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What is DNA Fingerprinting?

Each of us has the same chemical structure of DNA. But there are millions
of differences in the DNA sequence of base pairs symbolized as A, T, C, G.
This makes the uniqueness among us so that each of us except twins is
different from each other
genetically. DNA sequence
thus can be just like a
fingerprint to identify every
person entirely. But there are
so many base pairs in DNA
sequences that it is hard and
time-consuming to read out all
the sequences. Scientists
have developed many
techniques in order to quicken
the identification process.
Restriction Fragment Length Polymorphism (RFLP)
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. RFLP, as a molecular
marker, is specific to a single clone/restriction enzyme combination. Most
RFLP markers are co-dominant (both alleles in heterozygous sample will
be detected) and highly locus-specific.
Every strand of DNA has pieces that
contain genetic information which
informs an organism's development
(exons) and pieces that, apparently,
supply no relevant genetic
information at all (introns). Although
the introns may seem useless, it has
been found that they contain
repeated sequences of base pairs.
These sequences, called Variable
Number Tandem Repeats (VNTRs),
can contain anywhere from twenty to
one hundred base pairs.

An example of Variable Number


Tandem Repeats (VNTRs) on
three chromosome pairs.

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,
with an RFLP probe
through a hybridization
reaction, with a
radioactive version of the
VNTR in question. The pattern which results from this process is what is
often referred to as a DNA fingerprint.
An RFLP probe is a labeled DNA PCR primer sequence that hybridizes
with one or more
fragments of the digested
DNA sample after they
were separated by gel
electrophoresis, thus
revealing a unique blotting
pattern characteristic to a
specific genotype at a
specific locus. Short, single
- or low-copy genomic
DNA or cDNA clones are
typically used as RFLP
probes.
Isolation of sufficient DNA
for RFLP analysis is time-consuming and labor intensive. However, 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.
A given person's VNTRs come from the genetic information donated by his
or her parents; he or she could have VNTRs inherited from his or her
mother or father, or a combination, but never a VNTR either of his or her
parents do not have.

Because VNTR patterns are inherited genetically, a given person's VNTR


pattern is more or less unique. The more VNTR probes used to analyze a
person's VNTR pattern, the more distinctive and individualized that pattern,
or DNA fingerprint, will be.
Criminal Identification and Forensics
DNA isolated from blood, hair, skin cells, or other genetic evidence left at
the scene of a crime can be compared, through VNTR patterns, with the
DNA of a criminal suspect to determine guilt or innocence. VNTR patterns
are also useful in establishing the identity of a homicide victim, either from
DNA found as evidence or from the body itself.
National DNA Databank: CODIS
The COmbined DNA Index System, CODIS, blends computer and DNA
technologies into a tool for fighting violent crime. The current version of
CODIS uses two indexes to generate investigative leads in crimes where
biological evidence is recovered from the crime scene. The Convicted
Offender Index contains DNA profiles of individuals convicted of felony sex
offenses (and other violent crimes). The Forensic Index contains DNA
profiles developed from crime scene evidence. All DNA profiles stored in
CODIS are generated using STR (short tandem repeat) analysis.
CODIS utilizes computer software to automatically search its two indexes
for matching DNA profiles. Law enforcement agencies at federal, state, and
local levels take DNA from biological evidence (e.g., blood and saliva)
gathered in crimes that have no suspect and compare it to the DNA in the
profiles stored in the CODIS systems. If a match is made between a
sample and a stored profile, CODIS can identify the perpetrator.
This technology is authorized by the DNA Identification Act of 1994. All 50
states have laws requiring that DNA profiles of certain offenders be sent to
CODIS. As of August 2007, the database contained over 5 million DNA
profiles in its Convicted Offender Index and about 188,000 DNA profiles
collected from crime scenes but not connected to a particular offender.
As more offender DNA samples are collected and law enforcement officers
become better trained and equipped to collect DNA samples at crime
scenes, the backlog of samples awaiting testing throughout the criminal
justice system is increasing dramatically. In March 2003 President Bush
proposed $1 billion in funding over 5 years to reduce the DNA testing

