Paper No.

: 07 Forensic Anthropology
Module : 34 DNA fingerprinting, VNTR, Mini satellite and Micro Satellite

Development Team
Prof. Anup Kumar Kapoor
Principal Investigator
Department of Anthropology, University of Delhi

Paper Coordinator Prof. Anup Kumar Kapoor

Department of Anthropology, University of Delhi

Prof. A.K.Kapoor and Ms. Sangeeta Dey

Content Writer
Department of Anthropology, University of Delhi

Prof. R.K.Pathak
Content Reviewer
Department of Anthropology, Panjab University, Chandigarh

Forensic Anthropology
Anthropology
DNA fingerprinting, VNTR, Mini satellite and Micro Satellite
 Description of Module

Subject Name Anthropology

Paper Name 07 Forensic Anthropology

Module Name/Title DNA fingerprinting, VNTR, Mini satellite and Micro Satellite

Module Id 34

Forensic Anthropology
Anthropology
DNA fingerprinting, VNTR, Mini satellite and Micro Satellite
 Contents:
1. Introduction.
1.1 DNA fingerprinting with RFLP (Restriction fragment length polymorphism)
1.2 DNA fingerprinting with PCR (Polymerase chain reaction)
1.3 DNA fingerprinting with AmpFLP (Amplified fragment length polymorphism)
1.4 DNA fingerprinting with STR (Short tandem repeats)
2. Principle of DNA fingerprinting
2.1 Mini satellite DNA & VNTRs
2.2 Micro satellite DNA
2.3 Significance of Mini & Micro satellite DNAs
3. Technique of DNA fingerprinting
4. Application of DNA fingerprinting
5. Summary

Learning objectives:
· Students will be able to identify the steps to DNA fingerprinting.
· Students will be able to recognize repeated patterns in DNA fingerprinting
· Students will be able to explain the ways that by which DNA fingerprinting can be used to
apply in public domain.
· Students will be able to match unknown DNA fingerprints to the recorded or suspected DNA
fingerprint in the Bureau of crime investigation or in police investigation or concealing personal
identification.

Forensic Anthropology
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DNA fingerprinting, VNTR, Mini satellite and Micro Satellite
 1. Introduction

DNA (Deoxyribonucleic acid) fingerprinting is a well known method of identifying criminals. It is

considered as recent trend in personal identification. It is a technique to find out variations in
individuals of a population at DNA level. The technique of DNA fingerprinting was pioneered and
perfected by British geneticist Dr. Alec Jeffreys. In 1984, he had developed this process to visually
identify DNA found between the genes in the hope that it will reveal markers for inherited diseases and
lead to early treatment. He had no idea at that time that his new technique would help to solve cases
related to identity by comparing DNA.
Human “DNA prints” or “DNA fingerprints” are known to possess greater specificity. Like every
human organism is unique in terms of their fingerprints, likewise each individual has a unique DNA
fingerprint. Unlike conventional fingerprint that occurs only on the fingertips and can be altered by
surgery, a DNA fingerprint is the same for the every cell, tissue and organ of a person. It cannot be
altered by any known treatment. It represents the blueprint of the individual’s entire genomic makeup.
The “DNA fingerprints”: the specific set of DNA fragments produced by cutting the DNA with a
specific set of sequence-specific enzymes, depends on the exact sequence of subunits called
nucleotide-pairs in the DNA of the individual. Nucleotides are the molecules that make up DNA. Each
nucleotide has a nitrogenous base, a pentose sugar (deoxyribose) and inorganic phosphate. There are
double ring purine bases called adenine (A) and guanine (G) and two single ring pyrimidine, cytosine
(C) and the thymine (T). This sequence of nucleotide pairs differs in each and every individual. As
human genome contains about 3 X 109 nucleotide pairs, it is extremely unlikely that any two
individuals other than identical twins will have totally identical “DNA fingerprints.”

Source: https://c1.staticflickr.com/5/4042/5074826281_3131215f88.jpg
Figure 1: DNA fingerprinting
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 DNA fingerprinting can be done by various approaches such as DNA fingerprinting with RFLPs, with
PCR, with AmpFLP and with STR. These are the well known methods of DNA finger printing.
1.1 DNA fingerprinting with RFLP (Restriction fragment length polymorphism)

DNA molecules are long double stranded structure tightly packed in chromosomes within the nucleus
of each human cell. Within each DNA strand, number of genes are present which determine the
specific characteristics of the individual. The genetic information contained in gene composition of
DNA fragments are only 5% which are known as coding sequences, and the rest 95% do not contain
any genetic information but possess identifiable repetitive sequences of base pairs referred as non-
coding sequences. RFLP analyzes the length of the stands of DNA molecules with repetitive nucleotide
bases by determining a specific pattern of repeat and then cut those into fragments with specific
restriction enzymes. These fragments are then separated by electrophoresis which sorts fragments with
respect to length. The segments are than radioactively tagged to produce visual pattern or “DNA
fingerprint” on X-ray film. The drawback of this method is that it requires considerable amount of
DNA to sort fragments by length.

