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DNA Fingerprinting
Introduction
? DNA fingerprinting is science of identifying a person based on fact that each individual has
unique DNA, like unique finger prints. Exception being twins and fact that part of this DNA
uniqueness been contributed from parent.
? DNA fingerprinting is known as DNA profiling, genetic fingerprinting, DNA typing.
? It has been successful y used worldwide in solving many crime and paternity cases.
? DNA Barcoding - DNA analysis intended to identify a species, rather than an individual.
Introduction
? DNA fingerprinting discovered by Professor Alec Jef reys at Leicester University
in1984 discovery by while studying genetic variations in myoglobin.
? In India DNA fingerprinting was started by Dr. V K Kashyap and Dr. Lalji Singh.
? Dr. Lalji Singh: the "Father of Indian DNA fingerprinting"
Character of DNA
? DNA can tolerate wide range of temperature, pH and other factors.
? DNA can be denatured easily but denaturing doesn't change its property-
specificity(length/ weight), VNTR`s which is used in DNA fingerprinting.
? This property can be altered if target DNA`s length is altered by DNase, bacteria, fungi, UV
rays.
? DNA mixed with detergents, oil, gasoline and other adulterants does loose its typing
characteristics
Various Methods for DNA fingerprinting
? Restriction Fragment Length Polymorphism (RFLP)
? Amplified Fragment Length Polymorphism (AFLP)
? Random Amplification Polymorphic DNA
? Single Sequence Repeats
? Inter Simple Sequence Repeats
? Ligase chain reaction: Method of DNA amplification
Various Methods for DNA fingerprinting
? DNA sequencing: Biochemical methods for determining the order of the nucleotide
bases in a DNA oligonucleotide
? Polymerase chain reaction(PCR).- Mainly used for amplification of target DNA
sequence (when test sample has smal quantity of DNA).
? Most commonly used are- RFLP & PCR
RFLP vs PCR
Features
RFLP
PCR
Amount of DNA
Large (300-500 ng),
Smal (25 ng) (because smal
sample required
e.g. Col ecting sample for
quantity been amplified).
Paternity test
e.g. limited sample from crime
scene
DNA degradation
Useless when degradation is
Useful (because smal quantity of
present (because smal quantity target DNA been amplified)
of target DNA left)
Time required
More
Less
RFLP vs PCR
Features
RFLP
PCR
Decomposed sample
Not useful (because smal Useful (because smal quantity
quantity of target DNA left) of target DNA been amplified)
Sensitivity
Less
More(because smal quantity
been amplified)
Tedious
More
Less
Labor intensive
More
Less
Sensitivity to
Less sensitive
More sensitive
contamination
Result of the test
Non-discrete
Discrete (binary `yes/no')
Specimen col ection
vSamples Col ected from Living Subjects:-
I. Blood (most common, preferred)- .
?5ml venous blood in EDTA Tube
?mixed thoroughly without shaking
?Heparin not to be used as it interferes PCR
?Sample to be preserved at 2-8oC (not frozen)
Specimen col ection
vSamples Col ected from Living Subjects:-
II. Buccal epithelial cel s by sterile swabs:-
? easy to col ect,
? swab tip should not be touch by hand,
? fresh swab immediately taken from pack should be stabbed to the subject`s
mouth for 10 second.
? Sampled swab should be air dried before and packed in sealed container
Specimen col ection
vSamples Col ected from Living Subjects:-
III. Hair fol icles with roots (plucked hair)-
? 10-20 plucked head hairs with roots,
? roots contain keratinocytes which contain nuclear DNA,
? Shaft may be source of mitochondrial DNA.
? Cut or natural y fal hair should be avoided, but may contain nucleated
corneocyte (keratinocyte in last stage of dif erentiation) may be present
Specimen col ection
vSamples Col ected from Dead Bodies:-
? Postmortem material is inferior to live blood and tissue for DNA testing as DNA
progressive undergo degradation by autolytic and bacterial enzymes.
