BANGABANDHU SHEIKH MUJIBUR RAHMAN SCIENCE

AND TECHNOLOGY UNIVERSITY, GOPALGANJ-8100.

Assignment on
Application of DNA fingerprinting in immigration

COURSE TITLE: Molecular Markers and Diagnostics

COURSE CODE: BGE-413

Submitted to: Submitted by:

Dr. Md. Sarafat Ali Md Estiarul Haque Pran
Assistant Professor, Student ID: 16BGE015
Dept. of Biotechnology & Session: 2016-17
Genetic Engineering

1
Contents
Introduction..............................................................................3
Background..............................................................................4
History of DNA fingerprinting................................................5
Principle of DNA fingerprinting..............................................6
Methods of DNA fingerprinting..............................................8
1. RFLPS (Restriction fragment length polymorphisms).....8
2. VNTRs (Variable number tandem repeats)......................9
3. STRs (Short Tandem Repeats).......................................10
STR profiling for relationship analysis.............................11
Application of DNA fingerprinting in immigration...............12
Standard DNA fingerprints in immigration dispute............13
Conclusion.............................................................................15
Reference...............................................................................16

2
 Application of DNA fingerprinting in immigration

Introduction

DNA fingerprinting is a chemical test that shows the genetic makeup of a person or other
living things. It’s used as evidence in courts, to identify bodies, track down blood relatives,
and to look for cures for disease.

DNA is short for deoxyribonucleic acid, which is inside of every cell in your body. It’s a
chain of chemical compounds that join together to form permanent blueprints for life. These
compounds are called bases, and there are 4 of them. They pair up with another to form what
are called base pairs. Your DNA has about 3 billion of these couples. The way they’re strung
together tells your cells how to make copies of each other. The complete set of your
compounds is known as a genome. More than 99.9 % of everyone’s genome is exactly alike
(100% if you are identical twins). But the tiny bit that’s not is what makes you physically and
mentally different from someone else.

DNA fingerprinting uses chemicals to separate strands of DNA and reveal the unique parts of
your genome. The results show up as a pattern of stripes that can be matched against other
samples.

The discovery of minisatellite deoxyribonucleic acid (DNA) fingerprinting by Alec Jeffreys

et al. (1985b) is most often reviewed in the context of the way in which it has revolutionized
forensic science and police casework. Equally, however, it has been the touchstone for a
transformation in our capabilities of determining paternity issues. It has also provided, for the
first time, a mechanism by which to dissect complex relationships where direct family lineage
(parent to child) is not necessarily available. The technology and applications have continued
to evolve since 1985. Through the years there has been a logical progression in methodology
from DNA fingerprinting involving multilocus probes, to DNA profiling involving variable
number tandem repeat (VNTR) single locus probes (SLPs), to polymerase chain reaction
(PCR)-based short tandem repeat (STR) profiling, which is in use today.

3
Background

Our bodies are composed of tiny functional units called cells, each of which contains
information packaged as deoxyribonucleic acid, or DNA. This enormously long molecule
carries information for a cell much the same way magnetic tape carries information for a
stereo system. DNA interacts with cellular machinery just as the tape interacts with a tape
deck. Rather than recordings of music or words, though, our DNA molecules carry
instructions on how to construct and operate a human body. Information is often carried most
efficiently in a code. Morse code carries words as dots and dashes; computer memories carry
software as binary digit code. DNA also carries its information in coded form. DNA is
composed of two parallel chains of bases. The four different bases, designated A, T, C, and
G, encode information for the cell. The sequence of the bases in a DNA chain carries
instructions for the cell in the same way dots and dashes carry words in Morse code. The
physical shape of the DNA molecule is a "double helix" structure. This may be thought of as
a sort of twisted ladder, with the rungs corresponding to base pairs. Some have compared the
DNA structure to that of a zipper: two parallel strands, with teeth or bases pairing in the
middle. DNA base pairing is very specific, however: A will pair only with T, and C will pair
only with G. A DNA strand can only be "zipped up," or hybridized with another strand that
has a matching, complementary base sequence.

