methylated in upto 3% ofthe total population. This methylation will

interfere with RFLP profiling when the

Restriction Endonucleases used identify apalindrome having CG but cannot lyse the

phosphodiester bonds after methylation.


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B.2) SHORTCOMINGS OF RFLP

For obtaining statistically relevant

information, RFLP requiresthousands of cells from the clinicalforensic specimen, which may not bepresent in trace evidences.


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The DNA should be in undegraded

and fresh condition in order to obtainan unequivocal result.


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The exact sizes of the bands are unknown and

comparison to a molecular weight ladder isdone in a purely qualitative manner. Many labs

developed policies that described what they

considered a unique band, but it was not

standardized and led to DNA fingerprintingcoming under harsh attack in People v. Castro

545 N.Y.S. 2d. 985 (Sup. Ct. 1989).


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B.3) SHORTCOMINGS OF PCR

Contamination

Technical faults can be magnifiedtremendously to alter the result

completely

Selection of wrong primers inshallow gene pool areas or in

people of same family, etc.


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A robot used in DNA profiling adds solution and stirs DNAsamples from tissues


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B.4) SHORTCOMINGS OF SOUTHERN

BLOT

The Southern blot constitutes the basis

for RFLP. It can be poor in quality when: Stale sample is used

Forensic sample is poorly hybridised

Improper selection of radioactive probesfor people who may have genetic

similarity


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B.5) mt-DNA IN MATERNALLY RELATED

Since it is the nucleus only of the sperm

that penetrates the ovum,cytoplasmic and cell organelle

inheritance is entirely maternal.

Hence persons who are maternally

related may have several similar

STRs and VNTRs.


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B.6) STANDARDISATION

It has been about two decades since

the system was perfected and thereremains a lot to be standardised. Of

this, one is the regions to look for the

individual characters.


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The US FBI usethe CODIS(Combined DNAIndexingSystem) whichrecognises 13

sites and by farremains themost popularlyused:


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The loci should have maximum variability

and span throughout the genome The UK uses the SGM+ system of DNA

profiling, where 10 sites and the sex

chromosomes are utilised. There remains no global standard for

obtaining and uniquely interpreting PCR

based methods.


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B.7) SIMILARITY AMONG RELATIONS

The relatives, like maternally related, parents

and offsprings, etc. have direct genetic analogy

and can be often confusin with several commonsites in STRs and VNTRs.

The Leary case is landmark in this kind of a

genetically related problem, where the fatherwas wrongly prosecuted of statutory rape of

his 14 year old daughter. The verdict was

reversed after the DNA profiling of the

brother was done.


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The Leary case


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C. FALLACIES OF METHODOLOGY

1. Cost efficiency

2. Comparative analysis3. To determine acceptability

4. Stutter Bands

5. Shallow gene pool

6. Jeffrey Gafoor Case

7. The O. J. Simpson case

8. The Dr. Scheenberger case


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C.1) COST EFFICIENCY

The process of PCR

based methods arecostly. To reduce the

cost:benefit ratio, newer

instruments are used

which are morecapacious and can have

a greater output to lower

the total and average

expenses.

The Applied Biosystems 3130xl Genetic

Analyzer is a popular instrument that can

perform capillary electrophoresis to type DNA


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C.2) COMPARATIVE ANALYSIS

This method of spotting the offender byDNA profiling needs the DNA of the suspectto match with forensic sample or to matchthe forensic sample findings with a DNAdatabase archive. This has several

drawbacks from the ethical viewpoint. Thiswas seen when the rape accused could notbe identified and nor a suspect pinned.Norm Gahn issued a John Doe DNA

warrant for the man.


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C.3) TO DETERMINE ACCEPTABILITY

To determine the credibility of a DNA Fingerprintingresult, the following query method is to be adopted:

Could it be an accidental random match?

If not, could the DNA sample have beenplanted?

If not, did the accused leave the DNA sampleat the exact time of the crime?

If yes, does that mean that the accused isguilty of the crime?


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C.5) SHALLOW GENE POOL

Alan Legere of Canada was convicted of four

murders while he was an escaped convictin 1991 based on the DNA profiling. The

prosecution had to undertake multilocus

DNA testing repeatedly to rule out the

defences point that the DNA sample

could belong to many people due to the

close sub ethnicity of the inhabitants.


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C.6) JEFFREY GAFOOR CASE

Paradoxically, Welshman Jeffrey

Gafoor was convicted in 2003 ofthe murder of Lynette White in 1988

by comparing the crime scene DNA

evidence to that of his nephewa

maternal relationutilising mtDNA.


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C.7) O. J. SIMPSON CASE

Probably the msot celebrated DNA profiling

case in the criminolgical history, this brought

the system to legal prominence in USA. Due to

faulty technique, O. J. Simpson was convicted

of a double murder but the verdict was

overturned after a repeat, with set standardswas done. This also shows the necessity of

standardisation to interpret the results.


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C.8) DR. SCHEENBERGER CASE

Probably the most interesting of them all,Dr. John Scheenberger of Canada raped

one of his sedated patients. The semensample was collected from the victim. Onthree occasions, the police tried to matchthe doctors DNA sample with that of the

crime scene DNA but was in vain. It turnedout that he had surgically inserted aPenrose Drain in his arm and filled it withforeign blood and anticoagulants.


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D. THE STATISTICAL FALLACY


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INTERROGATORS FALLACY

This is also known as the INTERROGATORS FALLACY. Tomasde Torquemada, the Spanish Grand Inquisitor consideredconfession to be them ultimate proof of guilt. Nowadays,confessions can be obtained in ways that can bypass the truth.Robert A. J. Matthews published his work on conditionalprobabilities based on Bayesian Probability to determine theprobability of an accused being guilty given that he hasconfessed.