Source: http://homepage.smc.edu/hgp/tools.html
Figure 2: DNA fingerprinting with RFLP (Restriction fragment length polymorphism)

Forensic Anthropology
Anthropology
DNA fingerprinting, VNTR, Mini satellite and Micro Satellite
 1.2 DNA fingerprinting with PCR (Polymerase chain reaction)
DNA fingerprinting with PCR is most widely used method worldwide. PCR technique was formulated
in 1985 by Karry Mullis. The method is designed to permit selective amplification of a specific target
DNA sequence within a heterogeneous mixture of DNA sequences. The purpose of a PCR is to make a
huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize
DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from
an extinct dinosaur. Starting with one original copy an almost infinite number of copies can be made
using PCR. “Amplified” fragments of DNA can be sequenced, cloned, probed or sized using
electrophoresis. Using PCR, defective genes can be amplified to diagnose any number of illnesses.
Genes from pathogens can be amplified to identify them (i.e., HIV, Vibrio sp., Salmonella sp. etc.).
Amplified fragments can act as genetic fingerprints. PCR technique requires some prior DNA sequence
information from the target sequence. From this information, two DNA oligonucleotides primers
(amplimers) of about 15 – 25 nucleotides long and specific for the target sequence are designed. The
Primers are then added to the denatured template DNA which bind specifically to complementary
DNA sequences at the target sites. In the presence of a suitably heat – stable DNA polymerases and
DNA precursors (the four deoxynucleoside triphosphates, dATP, dCTP, dGTP, dTTP), primer initiate
the synthesis of new DNA strands which are complementary to the individual DNA strands of the
target DNA segment. The PCR has a drawback that it cannot discriminate DNA fragments on certain
basis as RFLP. PCR technique is very helpful to produce multiple copies of limited DNA molecules.

Source: http://en.wikipedia.org/wiki/Polymerase_chain_reaction
Figure 3: DNA fingerprinting with PCR (polymerase chain reaction)

Forensic Anthropology
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DNA fingerprinting, VNTR, Mini satellite and Micro Satellite
 1.3 DNA fingerprinting with AmpFLP (Amplified fragment length polymorphism)
Amplified fragment length polymorphism or AmpFLP is an attractive version of RFLP as it includes
relatively less complicated operation and cost – effective procedure. The method uses PCR technique
to amplify the minisatellite loci of the human genome, thus become quicker in recovery than the RFLP.
However there are issues to this method about the bunching of the VNTR sequences which is due to
use of gel in its analysis phase which ultimately results in misidentification of the DNA finger printing.

1.4 DNA fingerprinting with STR (Short tandem repeats)

STR or Short tandem repeats is another method of DNA fingerprinting which analyzes DNA fragments
for the number of repetitions at 13 specific DNA sites. It actually triggers specific areas that have short
mini satellites DNA or repeated DNA base pair sequences. STR thus can able to compute how many
times a particular sequence of base pair repeat themselves on a particular location within the strand of
DNA. This advantage makes the DNA comparisons matching to an almost positive range.

Thus, “DNA fingerprinting” now provide a powerful new tool for establishing identity or non-identity
in paternity, rape, and assault cases, as well as other identity cases where tissue samples might be
available linking a criminal to the crime. The “DNA fingerprinting” approach has become particularly
powerful with the recent development of techniques by which small amount of DNA can be
extensively amplified in vitro. This allows “DNA prints” to be made even when only very minute
amounts of tissue are available.
2. Principle of DNA fingerprinting
The ideal way to distinguish an individual from other people on Earth would be to describe his or her
entire genomic DNA sequence. This idea takes lots of effort, cost, time and thus clearly not practical.
So, we look for genes that are highly polymorphic, that is DNA sequences that have multiple alleles in
the human population, and therefore different in different individuals. This becomes the principle of
DNA fingerprinting or in other words DNA fingerprinting works on the principle of polymorphism in
DNA sequences. Important to DNA fingerprinting for determining identity are short nucleotide repeats
that vary in number from person to person and are inherited. In human genome about 10% of the entire
DNA is formed of repetitive sequences. These sequences are formed of few hundred nucleotides in
which a specific sequence of nucleotide bases repeated again and again without interruption and result
in the formation of tandem arrangement. These highly repetitive sequences can be categorized as Mini
Satellites and Micro Satellites.