DNA in the cell is contained in packages called chromosomes. An individual inherits half of
his or her chromosomes from each parent. The combined information encoded in the base
sequences of the inherited chromosomes is called the genome; this information determines
the individual's physical characteristics. Each body cell contains a complete set of
chromosomes, a complete DNA "blueprint" for the entire person. No cell uses the entire
"blueprint," however. Cells in different parts of the body read only the sections of DNA that
they need to perform their functions.

In a laboratory, DNA may be examined by cutting the long chromosomal chains into short
pieces. The DNA is cut using protein molecules called restriction enzymes. These enzymes
will cut DNA only at very specific points. The enzyme acts as a "magic pair of scissors"; it
recognizes a specific base sequence in the DNA and cleaves the DNA only at that place.'3
Different restriction enzymes recognize different sequences. The sequences that the enzyme
will recognize may be from 4-8 bases long. Such sequences are scattered at random

4
throughout the genome. Because the restriction enzymes cut only at their specific recognition
sequences, digesting a person's DNA with a certain restriction enzyme will produce the same
pieces every time.

History of DNA fingerprinting

Historically, identity testing in the forensic field started with the analysis of the ABO blood
group system. Later, new markers for identity and paternity identification were based on
variations of serum proteins and red blood cell enzymes; eventually the human leukocyte
antigen system was used. It was not until 20 years ago that Sir Alec Jeffreys, professor and
geneticist at the University of Leicester in the United Kingdom (UK), pioneered DNA-based
identity testing.

Professor Jeffreys was interested in studying the genetic variation between individuals and
had done some of the early work to detect genetic differences in humans. However, the
answer did not come to him on the initial project he was interested in but rather on an
unrelated project: analysis of the myoglobin gene in seal meat at the headquarters of the
British Antarctic Survey. When he and his colleagues compared the myoglobin gene in seals
with the human counterpart, they found that some short repeating sequences were
homologous between seals and humans. When they compared those sequences with the
published sequences of tandem repeats called minisatellites, they found that they were the
same. Professor Jeffreys recognized that the repeating sequences “could be highly variable,
informative genetic markers”. His group developed a radioactive probe, made up of short
sequences that could latch onto those repeating sequences and ultimately reveal patterns that
were unique to each individual: a DNA “fingerprint”.

The steps involved in DNA fingerprinting are as follows. First, the DNA is extracted from the
specimen (i.e., blood, semen, skin, hair). After DNA extraction, restriction enzymes are
added, which work like scissors to cut the DNA into the smaller segments that are different
between individuals. The DNA seg-ments are sorted by agarose gel electrophoresis and
visualized by staining with ethidium bromide. A Southern blot is performed to transfer the
DNA onto a membrane. A radioactive probe is applied to the membrane, and the pattern of

5
DNA is detected by exposing the membrane to x-ray film. The result is a pattern of DNA
bands that looks like a supermarket bar code. Each individual has a signature fingerprint.

Professor Jeffreys looked at a DNA fingerprint of a human family; he also looked at the
fingerprint of a cow, a baboon, a mouse, and a tobacco plant. The pattern of DNA segments,
composed of perhaps 15 to 20 bands, was different for each one. However, closer inspection
of the patterns of the human family revealed that the mother and the father each had their
own pattern and that the child had a composite of both, having inherited an allele from the
father and the mother.

In the spring of 1985, Professor Jeffreys and his colleagues published their first article on
DNA fingerprinting and saw the utility of it in the forensic sciences and in paternity
identification. Newspapers publicized the findings, and shortly thereafter a lawyer became
interested in the test and saw its applicability in one of the cases she was representing.

Principle of DNA fingerprinting

 The DNA of every human being on the planet is 99.9% same. However, about 0.1%
or 3 x 106base pairs (out of 3 x 109 bp) of DNA is unique in every individual.

 Human genome possesses numerous small non-coding but inheritable sequences of

bases which are repeated many times. They do not code for proteins but make-up 95%
of our genetic DNA and therefore called the ―junk DNA.

 They can be separated as satellite from the bulk DNA during density gradient
centrifugation and hence called satellite DNA.

 In satellite DNA, repetition of bases is in tandem. Depending upon length, base

composition and numbers of tandemly repetitive units, satellite DNAs have subcat-
egories like microsatellites and mini-satellites.

 Satellite DNAs show polymorphism. The term polymorphism is used when a variant
at a locus is present with a frequency of more than 0.01 population.