? In relatively fresh dead bodies- unclotted 10 ml of blood in EDTA sterile tube is
used. Buf ers present along with EDTA inhibit the activity of nucleases so it is
added advantage.
Specimen col ection
vSamples Col ected from Dead Bodies:-
? Clotted blood is not a good source of DNA as WBC settled down
? In intermediate postmortem intervals- Brain tissue is a good source of DNA.
? If decomposition is establishing- muscle, spleen, femur bone marrow and teeth
(morals) are preferred.
? In advanced decomposition- Hard tissue (bone and vascular pulp of teeth) is the
best source of DNA.
Specimen col ection
vFrom traces of organic matter/ DNA:- Sample from these source are so little that
often PCR is required before fingerprinting e.g.-
? Blood stain on cloth, newspaper, wood or tiles
? Semen stain on cloth, paper, floor
? Hair- body/pubic
? Fingernail scrapings
? Saliva stain on cigarette buds/ licked envelope
DNA Storage
There are 4 broad strategies for long term preservation of DNA:-
? Room temperature on a dry solid matrix.
? -20?C
? -80?C
? -196?C (in liquid nitrogen)
? Blood and other biological liquid can be stored on FTA card after drying at
room temperature
FTA Card
? FTA stands for Fast Technology for Analysis of nucleic acid.
? It chemical y treated filter paper designed for the col ection, preservation and
shipment of biological samples at room temperature.
? Liquid sample containing DNA material made to absorb on FTA card can
preserve DNA material long time, claimed over 50 years
FTA Card
? It has weak base, chelating agent, anionic surfactant or detergent and uric
acid on cel ulose base filter paper.
? Nucleic acid damage from nuclease, oxidation, UV light, microbes, fungus is
reduced on FTA card.
? When analyzing nuclear material, a smal piece containing sample is punched
from card, it is washed and DNA material extracted is use for PCR or RFLP.
FTA Card
? Other benefits:-
? No risk for spreading infectious agents due to the chemicals included.
? Rapid isolation of pure DNA.
? Transportation at room temperature.
? Reduces potential of cross contamination.
? Submitting samples via regular post
? Low transportation costs.
DNA Fingerprinting
v DNA extraction-
? Before the DNA can be analyzed, it must be extracted from the cel s and purified.
? The most common methods of DNA extraction include organic extraction (also cal ed
phenol chloroform extraction), Chelex extraction and solid phase extraction.
? Depending on quantity, extraction procedure is chosen.
? RFLP was preferred but if sample (DNA) is less then PCR is preferred
RFLP Principle
? Restriction fragment length polymorphism (RFLP) is a technique that uses
variation in homologous DNA sequences(known as polymorphism) in order to
distinguish individuals, populations, or species or to pinpoint the locations
of genes within a sequence.
? The term may refer to a polymorphism itself, as detected through the dif ering
locations of restriction enzyme sites
RFLP Principle
? RFLP analysis was the first DNA profiling technique testing.
? RFLP analysis is basic however slow and cumbersome.
? It requires a large amount of sample DNA combined process of probe labeling, DNA
fragmentation, electrophoresis, blotting, hybridization, washing
and autoradiography can take up to a month to complete.
Basic technique for the detection of RFLPs
? RFLP is being discussed to understand basic principal-
? Sample DNA is fragmented by restriction enzyme (endonuclease) which breaks DNA
at specific sites (where specific sequence is recognized by enzyme) process cal ed as
restriction digest.
? The specific DNA fragments thus produced are then separated in sequence
(sequence according to their length/weight) on agarose gel by process gel
electrophoresis.
Basic technique for the detection of RFLPs
? Sequenced DNA fragment bands on agarose gel are then transferred to a membrane-
Southern blot technique
? Now membrane contain sequential band of DNA fragment as on agar gel.
? Membrane is then soaked in solution containing specific label ing probe (designed
to hybridize with complementary target DNA fragments- target DNA)
Basic technique for the detection of RFLPs
? After soaking Membrane is then wash.