backlog, build crime lab capacity, stimulate research and development,


support training, protect the innocent, and identify missing persons.
CODIS is a computer software program that operates local, State, and
national databases of DNA profiles from convicted offenders, unsolved
crime scene evidence, and missing persons. Every State in the Nation has
a statutory provision for the establishment of a DNA database that allows
for the collection of DNA profiles from offenders convicted of particular
crimes. CODIS software enables State, local, and national law
enforcement crime laboratories to compare DNA profiles electronically,
thereby linking serial crimes to each other and identifying suspects by
matching DNA profiles from crime scenes with profiles from convicted
offenders. The success of CODIS is demonstrated by the thousands of
matches that have linked serial cases to each other and cases that have
been solved by matching crime scene evidence to known convicted
offenders.

How to perform DNA Fingerprinting?


DNA contains two strands which are
joined together by the forces between the
two nitrogenous bases on both strands.
We can imagine that DNA is a long
ladder with many rungs. Each rung
contains a pair of nitrogenous bases
which are distinguished into four different
types: adenine, guanine, cytosine and
thymine. They are in turn symbolized as
A, G, C, T respectively. The sequence of
rungs is different for different persons
and it is just like paragraphs of alphabets.
Most of the sequence can function to
regulate protein synthesis and to regulate
cell activities. The rest of the sequence,
in fact, have no specific function but with
high variability among people. So this
variability can be used to identify each
person.
One common technology used in DNA fingerprinting is the RFLP (restriction
fragment length polymorphism) analysis. The first step is to extract genomic
DNA (entire DNA pool of a person) from a person. This can be done by using
one's hairs or blood. The DNA molecules are enclosed in the nucleus of the
cells, so the extract of
cells needs to be lysed by
breaking the cell
membrane and the
nucleus envelope to
release the DNA
molecules. The DNA
molecules are now still
packed in the form of
chromatin by proteins.
They are then treated
with buffer, enzymes and
chloroform so as to
destroy the proteins. The
pure DNA molecules can
then be separated from
the destroyed proteins by
centrifugation.

The extracted DNA


molecules are now in a
very long chain and need
to be cut into many
fragments which are
easier to be recognized.
The DNA long chains are
cut by restriction
enzymes. A specific
restriction enzyme can cut
a specific locus on the
DNA chain. For example,
EcoR1 will only cut the
chain at the sequence of GAATTC. The mixture of fragments is then allowed
to run in the gel electrophoresis. The electrophoresis can separate the
fragments by electrical charges and a pattern of the fragment can be obtained
eventually. This pattern is different for different persons and hence can be
used for distinguishing people. The DNA fragments are then transferred from
the gel to a nylon membrane which is called a Southern Blot.
The separated DNA molecules are now still colorless and so it is hard to see
them for identification.
Probes have to be added
to make them visible on a
photographic film. The
probe is a small
radioactive fragment of
DNA which is
complementary to and can
match with the sequence
of the DNA on the nylon
membrane. This matching
is called hybridization. The
radiation from the probes
can cause darkening on
the photographic films.
This results in the
appearance of a pattern of
dark bars on the film,
which is the DNA
fingerprint of a person.

Applications
Forensic Uses
Blood or fragments of tissues such
as hair and skin cells may be left in
the scene of crime. The DNA
fingerprint of these cells from the
scene can be revealed and are
compared with the DNA fingerprint
obtained from criminal suspects. If
somebody's DNA fingerprint
matches with that from the scene
this will be a strong evidence
showing that he has committed the
crime. Also in the case of rapes,
the semen left in the victims'
vagina can also be extracted and
be used to reveal the DNA
sequence which is later compared
with the criminal suspects' DNA
fingerprints so as to find out who
did the rape.
Paternity
A child can inherit most of the DNA fragment
pattern from his parents. By comparing the
DNA fingerprints between the child and the
suspect parents, it is possible to identify who
the parent of the child is.