6
 Variations occur due to mutations. These mutations in the non-coding sequences have
piled up with time and form the basis of DNA polymorphism (variation at genetic
level arises due to mutations).

 The junk DNA regions are thus made-up of length polymorphisms, which show
variations in the physical length of the DNA molecule.

 At specific loci on the chromosome the number of tandem repeats varies between
individuals. There will be a certain number of repeats for any specific loci on the
chromosome.

 Depending on the size of the repeat, the repeat regions are classified into two
groups. Short tandem repeats (STRs) contain 2-5 base pair repeats and variable
number of tandem repeats (VNTRs) have repeats of 9-80 base pairs.

 Since a child receive 50% of the DNA from its father and the other 50% from his
mother, so the number VNTRs at a particular area of the DNA of the child will be
different may be due to insertion, deletion or mutation in the base pairs.

 As a result, every individual has a distinct composition of VNTRs and this is the main
principle of DNA fingerprinting. 

 As single change in nucleotide may make a few more cleavage site of a given
nucleotide or might abolish some existing cleavage site.

 Thus, if DNA of any individual is digested with a restriction enzyme, fragments

pattern (sizes) will be produced and will be different in cleavage site position. This is
the basics of DNA fingerprinting.

7
 Figure 1: Principle of DNA fingerprinting

Methods of DNA fingerprinting

There are three methods of DNA fingerprints:

1. RFLPs,

2. VNTRs, and

3. STRs.

1. RFLPS (Restriction fragment length polymorphisms)

Restriction fragment length polymorphisms, or RFLPs as they are commonly known, were
the first type of DNA fingerprinting which came onto the scene in the mid1980’s. RFLP’s
focus on the size differences of certain genetic locations. The first step in creating an RFLP

8
fingerprint is obtaining and isolating the DNA. DNA can be obtained from almost any of the
cells or tissues in the human body.

You do not need a large amount of tissue or blood to provide enough DNA for analysis. The
DNA is then extracted from the blood or tissue sample, and from here we carry out our
second step in the process which is the cutting, sizing, and sorting of the DNA sample. DNA
is cut using restriction enzymes, which cut the DNA stand at specific places. Restriction
enzymes are usually isolated from bacteria that use them to degrade foreign DNA like viral
DNA. Each type of restriction enzyme recognizes and cuts a particular DNA sequence. The
DNA at this point is cut into a various array of pieces which are sorted according by size
through a process called electrophoresis. In this process the DNA particles are mixed into a
buffer solution and applied to a gel made from seaweed agarose. Each side of the gel is
connected to an electrical current.

The DNA is negatively charged due to its phosphate groups, so it migrates towards the
positive electrode or anode. The smaller pieces of DNA move faster (sieve) through the gel
than the larger ones, so this provides the basis of the fragment separation. “This technique is
the DNA equivalent of screening sand through a progressively finer mesh screens to
determine particle sizes”.

The band pattern that the DNA creates in the agarose gel is then transferred to a nylon sheet.
To complete this transfer a nylon sheet is placed on the gel and left to soak overnight in a
high salt solution. After the soaking procedure is completed, the nylon membrane contains
the same pattern of DNA as occurred in the original gel. The membrane is now prepared to
undergo its probing phase.

Radioactive or fluorescently labelled probes are hybridized onto the nylon membrane, which
bind to specific DNA sequences present in the pattern to produce a pattern of bands which
create the DNA fingerprint. This process can be performed with several different probes
simultaneously to make the final product which looks very similar to the bar codes you see in
retail stores.

2. VNTRs (Variable number tandem repeats)

9
Variable number tandem repeats, or VNTRs represent specific locations on a chromosome in
which tandem repeats of 9-80 or more bases repeat a different number of times between
individuals. These regions of DNA are readily analyzed using the RFLP approach and a
probe specific to a VNTR locus. The fragments are a little shorter than RFLPs (about 1-2 kilo
base pairs), but are created through the exact same process.