2.1 Mini Satellites DNA & VNTRs

DNA fragments can be categorized as exons or coding sequences that contain genetic information and
introns or non-coding sequences which don’t contribute any genetic information about the
development of the individual. These introns which seem useless, has been found containing repeated

Forensic Anthropology
Anthropology
DNA fingerprinting, VNTR, Mini satellite and Micro Satellite
 sequences of DNA base pairs. These repetitive sequences are called Mini Satellites. Mini Satellites can
be recognized as repetitive DNA fragments that possess 10 – 60 base pair short series of nucleotide
bases. In human genome, minisatellites are known to occur at more than 1000 locations. Mini Satellites
generally GC rich repetitive sequences, that is sequences with lot of guanine and cytosine base pairs.
Length of repeats ranges from 10 to 100 base pair and repeats are tandemly intermingled. When the
number of repeats in a given minisatellite varies, we referred it as variable number tandem repeats or
VNTRs.

Source: http://www.slideshare.net/hhalhaddad/the-human-genome-project-part-iii
Figure 4: Minisatellite DNA
Variable Number Tandem Repeats or VNTRs:

· VNTRs are minisatellite DNA with varied repetitions of specific sequences. In one individual, the
specific sequence of mini satellite DNA repeats 5 times whereas in another it may repeat itself
1000 times.
· This makes VNTRs ideal for studying polymorphisms, for linkage analysis as genetic marker, can
be implemented as regulators for gene expression, and also used for population studies as VNTRs
greatly varies among individuals.
· The VNTRs of two persons may be of the same length and sequence at certain sites, but vary at
others sites. This is due to the reason, a child might inherit a chromosome with six tandem repeats
from the mother and the same tandem repeated four times in the homologous chromosomes
inherited from the father. Thus child’s VNTR alleles resemble half to the mother and half to the
father.
· Every human being has a unique VNTR sequence. A Persons particular VNTR can be determined
by performing southern blotting through a hybridization process with radioactive probes that target
specific VNTR sequences of DNA fragments will be discussed briefly later in the module.
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 · VNTR sequences are inherited genetically and an individual possess unique VNTR pattern. While
analyzing a individuals VNTR pattern, the use of more and more VNTR probes result in more
distinctive and unique individualized pattern of DNA fingerprint which will lead to personal
identification of individual in all domain of sciences.

Source: http://www.biology.arizona.edu/human_bio/problem_sets/dna_forensics_2/02t.html
Figure 5: VNTRs (Variable number tandem repeats)
2.2 Micro Satellites
Micro satellites are repetitive DNA fragments that possess 2 – 6 base pairs of nucleotide bases. Micro
satellites are generally CA rich repeat tandems with variable number of repeats between alleles which
makes it unique as CA nucleotide repeats found very frequently in human genomes and are
encountered every thousand base pairs.

Forensic Anthropology
Anthropology
DNA fingerprinting, VNTR, Mini satellite and Micro Satellite
 Source: http://www.slideshare.net/hhalhaddad/the-human-genome-project-part-iii
Figure 6: Micro Satellite DNA
Micro satellites are ideal as molecular markers, for determining disputed paternity, for studying gene
duplication or deletion, for recombination mapping, for population genetic studies as micro satellites
possess locus of many alleles, thus the progenitor of a particular allele or genotypes within pedigree are
often be identified. It also provides information regarding closely related alleles. Micro satellites are
associated with human diseases. For example, a gene FMR1 has 5 – 60 repeats of a triplet CCG under
normal condition in human genome but if the repeat rises above 200, the individual suffered from
fragile X syndrome and the individual becomes mentally retarded due to the microsatellite in the
fragile site on X chromosome. Some of the other diseases associated with micro satellites are as
follows:

· Huntington disease (Gene – HD)

· Fragile X syndrome (Gene – FMR1/FMR2)
· Kennedy disease (Gene – AR)
· Myotonic dystrophy (Gene – DMPK)
· Friedreich ataxia (Gene – X25)

2.3 Significance of Mini & Micro Satellites

Mini satellites and Micro satellites DNA repeat fragments do not have any specific function in DNA
structure but their existence is of great importance. Satellite DNAs has crucial role in the construction
of genetic maps, isolation of those genes which are responsible for causing human diseases. Technique
of DNA finger printing would not have been developed without these satellite DNAs.