The fallacy that confession=guilty was knownas the INTERROGATORS FALLACY.


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THE STATISTICAL CONSIDERATIONS: Let, A= the prior probability that the accused is guilty,

that is, the probability of guilt assessed from apositive DNA fingerprinting result. A= event of negation of A, that is, accused is

innocent

B= event where DNA fingerprinting is positive.


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From Bayes Theorem: P(C)=P(AUC)/P(A1C) Where P(C)= probability that the

accused has DNA +ve P(AUC)= probability that the accused is

guilty and has DNA +ve

P(A1C)= probability that the accused isnot guilty but has DNA +ve Now, AUC=CUA So, P(A1C)= P(AUC)/P(C)

Now, P(C1A)=P(CUA)P(A)


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From Bayes Theorem,

P(A1C)=P(CUA) P(A)=P(C1A)

P(A)1

P(A) P(C) P(C)


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The probability that the accused is guilty is

hinged upon two facts:

Accused is guilty and has DNA +ve i.e.

P(CUA)

Accused is not guilty and has DNA +ve

i.e. P(CUA)


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In all other cases, the accused has DNA ve

and is ruled out.

Thus, probability that the accused has

DNA+ve is P(C)= P(CUA)+P(CUA)=P(CUA)P(A) +

P(CUA) P(A)

P(A) P(A)


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From Bayes Theorem, P(C)= P(C1A) P(A) + P(C1A) P(A)

Also P(A)= 1P(A)


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Therefore, from equation 1 we have putting P(C): P(A1C)=P(C1A) P(A)= P(C1A) P(A) .

P(C) P(C1A) P(A) + P(C1A)P(A)

= P(A) .

P(A) + P(C1A) P(A)

P(C1A) Let p= P(A) and r= P(C1A) , then

P(C1A) P(A1C)= p .= p .

p + r.P(A) p + r(1-P(A))


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So,

P(A1C)= p/ {p + r(1-p)}

This is known as Matthews

confessional probability.


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THE STATISTICAL CONSIDERATIONS:

Now, for a DNA positiveaccused to be guilty,

Probabilty that the accused isGuilty given that DNA is +ve>probability that the accused isinnocent given that DNA is

+ve For the accused to be guilty,

then, the following probabilityhas to be satisfied:

P(A1C)>P(A) This, in turn implies that r


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Thus we see that the 50%

assumption of guilt tallies with the

accepted frequency of the DNA

profile in the population.

THUS IT IS STATISTICALLYESTABLISHED THAT THE

POSITIVE DNA RESULT

CANNOT BE TAKEN AS FINALVERDICT OF GUILT!!!


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RECOMMENDATIONS

SAMPLE COLLECTIONS:

FRESH WHOLE BLOOD:

5-10 ml of blood is collected on EDTA

5-10 volumes lysis buffer (0.32 M sucrose, 1% triton

X100, 5mM tris-HCl, pH 7.6) adde4d and

centrifuged at 2500x 15 min at 4o C

Supernatant discarded, 5ml of NaCl and NaEDTAadded also 1% Sodium Dodecyl Sulfate added,

Proteinase K follows, incubates overnight at 37 or

4-5 hrs at 50o C

Add equal volume of phenol, centrifuge


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SAMPLE COLLECTIONS:

FRESH WHOLE BLOOD:

Supernatant contains DNA transferred to fresh tube Phenol, chloroform, isoamylalcohol (24:24:1) of equa

volume added, extract supernatant as in step e.

Equal volume of Chloroform and Isoamylalcohol (24:1)

added Extraction of supernatant, followed by addition of 0.1

vol of 3M Na acetate and 2.5 mlvol of ice cold absolute

ethanol

DNA preci[pitates as compact strings and pellets

Discard su[pernatant as much as possible, rnse DNA

pellets with 70 % ethanol and dry in air

Resuspend in appropriate buffer


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RECOMMENDATIONS

SAMPLE COLLECTIONS

Blood stains: Stained fabric cut, put in polypropylene tubes and

mixed with lysis buffer. Incubated for over 30 min at

room temperature.

Centrifuge at 1000 rpmx5 min. discard supernatant Add TNE buffer (tris HCl, NaCl, EDTA), SDS,

Proteinase K, incubate ocernight at at 37 or 4-5 hrs at

50o C

Follow from step d above onwards


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RECOMMENDATIONS

SAMPLE COLLECTIONS

Sperm:

1oo l semen taken, centrifuged at

5000rpmx 5 min

Supernatant discarded, TNE buffer

added, Dithioerithritol and SDS

added ProteinaseK added before

overnight incubation at 37o C Follow from step d of fresh whole

blood sequence

RECOMMENDATIONS


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RECOMMENDATIONS

Tissues:

Tissues frozen in liquid nitrogen andhomogenised TNE buffer added, followed by SDS and

Proteinase K. overnight incubation at 55o

C Follow from step d of fresh whole blood

sequence

CO O S


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RECOMMENDATIONS

Hair:

Hair roots are taken and crushed gently

TNE buffer with SDS and Proteinase K added

and incubated overnight at 55oC Follow from step d of fresh whole blood

sequence

RECOMMENDATIONS


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RECOMMENDATIONS

Bone and teeth:

Bone marrow or dental pulp are frozen in

liquid nitrogen

TNE buffer with SDS and Proteinase K areadded and incubated overnight at 55oC

Follow from step d of fresh whole blood

sequence

RECOMMENDATIONS


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RECOMMENDATIONS

Limit PCR contaminations:

Use robotic technology for procedures

Process sample as quickly as possible.


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THANKS

[email protected]

Lacunae of DNA Fingerprinting

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