Since RFLPs and VNTRs are created in the same fashion, they exhibit the same overall
advantages and disadvantages. Some of the advantages of these types of DNA fingerprints
are that they are the most stable and reproducible, which is a valuable trait to have when you
are trying to determine an exact match of a person’s DNA, which must exclude billions of
other people’s DNA with a certain degree of confidence. They are also easier to prevent
contamination since the DNA sample is larger than with other types of DNA fingerprints, and
small amounts of DNA contamination does not alter the analysis. Some of the disadvantages
of RFLPs and VNTRs include they are very time consuming (especially the probe
hybridization step), relatively large amounts of DNA must be used to obtain an adequate
sample, too many polymorphisms may be present for a short probe, and the cost is very high
due to labor and time requirements.

3. STRs (Short Tandem Repeats)

Currently, the most popular method of DNA fingerprinting are short tandem repeats, or STRs
for short. Unlike VNTRs which analyze minisatellites that have repeat sequences of 9-80
base pairs, STRs use microsatellites which have repeat sequences of only 2-5 base pairs,
introducing the “less is more” philosophy to the world of DNA fingerprinting. This was a big
step forward in forensic science since the length of DNA fragment being analyzed is short
enough to be amplified by polymerase chain reaction (PCR), so now we are able to analyze a
very small sample of DNA that is quicker and easier than any previously known method and
match it to a person’s identity. PCR was developed in the mid 1980’s and used the same
principles that cells use to replicate DNA to amplify the specified region, which is usually
between 150-3,000 base pairs in length.

In order to amplify the DNA sequence, a pair of short priming sequences (which are
complimentary to the ends of the targeted sequence), a special heat-resistant DNA

10
polymerase called Taq polymerase, and a solution of the four DNA bases are all mixed
together in a test tube which contains a few copies of the targeted DNA sequence. The DNA
is then amplified (or replicated) by the repetition of a cycle which contains three vital step:

 The solution is heated to 95˚C to unzip the double helix DNA structure.
 The solution is cooled to 55˚C to allow the primers to bind to the ends of the DNA.
 The solution is then reheated to 75˚C which is the optimal temperature for the Taq
polymerase to create new copies of each DNA strand.

One PCR cycle takes approximately 2 minutes to complete. Each cycle doubles the amount
of the previous amount of targeted sequences in the test tube, so it only takes about 50 cycles
to produce hundreds of thousands of DNA copies.

So long as primers are chosen to flank an STR site, the band amplified will represent the STR
locus, and a simple gel or column will determine the band length. Thus this procedure avoids
the lengthy probe hybridization step to membrane of the RFLP/VNTR approaches.

STRs are currently the most popular type of DNA fingerprint, since the whole PCR process
takes only a few hours, compared to RFLP/VNTR probe hybridization and film exposure
which can take several days. STRs can use much smaller samples of DNA than
RFLPs/VNTRs, and can even use partially degraded DNA to create a fingerprint. Thus, the
integrity and quality of the DNA sample is not as great a factor with STRs than with the
traditional methods of DNA fingerprinting.

The current standard forensic protocol analyses 13 core STR loci which have been carefully
chosen for their uniqueness. The only disadvantage of the STR approach is it is sensitive to
contaminating DNA, so usually the STR approach is used first, followed by a VNTR analysis
if contamination is suspected, and enough DNA is available.

STR profiling for relationship analysis

Single locus probe profiling was the preeminent method of forensic DNA typing from 1989
to 1994, at which point a new generation of tests took over completely. These new DNA
profile tests came about through the invention of PCR, although relationship testing with all
its added complexity took several more years to change over to PCR-based methods. STRs

11
are a simpler version of minisatellites that comprise short repeating sequence motifs of only
2–6 base pairs.

These loci are thus known as microsatellites. The attraction of the microsatellite systems for
DNA identification analysis is that, in principle, all the logic, history and experience of
minisatellite analyses can be directly transposed into a repeat variation that will be contained
in a small fragment size that is ideally suited for PCR methodology. Paternity testing has
shifted predominantly to STR profiling, with the 4–6 SLP tests being replaced by about 12
STR tests.

More tests are required with STR profiling because the STRs have fewer allelic variants and
are thus less able to distinguish one person from the population. In fact, the complexity of
casework now addressed by STR profiling has extended to encompass cases that involve
establishing sibling status without parents and cases that skip a generation, such as uncle/aunt
to nephew/ niece.