? If target (complementary) DNA fragment is present on membrane then probe
hybridizes(sticks) to membrane and wil not wash away,
? If hybridization of probes does not occur then probe wil washes away.
? Traces probe hybridized to trace complementary DNA fragment on membrane can be
detected by florescent/ enzymatic or radioactive property.
RFLP- VNTR
? A restriction fragment length polymorphism is said to occur when the length of a
detected fragment varies between individuals, it indicating non-identical sequence
homologies.
? Each fragment length is considered an al ele(polymorphism)
VNTR
? In the schematic above, the rectangular blocks represent each of the repeated DNA
sequences at a particular VNTR location
? The repeats are in tandem i.e. they are clustered together and oriented in the same
direction.
? Flanking regions are segments of non-repetitive sequence (shown here as thin lines),
al owing the VNTR blocks to be extracted with restriction enzymes(endo nuclease) and
analyzed.
Polymorphism VNTR
? The exact number and size of fragments produced by a specific restriction
enzyme(endonuclease) varies from individual to individual, i.e. they are
individualistic in nature and establish nearly 100% identity.
Procedure
I. Isolation/extraction of DNA: DNA must be
recovered from the cel s or tissues of the body.
Only a smal amount of tissue--blood, hair or skin
--is needed. For example, the amount of DNA
found at the root of one hair is usual y sufficient
I . Cutting and sizing: Special enzymes cal ed
restriction enzymes(endo nuclease) cut the
DNA at specific site. For example, an
enzyme EcoR1 cuts DNA only at sequence
GAATTC.
I I. Sorting By Gel Electrophoresis
? Sorting VNTR fragments are separated on the basis of size using a process cal ed gel
electrophoresis.
? DNA fragments are injected into wel s and an electric current is applied along the gel.
? DNA is negatively charged so it is attracted to the positive end of the gel.
? The shorter DNA fragments move faster than the longer fragments. Thus DNA is
separated on basis of size.
IV. Transfer of DNA to nylon (Southern blotting)
? Specific DNA can be identify if it can be label ed / tagged/ hybridize(with a
complimentary genetic probe.)
? It is not possible in gel, so DNA is denatured and then transferred to nitrocel ulose or
nylon membrane, which picks DNA like a blotter picks up ink by capil ary action, it is
then fixed by heating
? The resulting blot formed is essential y a replica of the gel. This is cal ed Southern blot
named after Dr Edward Southern
Southern blot
V. Hybridization
? Adding known radioactive DNA probes (short sequence probe, complimentary to the
region of DNA which one wishes to detect) to the nylon sheet leads to fragment
location.
? The nylon membrane is immersed in a solution that contains DNA probe impregnated
with radioactive P32. Each probe typical y sticks in only one or two
specific/complementary sequences on the nylon sheet. This process is termed as
hybridization
Radioactive probe in solution
Revealing a pattern of
binds to DNA
bands
X-ray film
VI. Washing
The membrane is washed to remove excess or unbound probe and exposed to an X-
ray film.
? Spots on the X-ray film correspond to the locations of the fragments in the gel that
are complimentary to the probe (autoradiography).
? Nowadays, many radioactive probes are detected by chemical luminescence
which is analyzed by computer scanners
VI . DNA fingerprint- autoradiograph
The final print is known as an
autoradiograph or 'DNA fingerprint'
which appears as lines on the film.
Result Matching
? Two-step process:- It determine if two samples arose from one source.
? First- DNA-banding pattern of test sample is compared with known DNA sample
visual y. If matching not found result is declared, if matching is observed then bands
are forwarded for second step.
? Second step ? matching verified by computer assisted al ele sizing. Match should
fal within 2.5% of each other, if not found it should be considered `nonmatching.'
Result Matching
? If the DNA-banding pattern of a sample cannot be positively determined due to
technical problems, the results should be considered `inconclusive Now a days
highly polymorphic single locus genes such as the VNTR genes are utilized
instead of multilocus DNA analysis
? Due to the large number of distinguishable al eles in most populations, it is
possible to establish a `DNA signature' for almost any individual
Limitations of RFLP
? Slow- It takes few weeks to perform.