Southern Blot
A Southern blot is a method routinely used in molecular biology to check for the
presence of a DNA sequence in a DNA sample. Southern blotting combines agarose
gel electrophoresis for size separation of DNA with methods to transfer the sizeseparated DNA to a filter membrane for probe hybridization. The method is named
after its inventor, the British biologist Edwin M. Southern. Other blotting methods (i.e.,
western blot, northern blot, southwestern blot) that employ similar principles, but
using RNA or protein, have later been named in reference to Southern's name. As the
technique was eponymously named, Southern blot should be capitalized, whereas
northern and western blots should not.
Method
Restriction endonucleases are used to cut high-molecular-weight DNA strands into
smaller fragments. The DNA fragments are then electrophoresed on an agarose gel
to separate them by size.
A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or
below, depending on the direction of the transfer) the gel. Pressure is applied evenly
to the gel (either using suction, or by placing a stack of paper towels and a weight on
top of the membrane and gel), to ensure good and even contact between gel and
membrane. Buffer transfer by capillary action from a region of high water potential to
a region of low water potential (usually filter paper and paper tissues) is then used to
move the DNA from the gel on to the membrane; ion exchange interactions bind the
DNA to the membrane due to the negative charge of the DNA and positive charge of
the membrane.

The membrane is then exposed to high temperature (60 to 100 C) (in the
case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to
permanently and covalently crosslink the DNA to the membrane.
The membrane is then exposed to a hybridization probe. The probe DNA is
labelled so that it can be detected, usually by incorporating radioactivity or
tagging the molecule with a fluorescent or chromogenic dye. In some cases,
the hybridization probe may be made from RNA, rather than DNA. To ensure
the specificity of the binding of the probe to the sample DNA, most common
hybridization methods use salmon testes (sperm) DNA for blocking of the
membrane
.
After hybridization, excess probe is washed from the membrane, and the
pattern of hybridization is visualized on X-ray film by autoradiography in the
case of a radioactive or fluorescent probe, or by development of color on the
membrane if a chromogenic detection method is used.
Result
Hybridization of the probe to a specific DNA fragment on the filter membrane
indicates that this fragment contains DNA sequence that is complementary to
the probe.
The transfer step
of the DNA from
the
electrophoresis
gel to a membrane
permits easy
binding of the
labeled
hybridization
probe to the sizefractionated DNA.
It also allows for
the fixation of the
target-probe
hybrids, required
for analysis by
autoradiography
or other detection
methods.

The High Rise Killer


DNA Fingerprint
Tampa, Fl - June 13, 2008

A maid, doing her daily cleaning at a posh


condo, found Sarah Elizabeth Jones, 35,
dead in her bathtub. The maid said that
the doors were locked when she arrived.

Ms. Jones, who worked for an import


company in St. Petersburg, had been on
vacation at the time her body was found. She
was not known to have a boyfriend but one
man had been by seen by neighbors entering
her condo on at least two occasions.

The victim appeared to have


been killed after a struggle at
her condo near the beach.
Police found items strewn
about the condo as well as
blood in three different places
including the bathroom where
the body was found.
The numerous areas of blood were
analyzed by crime scene technicians
and were found to have come from two
different people. Police speculate that
the killer was injured in the struggle with
the victim leaving his or her blood at the
condo. Some of the blood was matched
to the victim, but other samples came
from an individual other than the victim.

Police also found fingerprints belonging to at least three persons,


including the victim. The fingerprints were sent to the Integrated
Automated Fingerprint Identification System (IAFIS) run by the
FBI. IAFIS returned a match on two different
men.
The first set of fingerprints belonged to Gerald
Goss, 45, from New Jersey, who listed his
occupation as librarian.

The second set belonged to John Kennedy, 25,


of Denver, who listed his occupation as writer.

Search warrants were obtained for blood samples from each


man. The samples were then compared using Restriction
Fragment Length Polymorphism (RFLP) technology. The
samples were run on an electrophoretic gel for comparison.
From these samples police hoped to determine which mans
blood matched the blood found at the scene that would identify
the killer.
The DNA from four blood samples 1) the unknown blood found at
the site, 2) the blood of the victim, 3) the blood of John Kennedy
and 4) the blood of Gerald Goss were treated with the restriction
enzyme HindIII and placed in an electrophoresis gel in lanes 2
thru 5 respectively. Lane 1 was used for the DNA length
reference markers.