The PCR technology behind STR profiling allows pinpoint accuracy in determining the types
of microsatellite allele so that all the nomenclature can move from allele size to numbered
allele type. This in turn has greatly aided the computerization of analyses, the
intercomparability of data between different results and the simplification of population allele
frequency databases.

Application of DNA fingerprinting in immigration

The availability of DNA testing meant that many problems associated with applications for
entry clearance for dependent relatives could be conclusively settled. The validity of disputed
family relationships could be determined, eliminating doubts and the need for the exercise of
discretion. The Pilot Trial established the feasibility of the procedure.

It also indicated the extent of erroneous decisions previously made by immigration officials.
For the first time applicants had the means to challenge effectively both the past refusals and
the attitudes and prejudices harboured by those exercising control. The Government had the
opportunity to correct past injustices, to exercise flexibility and magnanimity to those family
members who were able to prove conclusively that they had been wrongly excluded.

12
Since the early 1990s, the use of DNA analysis for the resolution of disputed relationships
has continued to expand. In the United Kingdom, paternity testing and immigration probably
exceeds 10 000 cases per year. The predominant use of DNA testing for disputed paternity is
associated with child maintenance, and for immigration casework the major value of the
DNA tests is to establish that a child abroad is the offspring of a United Kingdom resident.

DNA evidence is the legal and court-recognized end point for resolving disputed
relationships and the parties involved normally accept the findings, so it is rare to find the
DNA evidence actually going to court. The use of PCR methodology has meant a welcome
move away from the needle and syringe to provide sufficient blood for testing.

Testing can now use a simple blood prick, mouth swab or hair root to obtain sufficient DNA
to analyze. The standard process for testing still involves the parties visiting their doctor to
provide the sample, however, because the ‘chain of custody’ that is associated with such
professionals removes any doubt regarding the authenticity of the samples tested.
Relationship testing is not normally associated with the same before and after counseling that
accompanies medical genetic tests. But if relationship tests are initiated correctly – that is,
with the involvement of a family law expert or appropriate advisory counsel – then most
parties should be well advised about the consequences of the outcome to themselves and to
the children involved.

In such procedures, the DNA evidence brings resolution to, rather than the creation of, a
dispute. Particularly worrying, however, is the rise in ‘motherless’ testing, whereby men who
doubt the paternity of their child engage a testing company over the internet to test their child
without the mother’s involvement, using samples such as hair from the child’s brush. To try
and bring some principles into the sector, the Department of Health published a Code of
Practice in March 2001 to bring the public’s attention to issues such as consent,
confidentiality, and the validation and quality accreditation of laboratories.

Standard DNA fingerprints in immigration dispute

 When the first paper on DNA fingerprinting was published in spring 1985, it was
covered in the press – and one report in the Guardian was read by Sheona York at the
Hammersmith and Fulham Community Law Centre. Her clients, a Ghanaian family

13
 who were UK citizens living in London, were stuck in an immigration dispute. The
youngest son had travelled back to Ghana, but on his return to the UK was detained
due to an allegedly forged passport – the question being whether the boy coming
back was in fact the son or was a substitute.
"We took the case on – and it was a tricky case," says Professor Jeffreys. "The
woman had sisters back in Ghana, so the boy could have been a nephew, and we
didn't have the father for analysis. All we had were three fully accepted children – so
we used these children to reconstruct the DNA fingerprint of the missing father.
When you compared mum, dad, and the boy, the results were clear-cut – the boy was
definitely the son."
The case against the son was dropped, and huge press coverage ensued. "It was a
good news story of 'science fighting bureaucracy and helping families'", says
Professor Jeffreys. The University of Leicester switchboards were soon jammed with
calls about immigration. "I didn't realise the scale of the problem, thousands of
families were trapped in exactly this sort of dispute," he adds. Indeed, DNA
fingerprinting led to a change in the Immigration Act.