? Cumbersome- For every probing, the membrane is stripped of the previous
probe and rehybridized and autoradiography performed again.
? Requires a large amount of sample DNA
? It is not useful where DNA is degraded--limits use in cadaveric tissue.
PCR
? PCR is technique for amplifying(increase in number of copies) of particular DNA
fragments in sample in vitro, so that it can be readily analyzed.
? PCR itself does not accomplish DNA typing, but increases the amount of
DNA(DNA amplification) available for typing.
? Developed by Karry Mullis of the Cetus Corporation in 1983.
Reaction requires:-
? Heat resistant DNA polymerase (Taq polymerase)- enzyme which attaches
nucleotides on the growing strand of DNA and thus helps to expand it.
? Template DNA ? target DNA segment which is required to be amplified(increase in
number of copies).
? DNA Primers ? it is short sequences of nucleotides which bind at the end of the
template DNA segment. It is specific according to target template DNA
Reaction requires:-
? Deoxynucleoside triphosphates - equal amounts of dATPs, dTTPs, dCTPs, dGTPs
? Other chemical in buffer like Mg, pH control etc.
Process
? Chemicals are mixed in sterile condition in thermal cycler (PCR Machine) and run
for certain cycles(20-30).
? A cycle in thermal cycler has 3 stages at three temperatures:-
I. Denaturation:-Heat samples to 94?C for a minute or so
I . Annealing: cooling 50 or 60?C
I I. Extension:- Maintain temperature at 72?C for a minute or two
I. Denaturation
? Heating samples to 94?C for a
minute or so - double stranded DNA
dissociates to two single strands.
I . Annealing
? Drop temperature to around 50 or 60?C- it
wil cause the primers to pair up with the
single-stranded template (annealing).
? On the smal length of double-stranded DNA
(the joined primer and template), the
polymerase attaches and starts copying the
template.
I I. Extension/ Elongation
? Maintain temperature at 72?C for a minute
or two- it al ow the polymerase to elongate
the new DNA strands which are
complementary to the template where
primers are attached making a double
stranded DNA.
? In thermal cycler the process is completely
automated.-The time and temperature can be
programmed for repetitive cycles.
? Usual y 20-30 cycles runs.
? Each separated strand can serve as a template for synthesis as long as
Deoxynucleoside triphosphates and primer is provided for each strand as new
primer binding sites are duplicated on each cycle.
? PCR amplification is logarithmic, meaning the number of copies of the target DNA
doubled every cycle.
? After 20 cycles, this exponential process yields 220 copies of target sequence
Other amplification based methods
? Dot blots- series of DNA probes to detect and record target such as for HLA DQ,
DR at chromosome 6q in pattern of colored dots Amplified fragment length
polymorphism (AmpFLP)
? Utilizing mitochondrial DNA.
? STR analysis is less susceptible to DNA degradation than other AmpFLPs. The
chance of misidentification in this procedure is one in several bil ion.
Applications of DNA Fingerprinting
? Establishing identity of person in-
? Linking suspect to biological evidence- blood, semen, hair etc. found at scene of
crime.
? Early identification of criminal by matching genetic material evidence found at
crime scene from data base of criminal`s gene data bank e.g. CODIS 13 data
bank.
? Accidents/ Mass disaster- by DNA pattern matching with relatives or old confirmed
material/ DNA band ladder print.
Applications of DNA Fingerprinting
? Skeletal remains/ Mutilated bodies- by DNA pattern matching with relatives or
old confirmed material/ DNA band ladder print.
? Estimating close relatives,.
? Matching of missing, unknown deceased.
? Archeological, Immigration cases
? Record keeping for future identification.(eg. Soldiers in US Army)
Applications of DNA Fingerprinting
? Establishing paternity.
? Race, gender identification of unknown
? Diagnosis of inherited disorders:- like cystic fibrosis, hemophilia, Huntington's
disease, familial Alzheimer's, sickle cel anemia and thalassemia. Genetic
counselors use DNA fingerprint information to help prospective parents
understand the risk of having an affected child.
