Lesson 8
DNA Fingerprinting
1
Insufficient evidence?
Read the following article.
Mr. Chan applied for permission for his son to
come to HK to live with him, he had to provide
evidence to prove his paternal relationship with his
son. He underwent the blood group typing tests
and obtained proof that his blood type matched
with that of his son. However, Mr. Chan was told
by the Immigration Department that the blood
group typing result was an insufficient evidence.
2
Insufficient evidence?
 Why was the blood group typing
result not sufficient evidence?
 Any other tests recommend?
3
Blood group typing is much less sensitive.
May use DNA fingerprint to provide
biological evidence as a proof in a
paternity test.
4
What is DNA?
5
Structural relationship among
chromosomes, DNA and genes
1. Cells are the basic building
blocks of all living things.
2. In a cell, DNA (deoxyribonucleic
acid) is packaged in
chromosomes within the nucleus.
3. DNA is a large, polymeric
molecule.
4. A gene is a segment of DNA
molecule of a chromosome. It is
the basic unit of heredity. Genes
determine the body
characteristics of an organism.
5. Each inherited characteristic is
controlled by one or several
genes.
6
Look into DNA
• DNA looks like an
incredibly long twisted
ladder. This shape is
called a double helix.
• The sides of the ladder
are a linked chain of 5carbon sugars and
phosphate (PO4) groups
(called the backbone).
• The rungs connected to
the 5-carbon sugars are
known as bases.
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DNA
• Each rung is made up of
two bases that link
together. There are four
bases - adenine (A),
thymine (T), guanine (G)
and cytosine (C).
• Because of their chemical
nature, A will only link
with T and G will only link
with C. No base can join
with itself (i.e. NO A-A /TT /G-G / C-C base-pairs).
8
Activity 8.1 What is DNA?
Refer to the Pre-lesson Reading, finish
Activity 8.1 to check your
understanding on DNA.
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Activity 8.1
1.Decide whether the following
statements are TRUE or FALSE.
a) There are 46 pairs of chromosomes in
each nucleus of a human cell.
b) Chromosomes are made of DNA and
proteins.
c) DNA determines the body characteristics
of an organism.
d) DNA may be extracted from red blood
cells found in a blood sample.
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Activity 8.1
e) The Hair and Teeth of the same
person are composed of same DNA
molecules.
f) Identical twins have different base
sequences of DNA .
g) In the DNA structure, adenine (A) will
only link with cytosine (C) and guanine
(G) will only link with thymine (T).
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Activity 8.1
Answers:
a. F
b. T
c. T
d. F
e. T
f. F
g. F
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Activity 8.1
2. DNA has two strands. And the
sequence of one strand determines the
sequence of the other. If the base
sequence on one strand of DNA is:
GATCCTCATA
What is the base sequence on the
other strand?
Answer:
GATCCTCATA
CTAGGAGTAT
13
Did you see DNA before?
14
Activity 8.2
DNA Extraction
1. Objective of experiment
To extract DNA from strawberries with
washing-up liquid.
2. Apparatus and Materials
•
•
•
•
washing-up liquid (detergent)
salt
ice-cold alcohol
ice bath
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Activity 8.2
3.
Follow the procedures to extract DNA.