 The family of Abdur Rob

Mr Rob came to Britain in 1963 and became a British citizen in 1971. His wife and 4
sons applied to come to join him in 1974, but the application was refused in 1975.
The family appealed this decision and the appeal was allowed in 1976 for Mr Rob's
wife and 2 younger sons.
His elder two sons, Shorif and Mozir Uddin were refused on the grounds that they
were not related as claimed to Mr Rob. Their mother and 2 brothers left Bangladesh
for Britain in 1977, hoping that the family would soon be able to arrange for Shorif
and Mozir to join them. The separation of the family caused exceptional distress and
anxiety to Shorif. His mother described how he wrote repeatedly, asking when he and
his brother would be able to join the rest of the family. He was so affected by the
separation that he became mentally sick. His mother recalled how when she returned
to Bangladesh his condition improved, only to deteriorate again after she had
returned to Britain. Meanwhile a fresh application lodged in 1978 by the two brothers
was refused in 1980, and the appeal against this refusal dismissed in 1982.
The family continued by every means at their disposal to establish that Shorif and
Mozir were genuine sons of the family. A village visit undertaken by a London
14
 solicitor, Graham Smith, and conventional blood tests on the two boys, both
supported the claimed relationship. As a result of a Tribunal decision, children
having a claim to British citizenship by descent could travel to Britain without entry
clearance to exercise their right of abode, and if entry were refused they had the right
to appeal that decision before removal. The family were advised to write Shorif and
Mozir that they should travel to Britain on that basis. The two sons came in 1986. On
arrival they were refused entry but granted temporary admission. Once in Britain
they underwent DNA testing which confirmed the relationship between themselves
and Mr Rob and his wife.
 The family of Mr Altab Ullah
Mr Ullah is about 60 years old. He is a Bangladeshi citizen but has applied for British
citizenship. He first applied for his family, his wife, 4 daughters and one son, to join
him. His application was refused as the ECO was not convinced that they were in fact
related to him as claimed.
A fresh application made in 1985 or 1986 resulted in entry clearance being granted
to his wife, Mrs C Bibi, and their youngest daughter. However his son, Abdul
Khalique was again refused entry clearance. The Home Office then requested the
family to submit to DNA testing. The tests were carried out in May 1988. The DNA
test report found that the claimed relationship was established.

Conclusion

DNA fingerprinting is the most sophisticated way to identify living organisms. DNA is a
unique piece of genetic material within biological organisms, which have characteristics that
are one of a kind. DNA cannot easily be altered once it is left at a crimescene or deposited
with a mummy, which makes it a strong forensic tool. RFLPs and VNTRs are the traditional
methods of fingerprinting DNA, which uses a relatively large sample that uses the method of
probe hybridization to detect polymorphisms in the DNA. STRs are the most current form of
DNA fingerprinting, which is PCR based and uses a very small sample of DNA. DNA
fingerprinting has many applications that range from criminal rape cases, paternity tests,
molecular archeology, sports memorabilia, etc. It also use to solve immigration problem. The

15
DNA molecule is like a snowflake in that there are no two exactly alike, but is one of the only
things in common that all biological organisms are created with.

Reference

1. Jeffreys AJ, Wilson V and Thein SL (1985b) Individual-specific fingerprints of

human DNA. Nature 316: 76–79.
2. Mullis KB (1990) The unusual origin of the polymerase chain reaction. Scientific
American 262: 36–43.
3. Peters C, Schneider V, Epplen JT and Poche H (1991) Individualspecific DNA
fingerprints in man using the oligonucleotide probe (GTG)5/(CAC)5. European
Journal of Clinical Chemistry and Clinical Biochemistry 29: 321–325.
4. Smith JC, Newton CR, Alves A, et al. (1990) Highly polymorphic minisatellite DNA
probes. Further evaluation for individual identification and paternity testing. Journal
of the Forensic Science Society 30: 3–18.
5. Thomson JA, Ayres KL, Pilotti V, et al. (2001) Analysis of disputed single-
parent/child and sibling relationships using 16 STR loci. International Journal of
Legal Medicine 115: 128–134.
6. Thomson JA, Pilotti V, Stevens P, Ayres KL and Debenham PG (1999) Validation of
short tandem repeat analysis for the investigation of cases of disputed paternity.
Forensic Science International 100: 1–16
7. Bernstein, David (2001) “Frye, Frye, Again: The Past, Present, and Future of the
General Acceptance Test.” Law and Economics Research Papers Series Paper No. 01-
07. http://papers.ssrn.com/paper.taf?abstract_id=262034
8. Betsch, David (2005) DNA Fingerprinting in Human Health and Society.
http://www.extension.iastate.edu/Publications/NCR550.pdf

16