DNA Profiling

1. Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. Introduction
 DNA Fingerprintingis is technique used by
scientists to distinguish between
individuals of the same species using only
samples of their DNA.
 We’re all (nearly) unique
 Most DNA is highly conservative from one
person to the next
 A few small domains (0.1%) are very
variable.
Historic examples
1800s – photography
Early 1900s – fingerprints, Palm prints
Retinal Scan
Footwear and tire impressions
Other – ears, lips, etc.
DNA Fingerprinting – 1984-85
Unique signature found in each
person’s genetic makeup
Forensic science – intersection of law
and science

3. Why Use DNA Fingerprinting?
DNA fingerprinting is a way of
telling individuals of the same
species apart
DNA sequences are variable and can
therefore be used as identifying
characteristics.
DNA fingerprinting has advantages
over other sources of evidence
(fingerprints, blood type etc.):
o Highly accurate.
o Can be gathered from trace crime scene
evidence.
A process or technique of analysis
revealing unique patterns of an
individual’s DNA involving non-
coding regions

4. DNA Fingerprinting/ ProfilingDNA Fingerprinting/ Profiling
 The genome in a particular species is more
of less same in every member of a species.
 Genome contains polymorphisms,
positions were the nucleotide sequences
are not same in every member of the
population.
1980 - American researchers discovered
non-coding regions of DNA
1984 - Professor Alec Jeffries developed
the process of DNA profiling
Sir Alec Jeffreys was the first to
comprehensively research on DNA
fingerprinting in 1984.
1987 - First conviction based on DNA
evidence

5. DNA Fingerprinting/ ProfilingDNA Fingerprinting/ Profiling
 Like several other significant discoveries in biotechnology genetic fingerprinting
was discovered by scientists who were searching for something else.
 Unlike many other advances in modern genetics DNA profiling has already had a
major effect on the lives of thousands of people throughout the world.
 In 1984 Prof Alec Jeffreys of the University of Leicester in England was studying
the gene for myoglobin, a protein that stores oxygen in the muscle. He found that
part of the gene did not carry instructions for the manufacture of myoglobin (non
coding DNA sequence). Instead, this bit of DNA consisted of an unusual sequence
of bases repeated several times.
 Jeffreys realised these aparantly useless & harmless pieces of DNA could act as
genetic markers for the myoglobin gene – helpful in tracking down its location on
a particular chromosome.
 To his surprise Jefferys found that the sequence of non coding DNA from the
myoglobin gene occurred in many different places throughout the human
chromosome.
 Further investigation showed that each of the variable regions shared a common
sequence of about 16 base pairs.
 The number of times this sequence occurred was aparantly unique to each
individual.
 Prof. Jefferys was quick to realise that the technique could be used to establish
with accuracy the identity and relatedness of individuals
 Legal history – made in Nov 1987 when a Bristol man was sentenced to 8 yrs in jail
for rape. First conviction based on evidence using the revolutionary new technique
known as DNA fingerprinting (matched blood with semen stains on the clothes of
his victim)

6. Restriction Fragment Length Polymorphism (RFLP),
Randomly Amplified Polymorphic DNA (RAPD)
Amplified Fragment length Polymorphism (AFLP)
Variable number tandem repeats (VNTR), particularly short
tandem repeats (STR)s,
Single nucleotide polymorphisms (SNPs)

7.  Restriction Fragment Length Polymorphism (RFLP) is a
difference in homologous DNA sequences that can be detected
by the presence of fragments of different lengths after digestion
of the DNA samples in question with specific restriction
endonucleases.
 The presence or absence of certain recognition sites in a DNA
sample generates variable lengths of DNA fragments, which are
separated using gel electrophoresis.
 RFLP is a technique for analyzing the variable lengths of DNA
fragments that result from digesting a DNA sample with a
restriction endonuclease

8. If two different samples show VNTRs of different lengths,
the samples could not have come from the same organism.
VNTRs of the same length could have come from the same
organism, or from two individuals who happen to have
VNTRs of the same length at that location.
By comparing enough VNTRs from two individuals,
however, the likelihood of a coincidental match can be
reduced to nearly zero.