Teacher Southern Blot Instructions


Prior to the experiment the teacher needs to fill the Gel Buffer, Fluor Tag and
Wash tubes with water. The Fluor Tag tube can be filled with water with a small
amount (1-5 drops per gallon) of blue food coloring (VERY light blue) if you wish.
--------------------------------------------------------------------------------------------------------------------The Students Should Always Wear Gloves
1. Wet the sponge with water from the sink faucet.
2. Remove the nylon membrane from its plastic bag. Handle the nylon with forceps using the end with
the label.
3. Place the sponge into the plastic tray.
4. With your fingers, carefully remove the gel from its plastic bag and place it on the sponge.
5. Place the wetted nylon membrane face-down onto the gel with the printed part at the bottom of the
gel (the wells are at the top of the gel). Smooth out bubbles with a gloved finger.
6. Pour 50 milliliters of buffer (Gel Buffer) into the tray.
7. Place a two inch stack of paper towels on top of the membrane.
8. Let the DNA transfer proceed for 10-20 minutes.
9. Remove the paper towels.
10. Remove the nylon membrane with forceps using the end with the label.
11. Place the nylon membrane face up on a paper towel.
12. Put the gel back into its container and plastic bag.
13. Remove the sponge and empty the plastic tray.
14. Pour the tube of fluorescent probes (Fluor Tag) into the plastic tray.
15. Place the nylon membrane face down in the plastic tray.
16. Wait 5 minutes.
17. Remove the nylon membrane with forceps using the end with the label.
18. Place the nylon membrane face up on a paper towel.
19. Empty the plastic tray.
20. Pour the tube of wash buffer (Wash) into the plastic tray.
21. Place the nylon membrane face down in the plastic tray.
22. Wait 5 minutes.
23. Remove the nylon membrane with forceps using the end with the label.
24. Place the nylon membrane face up on a paper towel.
25. Examine the nylon membrane with a fluorescent light and fill in the Southern Blot data

Student Southern Blot Instructions


Wear Gloves
1. Wet the sponge with water from the sink faucet.
2. Remove the nylon membrane from its plastic bag. Handle the nylon with forceps using the
end with the label.
3. Place the sponge into the plastic tray.
4. With your fingers, carefully remove the gel from its plastic bag and place it on the sponge.
5. Place the wetted nylon membrane face-down onto the gel with the printed part at the
bottom of the gel (the wells are at the top of the gel). Smooth out bubbles with a gloved finger.
6. Pour 50 milliliters of buffer (Gel Buffer) into the tray.
7. Place a two inch stack of paper towels on top of the membrane.
8. Let the DNA transfer proceed for 10-20 minutes.
9. Remove the paper towels.
10. Remove the nylon membrane with forceps using the end with the label.
11. Place the nylon membrane face up on a paper towel.
12. Put the gel back into its container and plastic bag.
13. Remove the sponge and empty the plastic tray.
14. Pour the tube of fluorescent probes (Fluor Tag) into the plastic tray.
15. Place the nylon membrane face down in the plastic tray.
16. Wait 5 minutes.
17. Remove the nylon membrane with forceps using the end with the label.
18. Place the nylon membrane face up on a paper towel.
19. Empty the plastic tray.
20. Pour the tube of wash buffer (Wash) into the plastic tray.
21. Place the nylon membrane face down in the plastic tray.
22. Wait 5 minutes.
23. Remove the nylon membrane with forceps using the end with the label.
24. Place the nylon membrane face up on a paper towel.
25. Examine the nylon membrane with a fluorescent light and fill in the Southern Blot data
sheet.

Teacher Key
The High Rise Killer
DNA Marker Unknown

1
115 bp

109 bp

103 bp

95 bp

84 bp

75 bp

62 bp

54 bp

43 bp

30 bp

Victim

Suspect #1

Suspect #2

Student Worksheet
The High Rise Killer
DNA Marker Unknown

1
115 bp

109 bp

103 bp

95 bp

84 bp

75 bp

62 bp

54 bp

43 bp

30 bp

Victim

Suspect #1

Suspect #2