Applications of DNA Fingerprinting
? Developing cures for inherited disorders
? Protecting genome of endangered species.
? Pedigree analysis of pets
Anlysis of sample from crime scene
Anlysis of sample from crime scene
CODIS 13
? CODIS is a DNA database funded by the FBI that
stores DNA profiles of convicted felons.
? Use 13 markers plus one to determine gender.
? If any two samples have matching genotypes at al
13 CODIS loci, it is a certainty same individual (or
an identical twin).
Paternity Testing
? This is possible because child receive its DNA( except mitochondrial DNA) only
from mother or father, that means that any bands a child has must also be
present one parent or the other.
? In the given example (see next slide) the highlighted bands that did not come from
the mother, must have come from the father. Father 2 matches every band with
the child that did not come from the mother.
Paternity Testing
Paternity identification
Q.-Identification of person from
suspects whose blood found at incident
spot
A.- John
Limitation of DNA fingerprinting
DNA fingerprinting is controversial in a number of areas
? Too sensitive- a tiny amount of contaminant DNA in a sample may become
amplified- leading to an erroneous conclusion.
? Time constraints.- The process is lengthy, with each of four or five loci exposed
sequential y, it usual y takes 10 weeks.
Limitation of DNA fingerprinting
? Touch DNA:- People leave microscopic level genetic material everywhere they go.
High sensitive DNA fingerprint technique can detect DNA as less as 20 cel s.
It is relatively common for innocent person`s DNA to be present on near by area
where he has not visited or on weapon which he might just touched much time
befoe. this could erroneously tag person as criminal.
Limitation of DNA fingerprinting
? Accuracy of the results:
? Non uniqueness of DNA fingerprinting: DNA segments(instead of complete DNA
strands) are 'fingerprinted', target segment may not be unique
? Laboratories that may not fol ow uniform testing standards and quality controls
Result may alter.
? Technical problems- Ambiguity in interpretation of the bands from
misinterpretation, actual shifts, degradation, missing bands, extra bands.
Limitation of DNA fingerprinting
? Forensic specimens are often contaminated, making the extraction of pure
DNA dif icult, chance of false positive or false negative are high
? Cost- expensive.
? Unlike fingerprints DNA profile cannot be enlarged and shown in the court of
law
Limitation of DNA fingerprinting
? Ethical issues and privacy:-
Many countries are creating genetic information database as-
? FBI has created Combined DNA Index System (CODIS 13) databank for criminals.
? US is also making database for its soldiers.
? Similar database is present in UK also.
These database contains DNA fingerprint of from convicted criminals and from evidence found
at crime scenes. misuse of the database, such as identifying individuals with stigmatizing
il nesses such as AIDS.
Mitochondrial DNA (mtDNA)
? It smal circular genome located in the mitochondria,
? It can be recovered from damaged, degraded or very smal biological samples.
? Cel s contain hundreds of copies of mtDNA genomes, as compared to two copies
of the DNA located in the nucleus.
? So increases chances of recovering sufficient mtDNA where nuclear DNA lacking
Mitochondrial DNA (mtDNA)
? mtDNA is maternal y inherited therefore-
? Individual's mother, siblings, as wel as al other maternal y-related family
members wil have identical mtDNA sequences. So comparisons can be made
from any maternal relative, ? Nucleic Acid Sequence Based Amplification
(NASBA), is used.
? mtDNA is not useful in paternity suit.
? mtDNA can`t be as specific as nuclear DNA
Famous cases
? Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence.
He was arrested in 1986 for the rape and murder of two girls and was sentenced
in 1988.
? In 2002 Elizabeth Hurley used DNA Fingerprinting to prove that Steve Bing was
the father of her child Damien.
Famous cases
? O.J. Simpson was cleared of a double murder charge in 1994 which relied heavily
on DNA evidence.
? The Bil Clinton? Monica Lewinsky scandal.
? Tandoor Kand Delhi
This post was last modified on 12 August 2021