9. It requires relatively large amounts of DNA.
Isolation of sufficient DNA for RFLP analysis is time
consuming and labor intensive.
In addition, samples degraded by environmental factors,
such as dirt or mold, do not work well with RFLP.
The vast majority of RFLP methods are qualitative and
perform best on pure animal tissue since mixtures can
produce complicated fingerprints that are not easily
interpreted.

10. Also called as PCR-RFLP
PCR can be used to amplify very small amounts of DNA,
usually in 2-3 hours, to the levels required for RFLP analysis.
Therefore, more samples can be analyzed in a shorter time.

12. Random Amplified Polymorphic DNA (RAPD)
Random amplified polymorphic DNA (RAPD) analysis takes
advantage of short arbitrary PCR primers and produces a range
of amplified products
Amplification products are generally separated on agarose gels
and stained with ethidium bromide.
RAPD does not require any specific knowledge of the DNA
sequence of the target organism.
The identical 10-mer primers will or will not amplify a segment
of DNA, depending on positions that are complementary to the
primers sequence.
At an appropriate annealing temperature during the thermal
cycle, oligonucleotide primers of random sequence bind several
priming sites on the complementary sequences in the template
genomic DNA, producing discrete DNA products if these
priming sites are within an amplifiable distance of each other .
Nucleotide variation between different sets of template DNAs
will result in the presence or absence of bands because of
changes in the priming sites.

13. Standard PCR RAPD Reaction/ Sample #1

14. Gel electropheresis

15. Advantages
Little or no information on the DNA sequence is required
RAPD is therefore not only relevant to domestic animals, but
also to rare species
Discrimination between pork, beef, lamb, chicken, and turkey
has been demonstrated in processed food (Saez, Sanz, & Toldra,
2004 )
RAPD is an inexpensive yet powerful typing method for many
bacterial species.

16. Disadvantages
It is not possible to distinguish whether a DNA segment is
amplified from a locus that is heterozygous (1 copy) or
homozygous (2 copies).
Mismatches between the primer and the template may result in
the total absence of PCR product as well as in a merely
decreased amount of the product. Thus, the RAPD results can be
difficult to interpret

17. Amplified fragment length polymorphism (AmpFLP)
Step 1: Preparing the AFLP Template
Step 2: Ligation Reaction with Restriction Fragments and
Adaptors
Step 3: Selective PCR Amplification
Step 4: Electrophoretic Separation of Amplified DNA Fragments
and Analysis of results

18. Step 1: Preparing the AFLP Template
Isolation and purification of DNA
Digestion of the DNA with a pair of restriction enzymes, often
MseI and EcoRI
MseI recognizes 5 -TTAA-3 , cleaves after the first 5 -T and′ ′ ′
generate DNA fragments with 5 overhangs (5 -TA-3 )′ ′ ′
EcoRI recognizes 5 -GAATTC-3 , cleaves after the 5 -G. generate′ ′ ′
DNA fragments with 5 overhangs (5 -AATT-3 )′ ′ ′
These overhangs are distinct from each other and are non-
complementary.

19. Step 2: Ligation Reaction with Restriction
Fragments and Adaptors
MseI adaptor and an EcoRI adaptor
Rather than containing a 3 -A after the 5 -TA-3 overhang, the′ ′ ′
MseI adaptor contains a 3 -C, which destroys the MseI′
recognition site once ligated.
Similarly, rather than containing a 3 -C after the 5 -AATT-3′ ′ ′
overhang, the EcoRI adaptor contains a 3 -G, which destroys the′
EcoRI recognition site once ligated.