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DNA fingerprinting, VNTR, Mini satellite and Micro Satellite
 3. Technique of DNA fingerprinting
DNA fingerprinting requires only a small amount of tissue like blood or semen or skin cells or even
follicle root of the hair. Typically DNA content of about 100,000 cells or about 1 microgram is
sufficient.

The procedure of DNA fingerprinting involves the following major steps:

a) The DNA is extracted from the nuclei of whatever evidence is available. For Example, from
WBCs in case of blood sample or from hair follicle cells that cling to the roots of hair that have
fallen or have been pulled out; or from spermatozoans in semen sample. Nuclei of the cells are
extracted in high speed refrigerated centrifuge.

Source: http://www.primarycareofappleton.com/ClinicServices/LabTests/ChemistryBloodTest.aspx
https://followthelemur.wordpress.com/2011/09/13/the-science-of-vampires/
http://mashable.com/category/dna/
Figure 7: DNA molecule extraction from blood sample collected from individuals.

b) If the content of the DNA is limited, DNA can be amplified by making many copies of it using
PCR or Polymerase Chain Reaction.

Source: http://swh.schoolworkhelper.netdna-cdn.com/wp-content/uploads/2011/06/PCR1.jpg?c71720
Figure 8: DNA amplification through PCR technique.

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 c) The DNA sample so extracted and amplified is digested by a restriction enzyme (EcoRI) which
functions to cut the DNA into fragments at specific sites. Thus, these enzymes are site recognizing
enzymes for restriction fragment length analysis. The number of these sites present in an
individual’s DNA dictates the number and size of DNA fragments generated by the enzymes.

Source: http://www.discoveryandinnovation.com/BIOL202/notes/lecture23.html
Figure 9: Function of Restriction enzyme

d) Chopped DNA fragments are introduced and passed through electrophoresis set up containing
agarose polymer gel. This is done for separation of DNA fragments with respect to their length.
The separated fragments can be visualized by staining them with a dye that fluoresces under
ultraviolet radiation.
e) In the preceding steps, DNA is in double stranded form. But now double stranded DNA is split
into single stranded DNA using alkaline chemicals.
f) These separated DNA sequences are transferred to a nylon or nitrocellulose sheet placed over the
gel. This is called “Southern Blotting.”

Source: http://dnafingerprinting19.tripod.com/sitebuildercontent/sitebuilderpictures/dna-evidence3.jpeg
Figure 10: Southern blotting of the DNA molecules.
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 g) The nylon sheet is then immersed in a bath and probes or markers that are radioactive, synthetic
DNA segments of known sequences are added. The probes target a specific nucleotide sequences
which is complementary to the VNTR sequences and hybridize them.

Source: http://media-3.web.britannica.com/eb-media/72/47672-004-4E16B61F.jpg
Figure 11: Agarose gel electrophoresis showing DNA movements.

h) Finally X-ray film is exposed to the nylon sheet containing radioactive probes. Dark bands
develop at the probe sites which look something like bar codes used to identify items at the
grocery store.

Source:http://science.nayland.school.nz/graemeb/images/class%20work/2005/yr10&11images/gene/DNA_finger
prnt.jpg
Figure 12: X-ray film showing bands of DNA molecules.
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 Overview of the steps of DNA fingerprinting method

Source: http://i.dailymail.co.uk/i/pix/2014/07/23/article-2702606-1FE749C000000578-30_634x465.jpg
Figure 13: Overview of the technique of DNA fingerprinting

Note:
Southern Blotting was named after its inventor Edward M. Southern who developed this method. It is
well known technique to detect specific molecules of DNA from a cluster of DNA molecules. It allows
investigators to specifically determine the relative molecular weight of a restriction fragment and also
it permits to estimate relative amount of DNA molecules in the sample. Scientists use this method to
identify sequences of sample DNA molecule by probe hybridization. Procedure of Southern blotting
performs by extracting DNA molecule from sample then it is chopped into small fragments with
restriction enzymes. Then the cut DNA fragments transferred into agarose gel and split into single
stranded DNA molecules by alkaline chemicals. These separated fragments are transferred to a nylon
sheet. The sheet is than incubated. Sequences of the single stranded DNA are exposed to known
synthetic florescent DNA segments and hybridization occurs and can be viewed through
autoradiography on the X-ray film. An adaptation Southern blotting is Northern Blotting where RNA
molecules are electrophoresed instead of DNA molecules. Southern blotting has several applications
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 such as identification of desired DNA probes; Detection of mutation; Genetic manipulation can be
done. Also it has implication in cloning by determining all the sequences of a sample of DNA or RNA.