Grind strawberries with a pestle in the mortar.
Add about 10 mL washing-up liquid, 1/2 teaspoon salt
and approximately 30 mL tap water to the grinded
strawberries. Stir the mixture gently for 1 minute.
Filter the mixture through a muslin cloth into a test
tube, about 1/3 full, to separate strawberries from the
clear liquid.
Double up the volume of the mixture by adding alcohol
slowly into the test tube.
Leave the mixture undisturbed for 2 – 3 minutes in an
ice-bath until no emergence of bubbles.
Swirl a toothpick around the interface of the two layers
formed inside the test tube. Long and stringy threads
can be picked up.
16
Activity 8.2 Questions (optional)
1.
What is the purpose of grinding the strawberries?
To break down the cell wall, cellular membranes and
nuclear membranes.
2.
Where is the DNA of the strawberries?
In the cell nucleus.
3.
What is the function of detergent in step 2?
Soap dissolves cell membranes so that DNA is
liberated.
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Activity 8.2 Questions (optional)
4.
What is the function of salt in step 2?
Salt solution helps the DNA strands come together.
5.
What happens when alcohol is added to the solution in
step 4?
DNA is soluble in water and will appear clear in water.
But DNA is insoluble in non-polar solvent, such as
alcohol. Therefore, DNA will precipitate (solidify and
appear) out of alcohol. Of course, this is not pure DNA.
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Follow-up Discussion
Write down your answers
on a piece of paper.
19
Activity 8.2
Follow-up Discussion Questions
1. Do you think your results would be different if
you use a vegetable or fruit other than
strawberries, say, onion, kiwi fruit, broccoli,
banana (without skin)? Explain.
2. Do different organisms have the same DNA?
3. At a crime scene what evidence would you
collect in order to extract DNA? List as many
as possible.
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Activity 8.2
Answers:
1. DNA can be easily extracted from many different plants.
The amount of DNA extracted depends upon many
factors, including the number of cells crushed, and
whether the cells can be easily broken apart.
2. Though all DNA molecules are made up of similar
chemical components, different organisms have different
DNA molecules due to the different base sequences of
the bases — A, T, G, and C, which makes a specific
organism have distinctive characteristics.
3. Blood (white blood cells), saliva, skin scalp, hair and etc.
Note that DNA is found in nearly every cell in the body. But
one significant exception is red blood cells (lack cell
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nuclei).
Where should you collect DNA
evidence?
22
Collection of DNA Evidence
1. Before the collection of evidence closeups of any biological evidence should be
photographed and its location relative to
the entire crime scene needs to be
recorded through notes, sketches, and
photographs.
2. All clothing from both the victim and
suspect should be collected and sent to
the laboratory for examination. Why?
23
Collection of DNA Evidence
Answer:
Because the criminal may have wiped his or
her hands on materials not readily
apparent to the investigator. In addition,
blood may exist in less-than-obvious
places.
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Activity 8.3 Collection &
Preservation of DNA Evidence
Guess (i) the possible locations of DNA on the collected evidence
from a crime scene and (ii) the possible sources of DNA.
Evidence
e.g. Eyeglasses
Possible locations of DNA on the
evidence
Ear pieces
Sources of DNA
Sweat, skin
Baseball bat
Facial tissue
Dirty laundry
Used cigarette
Blanket, pillow, sheet
Bite mark
Fingernail, partial fingernail
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Activity 8.3
Answers
Evidence
Possible locations of DNA
on the evidence
Sources of DNA
Eyeglasses
Ear pieces
Sweat, skin
Baseball bat
Handle
Sweat, skin, blood
Facial tissue
Surface area
Mucus, blood, sweat, semen
Dirty laundry
Surface area
Blood, sweat, semen
Used cigarette
Cigarette butt
Saliva
Bottle, can, glass
Sides, mouthpiece
Saliva, sweat
Blanket, pillow, sheet
Surface area
Sweat, hair, semen, urine, saliva
Bite mark
Person’s skin
Saliva
Fingernail, partial fingernail
Scrapings
Blood, sweat, tissue
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Preservation of DNA Evidence
• Paper bags or other “breathable” containers
should be used. Because biologic evidence
must never be packaged in airtight containers
because moisture can build up, which promotes
the growth of bacteria that can degrade DNA.
• All precautions against contamination must be
taken. These include
– wearing face marks, shoe covers and gloves;
– using tools such as tweezers to collect evidence.
27
Precautions in handling
biological evidence
• All body fluids must be assumed to be
infectious; hence, wearing disposable
latex gloves while handling the evidence is
required to prevent being infected.
28
Can DNA be cut?
29
Restriction Enzymes
• A common use of restriction enzymes is to generate
a "fingerprint" of a particular DNA molecule.
• DNA may be cut by some special naturally-occurring
proteins called restriction enzymes.
• Each restriction enzyme only identifies and “cuts” at
a very specific sequence (recognition site) in the
DNA strand.
• Restriction enzymes typically recognise a
symmetrical sequence of DNA, such as the site
GAATTC.
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Example 1

Read to the left
Read to the right

• The top strand is the same as the bottom strand
when you read backwards.
• When the enzyme EcoRI cuts the strand between
G and A, it leaves overhanging chains:
• These are termed "sticky ends" because the base
pairs formed between the two overhanging portions
will glue the two pieces together, even though the
backbone is cut.
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Example 2
Another symmetrical sequence of DNA, GGATCC:
GGATCC
CCTAGG
When the enzyme Bam HI cuts the strand
between G and G, it leaves overhanging chains:

G
GATCC
CCTAG
G

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Activity 8.4 Restriction Enzymes
•
In the worksheet, different DNA samples
with their restriction sites are given.
Answer the following questions:
 How many times does the specified base
sequence appear?
 How many fragments would result from a
restriction digestion of the DNA sample with the
given enzyme?
 List all the fragments formed.
33
Activity 8.4
Answers:
1.
a. The strand is cut into four pieces.
b. Fragments produced:
2.
Hae III,
Bam HI
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How to separate DNA fragments?
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Electrophoresis
• DNA fragments of different sizes can be
separated by using gel electrophoresis.
• DNA fragments carry negative charges.
When there is a current flow, DNA
fragments move towards the anode.
Source: Science Education Section,
CDI, EDB.
36
Outline of Electrophoresis
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Outline of Electrophoresis
1. DNA fragments are loaded into
“wells” in a gel. The gel floats in
a buffer solution within a
chamber between two
electrodes.
2. When an electric current is
passed through the chamber,
negatively charged fragments
move towards the positive
“wells”
terminal.
3. Shorter DNA fragments (smaller
size) move faster than the
longer ones (bigger size).
4. In a given time, DNA fragments
are separated into bands
according to their size.
Source: Science Education
Section, CDI, EDB.
Source: Science Education
Section, CDI, EDB.
38
Electrophoresis demonstration
• The steps for Electrophoresis are
demonstrated in the following video:
http://cd1.edb.hkedcity.net/cd/science
/biology/resources/L&t2/practical.htm
(Video Clips: DNA Gel Electrophoresis)
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DNA Analysis Technique
Restriction Fragment Length
Polymorphism (RFLP)
40
RFLP
•
•
It was the first commercial technique of
DNA analysis.
It can be used in paternity cases or
criminal cases to determine the source of
a DNA sample.
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RFLP
•
Steps
1. It uses restriction enzymes to cut DNA.
2. DNA fragments of different lengths are
produced.
42
RFLP
3. Using gel electrophoresis, the fragments are
separated so that fragments with different
lengths can be separated. This provides a
pattern of bands.
43
RFLP
4. Cover specific radioactive probes over the
gel. The probes contain a match for the
DNA sequence that the test is looking for.
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RFLP
5. Put a film under the gel to record. Allow signal
exposure in dark room.
6. DNA fingerprints are now ready for analysis and
comparison.
For animation, you may browse http://ihome.cuhk.edu.hk/~z045513/
(Virtual Lab.  Topic: 7. RFLP)
45
Another DNA Analysis Technique
To learn more, you may surf the following websites:
• Polymerase Chain Reaction (PCR)
（http://users.ugent.be/~avierstr/principles/pcr.html）
• Short Tandem Repeats (STRs)
（http://www.cstl.nist.gov/biotech/strbase/intro.htm）
• Mitochondrial DNA (mDNA)
（ http://ghr.nlm.nih.gov/handbook）
46
RFLP Vs PCR
Advantage of using RFLP (more often used in
forensic work):
 Can individualise a specimen to a narrow portion
of the population-possibly one person in billions
Disadvantage of using RFLP:
 Requires a larger sample than PCR; more timeconsuming and labour intensive; uses
radioactive reagents that require special lab.
Procedures.
47
PCR Vs RFLP
Advantages of using PCR:
Faster and simpler to use and may be
applied to exceedingly tiny samples-as
small as a billionth of a gram of DNA
Disadvantage of using PCR:
Results of PCR are less dramatic than
those of RFLP, being discriminatory on the
order of one individual in thousands rather
than the potential billions with RFLP
(Crime Science: Methods of Forensic Detection (p.203))
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What is the use of
DNA fingerprinting?
• More than 99% of the base sequence in
DNA is the same in all humans. Less than
1% is unique to each individual.
• Scientists make use of this difference to
generate DNA fingerprints which are
specific to different individuals.
49
Try to match two DNA fingerprints
50
Warm-up exercise
In the following simulation,
http://www.pbs.org/wgbh/nova/sheppard/la
b02.html
– decide which two fingerprints match each
other;
– drag the right-most DNA fingerprint over the
suspects’ fingerprints to find a match.
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Activity 8.5
DNA Fingerprint Analysis
Case 1
Mr. Chan’s family consists of mom, dad and four kids. The parents
have one daughter and one son together, another daughter is from the
mother’s previous marriage, and the other son is adopted. Here are
the DNA analysis results:
1. Which child is adopted? Why?
2. Which child is from the mother’s previous marriage? Why?
3. Who are the own children of Mr and Mrs Chan?
52
Activity 8.5
Answers:
• Child 4 is adopted.
• Child 2 is the child from the mother’s
previous marriage.
• Child 1 and Child 3 are own children of
Mr and Mrs Chan.
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Activity 8.5
Case 2
A blood sample from a crime
scene was collected. DNA
samples of the victim and the
potential suspects (June,
Scarlet and John) were also
collected for DNA analysis.
The DNA profile is shown.
Now, you should be able to
identify the potential murderer.
54
Activity 8.5
Answers:
• All of the DNA fragments of Scarlet can be
found in the crime scene sample making
her the most likely suspect.
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Activity 8.5
Extended discussions:
1. Both June and Scarlet have the same
DNA fragments (“8” and “12”), why?
2. Why DNA evidence must be combined
with the traditional forms of evidence
such as eyewitness accounts?
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Activity 8.5
1.
2.
Answers:
This pattern may arise if the two women are related or
if this pattern were common in population.
Someone’s DNA is found at a crime scene does not
mean that they committed the crime because of the
following reasons:
(i) The DNA sample may be contaminated by the
environment.
(ii) The sample may be a mixture of more than one
person’s DNA.
(iii) The DNA evidence may be degraded or broken
down.
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Activity 8.6
Ethical and Non-ethical Issues
Write down your answers
on a piece of paper.
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Activity 8.6 Group Discussions
1. Do white blood cells give more accurate test
result for paternity than cheek cells?
2. What are the legal and ethical issues on the
use of DNA fingerprinting in society?
3. DNA fingerprinting can be used to convict
someone of a crime. What would be the
difficulty in using this technique to identify
criminals if the suspects were father and son /
twin brothers?
4. Other than forensic science, list out the other
uses of DNA analysis. Describe each use
briefly.
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References
• Nickell, J. and Fischer, J.F. (1999). Crime
Science: Methods of Forensic Detection.
Kentucky: The University Press of
Kentucky.
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