20. Step 3: Selective PCR Amplification
Each unique genomic DNA fragment is sandwiched between the
MseI adaptor and the EcoRI adaptor and their associated unique
DNA sequences.
Although the MseI and EcoRI adaptor-ligated ends of each DNA
fragment will be identical, the sequences of the genomic DNA
fragments, which begin just after the original MseI and EcoRI
sites, will differ.
If PCR reactions are carried out using primers corresponding to
the MseI and EcoRI adaptor sequences, they would amplify
every single genomic DNA fragment, and therefore
indecipherable set of DNA fragments would be produced.
In order to selectively amplify a smaller number of genomic
DNA fragments, sets of primers are used that are
complementary to the MseI or EcoRI adaptor sequences
starting at their 5 ends but that add up to three unique′
nucleotides following the end of the original MseI or EcoRI
recognition site.

21. Primer design does not require any previous knowledge of the
genome under study.
Simply PCR reactions are set up using a variety of primer sets,
viz. one MseI-associated primer and one EcoRI-associated
primer.
AFLP-PCR reactions are carried out under stringent conditions,
permitting only the selective amplification of those genomic
DNA fragments that are perfectly complementary to the 3 ends′
of the primer sequences.
The stringent PCR conditions employed in AFLP-PCR reactions
lead to highly reproducible results that are readily comparable
among different samples.

22. Step 4: Electrophoretic Separation of Amplified
DNA Fragments and Analysis of Results
The band pattern after gel electrophoresis is analyzed to
determine the DNA finger print.

24. VNTR’S (variable number tandem repeats )
The non-coding genes contains identifiable repetitive sequences
of base pairs, which are called Variable Number Tandem Repeats
(VNTR’s).
Every human being has some VNTRs. To determine if a person has
a particular VNTR, a Southern Blot is performed, and then the
Southern Blot is probed, through a hybridization reaction, with a
radioactive version of the VNTR . The pattern which results from
this process is what is often referred to as a DNA fingerprint.

25. VNTR
 The introns contain repeated sequences of between 1 and 100 base
pairs
 Called variable number tandem repeats (VNTR’s)
 Some VNTR’s are inherited from mother and some from father
DNA fingerprinting is restricted to the detection of microsatellites
1 to 6 nucleotide repeats dispersed throughout the chromosomes
Probes used to identify the microsatellite surround the specific
microsatellite being analyzed
Also called short tandem repeats (STR)
FBI has chosen 13 unique STRs for testing
 Combined DNA Index System (CODIS)
DNA fingerprinting is a comparative process
Samples from crime scene must be compared to suspect DNA
Best sample from suspect DNA is fresh, whole blood
STR Analysis
o Use primers to amplify STR’s in DNA using PCR
o FBI uses 13 STR regions
o Odds that two individuals will have the same 13-loci DNA profile are
more than one in a billion

26.  Variable Number Tandem Repeats, VNTRs, are highly polymorphic, multi-allelic
DNA markers that contain tandem repeats of 11 to 60 base pair sequences, but
represent only a single locus.
 The highly polymorphic nature of VNTR loci makes them a useful tool for
human identification.
 VNTR loci are routinely detected by RFLP analysis. Using this technique, the
DNA sample is subjected to restriction enzyme digestion followed by gel
electrophoresis. The separated restriction fragments are then transferred to
a membrane in a Southern blot procedure and VNTR detection is performed
using specific probes that are labeled with radioactive or chemiluminescent
tags.
 VNTR loci are further characterized by length as well as how many times the
sequence is repeated.
 There are three different VNTR structures which are used: Restriction
Fragment Length Polymorphisms (RFLP), Amplification Fragment Length
Polymorphisms (AMP-FLP) and Short Tandem Repeats (STR).
 Note that the longer the repeat, the higher the heterozygosity. As the
heterozygosity increases so does the power of discrimination.
 AMP-FLP and STR analysis are performed using PCR technology.
 Currently STRs are in widespread use.
 Their main advantages is that their small size allows for the use of small
amounts of degraded DNA.
 PCR technology allows use of less than 1ng of DNA for identification.