Source: http://www.gene-quantification.de/mrna.html
Figure 14: Southern blotting Technique.

4. Application of DNA fingerprinting

· In Forensic Domain:
DNA fingerprinting and forensic science is basically pertains to intersection of law and science. DNA
fingerprinting is highly specific and unique. No two individuals have exactly same DNA fingerprints
except identical twins. DNA can be isolated from whatever evidence collected at the scene of crime
like blood, hair, skin cells, saliva, semen, body tissues. DNA fingerprints thus can be matched or
compared with suspected criminals through VNTR prototype.

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 Source: http://image.slidesharecdn.com/lab7-dnafingerprintingandgelelectrophoresisfall2014-140925074116-
phpapp01/95/lab-7-dna-fingerprinting-and-gel-electrophoresis-fall-2014-19-638.jpg?cb=1411630925
Figure 15: Matching of VNTR sequences to reveal criminal identification
· Identification of Paternity and maternity:
A child inherits his or her VNTR sequences from his or her parents. Parent- child VNTR sequence
analysis can used to solve disputed paternity and maternity cases. The sequences are so unique that
parental sequences can be reconstructed even if only the child VNTR sequences are known. This
information can also help solve inheritance cases, legal nationality, immigration cases, to verify
whether a hopeful immigrant is a really close relative of already an established resident, identity of
homicide victim, adoption cases. In 2002, through DNA fingerprinting Elizabeth Hurley prove that
Steve Bing was the father of his child Damein.

· Personal identification:
DNA fingerprinting act as a genetic bar code to identify individuals. It is very reliable technique by
which personal identification can be made with higher accuracy. It also requires to isolate and analyze
VNTR sequences of a particular individual to match it with already registered sequences to conceal
identity. Once the identity is assured by fingerprinting technique, identification is 100 percent
confirmed or denied.

· Identification of racial groups:

DNA fingerprinting help identify racial groups to rewrite biological evolution.

· Diagnosis of inherited disorders:

DNA fingerprinting is useful in diagnosis of inherited disorders which include Hungtington’s diseases,
sickle cell anemia, thalassemia, cystic fibrosis, hemophilia and many more.

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 · Development of cures for inherited disorders:
DNA fingerprint analysis of relatives who have a history of particular disorder makes it possible to
determine sequences of inherited disorders. DNA sequences associated with the disorder can be
ascertained and an alternative cure can be developed in the form of DNA sequences with advent
technologies and can be implanted to cure the inherited disorder. DNA finger printing was used to
identify Chinese medicine - Lingzhi by the Hong Kong Baptist University in 2000.

· Detection of AIDS:
Individuals suffering from HIV – AIDS can be identified by comparing the bands of HIV “RNA”
(converted to DNA by RT – PCR) with the bands of the individual DNA isolated from blood sample.

· Breeding program:
DNA fingerprinting permits us to determine the genotype or genome of the individual. Breeders
conventionally use DNA fingerprinting to assure homozygous or heterozygous dominance. Offspring
of the superior genotype are expected to inherit the highly adaptable characters and DNA
fingerprinting allows precise determination of dominant genotype.

5. Summary
DNA fingerprinting is a well known method to determine variation among individuals of population
at DNA level. The degree of variation is so high every individual with the exception of identical twins,
produce a unique band pattern as every individual has unique set of ordinary fingerprints. It works on
the principle of polymorphism in DNA sequences. DNA fingerprinting requires only a small amount of
tissue like blood or semen or skin cells or even follicle root of the hair. DNA fingerprinting can be
done by various approaches such as DNA fingerprinting with RFLPs, with PCR, with AmpFLP and
with STR. These are the well known methods of DNA finger printing. “DNA fingerprinting” is now
become a powerful new tool for establishing identity or non-identity in paternity, rape, and assault
cases, as well as other identity cases where tissue samples might be available linking a criminal to the
crime. The “DNA fingerprinting” approach has become particularly powerful with the recent
development of techniques by which small amount of DNA can be extensively amplified in vitro. This
allows “DNA prints” to be made even when only very minute amounts of tissue are available.

17

Forensic Anthropology
Anthropology
DNA fingerprinting, VNTR, Mini satellite and Micro Satellite