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LACUNAE OF DNA FINGERPRINTING

TECHNIQUES
Pranab Chatterjee

Second Professional M.B.B.S.


Medical College, Kolkata
pranabchatterjee@hotmail.com
Introduction
 DNA fingerprinting is
the newest technique
for identification in
medicolegal cases
Introuction: Pro-view

 Marine Jacot, UNESCO:


“Police and judges in Western countries all
agree 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 19th
century.”
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
THE LACUNAE

 There are four major classes of fallacies:


 LEGAL FALLACIES
 TECHNICAL FALLACIES
 STATISTICAL FALLACIES
 FALLACIES IN METHODOLOGY
A. THE LEGAL FALLACIES
1. THE FRYE CRITERIA
2. PROSECUTOR’S
FALLACY
3. THE DEFENDANT’S
FALLACY
A.1) THE FRYE CRITERIA
The introduction of the DNA fingerprinting in
the US legal system was based on the Frye
criterion, 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.
A.2) THE PROSECUTOR’S FALLACY
 This is in relation to the presentation
of the case by the prosecutor. A DNA
sequence 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.”
ELIMINATING THE PROSECUTOR’S
FALLACY

 However, if it was presented in the


following manner, this would cast less
detrimental effect on the defendant;
“The chance of obtaining this DNA
Profile from the forensic sample if it
originated from a person other that
the defendant is one in a million.”
A.3) THE DEFENDANT’S FALLACY

 The defendant also falls in the trap of biased


presentation to alter the complexion of
events. In the same DNA sequence of
occurrence, the defendant might say that:
“In the total population of 300 million, there
can be at least 300 people who can have
committed the crime…what about
investigating them before the
prosecution nails my client!!!!”
B. THE TECHNICAL FALLACIES

1. METHYLATED DNA
2. SHORTCOMINGS OF RFLP
3. SHORTCOMINGS OF PCR
4. SHORTCOMINGS OF SOUTHERN
BLOT
5. MT-DNA IN MATERNALLY
RELATED
6. STANDARDISATION
7. SIMILARITY AMONG RELATIONS
B.1) METHYLATED DNA

 The cytidylate residues in a 5’-CG-3’


residue can be methylated in upto 3% of
the total population. This methylation will
interfere with RFLP profiling when the
Restriction Endonucleases used identify a
palindrome having CG but cannot lyse the
phosphodiester bonds after methylation.
B.2) SHORTCOMINGS OF RFLP

– For obtaining statistically relevant


information, RFLP requires
thousands of cells from the clinical
forensic specimen, which may not be
present in trace evidences.
B.2) SHORTCOMINGS OF RFLP

– The DNA should be in undegraded


and fresh condition in order to obtain
an unequivocal result.
B.2) SHORTCOMINGS OF RFLP

– The exact sizes of the bands are unknown and


comparison to a molecular weight ladder is
done 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 fingerprinting
coming under harsh attack in People v. Castro
545 N.Y.S. 2d. 985 (Sup. Ct. 1989).
B.3) SHORTCOMINGS OF PCR

– Contamination
– Technical faults can be magnified
tremendously to alter the result
completely
– Selection of wrong primers in
shallow gene pool areas or in
people of same family, etc.
A robot used in DNA profiling adds solution and stirs DNA
samples from tissues
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 probes
for people who may have genetic
similarity
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.
B.6) STANDARDISATION

 Ithas been about two decades since


the system was perfected and there
remains a lot to be standardised. Of
this, one is the regions to look for the
individual characters.
B.6) STANDARDISATION

 The US FBI use


the CODIS
(Combined DNA
Indexing
System) which
recognises 13
sites and by far
remains the
most popularly
used:
B.6) STANDARDISATION

– 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.
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 common
sites in STRs and VNTRs.
 The Leary case is landmark in this kind of a
genetically related problem, where the father
was wrongly prosecuted of statutory rape of
his 14 year old daughter. The verdict was
reversed after the DNA profiling of the
brother was done.
The Leary case
C. FALLACIES OF METHODOLOGY

1. Cost efficiency
2. Comparative analysis
3. 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
C.1) COST EFFICIENCY

The process of PCR


based methods are
costly. To reduce the
cost:benefit ratio, newer
instruments are used
which are more
capacious and can have
The Applied Biosystems 3130xl Genetic
a greater output to lower Analyzer is a popular instrument that can
perform capillary electrophoresis to type DNA
the total and average
expenses.
C.2) COMPARATIVE ANALYSIS

This method of spotting the offender by


DNA profiling needs the DNA of the suspect
to match with forensic sample or to match
the forensic sample findings with a DNA
database archive. This has several
drawbacks from the ethical viewpoint. This
was seen when the rape accused could not
be identified and nor a suspect pinned.
Norm Gahn issued a “John Doe” DNA
warrant for the man.
C.3) TO DETERMINE ACCEPTABILITY