27. What are STRs?
Short Tandem Repeats (STR) are repetitive sequences:
Tetranucleotide: AAAG AAAG AAAG AAAG
Trinucleotide: CTT CTT CTT CTT CTT
Dinucleotide: AG AG AG AG AG AG
Tetranucleotides are favored in human identity
Good balance of “ease of interpretation” and “variability
found in nature”

28. STR Analysis
Intron regions of DNA (junk DNA) contain sequences that are
20-100 bp in length that are repeated at different locations (loci)
along the chromosome.
CGGCTACGGCTACGGCTA
Each person has some STRs that were inherited from mother
and some from father
No person has STRs that are identical to those of either parent
The number of repeats at each loci on chromosome is highly
variable in the population, ranging from 4 to 40.

29. Interpretation
The length of the DNA after cutting the chromosome with a
restriction enzyme, and its position after electrophoresis will
depend on the exact number of repeats at the locus.
Since the number of times sequence is repeated is different for
each organism, fragment size will be different.

30. Advantage
The odds that two individuals will have the same 13-loci DNA
profile is about one in a billion.
We do not need to sequence the entire DNA to distinguish it
from another sample DNA.

31. SNP
►A SNP is defined as a single base change in a DNA
sequence that occurs in a significant proportion (more
than 1 percent) of a large population.
In human beings, 99.9 percent bases are same.
Remaining 0.1 percent makes a person unique.
Different attributes / characteristics / traits
 how a person looks,
 diseases he or she develops.
These variations can be:
Harmless (change in phenotype)
Harmful (diabetes, cancer, heart disease, Huntington's disease, and
hemophilia )
Latent (variations found in coding and regulatory regions, are not
harmful on their own, and the change in each gene only becomes
apparent under certain conditions e.g. susceptibility to lung cancer)
►

32. SNP facts
►SNPs are found in
 coding and (mostly) noncoding
regions.
►Occur with a very high
frequency
 about 1 in 1000 bases to 1 in 100 to
300 bases.
►The abundance of SNPs and the
ease with which they can be
measured make these genetic
variations significant.
►SNPs in coding regions may
alter the protein structure made
by that coding region.
SNPs may / may not alter protein
structure

33. SNPs act as gene markers
►SNPs close to particular gene acts as
a marker for that gene.

34. SNP maps
►Sequence genomes of
a large number of
people
►Compare the base
sequences to discover
SNPs.
►Generate a single map
of the human genome
containing all possible
SNPs => SNP maps

35. SNP ProfilesGenome of each individual contains
distinct SNP pattern.
People can be grouped based on the
SNP profile.
SNPs Profiles important for
identifying response to Drug
Therapy.
Correlations might emerge between
certain SNP profiles and specific
responses to treatment.

36. Techniques to detect known Polymorphisms
Hybridization Techniques
Micro arrays
Real time PCR
Enzyme based Techniques
Nucleotide extension
Cleavage
Ligation
Reaction product detection and display

37. Techniques to detect unknown Polymorphisms
Direct Sequencing
Microarray
Cleavage / Ligation
Electrophoretic mobility assays

38. Mitochondrial DNA Analysis
mtDNA analysis uses DNA extracted from mitochondrion
All mothers have the same mitochondrial DNA as their offspring
mtDNA profile of unidentified remains with the profile of a
potential maternal relative can be an important technique in
missing-person investigations.

39. Advantage
mtDNA can be used to examine the DNA from samples that
cannot be analyzed by RFLP or STR
While older biological samples that lack nucleated cellular
material, such as hair, bones, and teeth, cannot be analyzed
with STR and RFLP, they can be analyzed with mtDNA.
In the investigation of cases that have gone unsolved for many
years, mtDNA is extremely valuable.

40. Disadvantages
Large variations of mitochondria in different tissue.
Not possible to perform a meaningful quantification based
on neither DNA nor meat content (w/w).

41. PCR Analysis
Polymerase chain reaction (PCR) is used to make millions of
exact copies of DNA from a biological sample.
DNA amplification with PCR allows DNA analysis on biological
samples as small as a few skin cells.
The ability of PCR to amplify such tiny quantities of DNA
enables even highly degraded samples to be analyzed.
Great care, however, must be taken to prevent contamination
with other biological materials during the identifying,
collecting, and preserving of a sample.
Gel electrophoresis has been widely used to separate PCR
amplicons and determine their presence, intensity, size, and
pattern through visualization with different staining dyes.
The PCR technique can, in theory, amplify one copy of target
DNA and the LOD is therefore often lower than observed in
protein based methods.
A low LOD and a large amount of DNA sequence data have
resulted in a shift in species determination from protein to DNA
analysis, and in particular PCR.

42. Applications
Forensics
 Human forensics
Animal forensics
Solving child disputes
Meat adulteration determination
As an epidemiological tool for determining particular sources
and causative agents of infectious disease

43. Advantage of Molecular Methods in Epidemiology
Aid in faster diagnosis of diseases.
Increased sensitivity and specificity.
Rapid detection of pathogen than conventional methods.
Identification of epidemiologically important strains.
Decrease the man power need for pathogen detection.
Give rapid answers to treatment options in life threatening
infections.
Adapted to instrumentation.

44. Uses of DNA Profiling
DNA profiling is used to solve
crimes and medical problems

45. Crime
Forensic science is the use of scientific knowledge in legal
situations.
The DNA profile of each individual is highly specific.
The chances of two people having exactly the same DNA
profile is 30,000 million to 1 (except for identical twins).

46. Biological materials used for DNA profiling
Blood
Hair
Saliva
Semen
Body tissue cells
DNA samples have been obtained
from vaginal cells transferred to
the outside of a condom during
sexual intercourse.

47. DNA Profiling can solve crimes
The pattern of the DNA profile is then compared with those of
the victim and the suspect.
If the profile matches the suspect it provides strong evidence
that the suspect was present at the crime scene (NB:it does not
prove they committed the crime).
If the profile doesn’t match the suspect then that suspect may
be eliminated from the enquiry.

48. ExampleA violent murder occurred.
The forensics team retrieved a blood sample from the crime scene.
They prepared DNA profiles of the blood sample, the victim and a suspect as
follows:
Suspects
Profile
Blood sample from
crime scene
Victims
profile
Was the suspect at the crime scene?

49. Solving Medical Problems
DNA profiles can be used to determine whether a particular
person is the parent of a child.
A childs paternity (father) and maternity (mother) can be
determined.
This information can be used in
• Paternity suits
• Inheritance cases
• Immigration cases

50. Example: A Paternity Test
By comparing the DNA profile of a mother and her child it is possible to
identify DNA fragments in the child which are absent from the mother and
must therefore have been inherited from the biological father.
Mother Child Man
Is this man the father of the child?

51. 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
profiling to prove that Steve Bing was the
father of her child Damien
O.J. Simpson was cleared of a double
murder charge in 1994 which relied heavily
on DNA evidence. This case highlighted
lab difficulties.

52. Applications
Diagnosing Disease

53.  Genealogy:
 Another application of analysis of RFLP and
VNTR loci in establishing familial relationships is
in the area of molecular genealogy.
 Traditional genealogy involves extensive search
of historical records. But our DNA also contains
a record of our familial relationships.
 DNA analysis can provide clues about what part
of the world our ancestors came from.
 As DNA is copied and passed down through
generations, it gradually accumulates more
mutations. These mutations account for human
genetic variation.
 RFLP and VNTR analysis of these mutations
show that people who are more closely related
will share more similarities in their DNA.
 These individuals are said to have the same
haplotype. The number of differences between
your haplotype and the haplotype of another
person can tell you the number of generations
you have to go back to find a common ancestor.
Applications

55. Thanks
Acknowledgement: All the material/presentations available online on
the subject are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source
and authenticity of the content.
Questions???