 To determine the credibility of a DNA Fingerprinting


result, the following query method is to be adopted:
– Could it be an accidental random match?
– If not, could the DNA sample have been
planted?
– If not, did the accused leave the DNA sample
at the exact time of the crime?
– If yes, does that mean that the accused is
guilty of the crime?
C.4) STUTTER BANDS

 STRs are prone to an artifact called


stutter bands or shadow bands which
occurs as the DNA repeats slip out of
identification during PCR and the
products are one repeat length shorter.
They maybe impossible to detect as it
cannot be interpreted whether the light
bands are due to stutter.
C.5) SHALLOW GENE POOL

Alan Legere of Canada was convicted of four


murders while he was an escaped convict
in 1991 based on the DNA profiling. The
prosecution had to undertake multilocus
DNA testing repeatedly to rule out the
defence’s point that the DNA sample
could belong to many people due to the
close sub ethnicity of the inhabitants.
C.6) JEFFREY GAFOOR CASE

 Paradoxically, Welshman Jeffrey


Gafoor was convicted in 2003 of
the murder of Lynette White in 1988
by comparing the crime scene DNA
evidence to that of his nephew—a
maternal relation—utilising mtDNA.
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 standards
was done. This also shows the necessity of
standardisation to interpret the results.
C.8) DR. SCHEENBERGER CASE

 Probably the most interesting of them all,


Dr. John Scheenberger of Canada raped
one of his sedated patients. The semen
sample was collected from the victim. On
three occasions, the police tried to match
the doctor’s DNA sample with that of the
crime scene DNA but was in vain. It turned
out that he had surgically inserted a
Penrose Drain in his arm and filled it with
foreign blood and anticoagulants.
D. THE STATISTICAL FALLACY
INTERROGATOR’S FALLACY
This is also known as the INTERROGATOR’S FALLACY. Tomas
de Torquemada, the Spanish Grand Inquisitor considered
confession 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 conditional
probabilities based on Bayesian Probability to determine the
probability of an accused being guilty given that he has
confessed.

The fallacy that confession=guilty was known


as the INTERROGATOR’S FALLACY.
 Let, THE STATISTICAL CONSIDERATIONS:
 A= the prior probability that the accused is guilty,
that is, the probability of guilt assessed from a
positive DNA fingerprinting result.
 A’= event of negation of A, that is, accused is
innocent
 B= event where DNA fingerprinting is positive.
 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 is
not guilty but has DNA +ve
 Now, AUC=CUA
 So, P(A1C)= P(AUC)/P(C)

 Now, P(C1A)=P(CUA)
P(A)
 FromBayes’ Theorem,
P(A1C)=P(CUA) P(A)=P(C1A)
P(A)………1
P(A) P(C) P(C)
 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’)
 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’)
 From Bayes’ Theorem,
 P(C)= P(C1A) P(A) + P(C1A’) P(A’)
 Also P(A)= 1—P(A’)
 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))
 So,
 P(A1C)= p/ {p + r(1-p)}
 This is known as Matthew’s

confessional probability.
THE STATISTICAL CONSIDERATIONS:
 Now, for a DNA positive Prior Odds defendant is
accused to be guilty, Assumption
innocent given the
frequency of the
 Probabilty that the accused is of Guilt DNA profile, and
incorporating prior
Guilty given that DNA is +ve> assumptions of
probability that the accused is guilt
innocent given that DNA is 0.000 1
+ve
 For the accused to be guilty, 0.001 1 in 1,002
then, the following probability 0.10 1 in 111,112
has to be satisfied:
0.50 1 in 1,000,0001
 P(A1C)>P(A)
 This, in turn implies that r<1 0.90 1 in 9,000,001
 The calculation of probable
0.99 1 in 99,000,001
guilt is tallied alongside:
Thus we see that the 50%
assumption of guilt tallies with the
accepted frequency of the DNA
profile in the population.
 THUS IT IS STATISTICALLY

ESTABLISHED THAT THE


POSITIVE DNA RESULT
CANNOT BE TAKEN AS FINAL
VERDICT OF GUILT!!!
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 NaEDTA
added 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
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
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
RECOMMENDATIONS

 SAMPLE COLLECTIONS
 Sperm:
 1oo µl 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
 Tissues:
 Tissues frozen in liquid nitrogen and
homogenised
 TNE buffer added, followed by SDS and
Proteinase K. overnight incubation at 55o
C
 Follow from step d of fresh whole blood
sequence
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

 Bone and teeth:


 Bone marrow or dental pulp are frozen in
liquid nitrogen
 TNE buffer with SDS and Proteinase K are
added and incubated overnight at 55oC
 Follow from step d of fresh whole blood
sequence
RECOMMENDATIONS

 Limit PCR contaminations:


 Use robotic technology for procedures
 Process sample as quickly as possible.
THANKS…

QUESTIONS?
pranabchatterjee@hotmail.com