The Chinese University of Hong Kong
Department of Biochemistry
DNA Fingerprinting and Agarose Gel Electrophoresis 1
Prof. S.K. Kong, Prof. Stephen K.W. Tsui and Patrick Law
OBJECTIVES
To generate and analyze DNA fingerprint patterns from DNA samples taken from members of a family.
After performing this experiment, students should be able to:
1. know that VNTR is a kind of genetic polymorphism;
2. explain how VNTR can be employed for paternity testing;
3. realize the power of PCR reaction; and
4. perform the agarose gel electrophoresis which separates DNA molecules based on molecular
weight differences.
A) Introduction
The Human Genome and Genetic Polymorphism
Genetic polymorphism refers to the differences in genome DNA sequences among
individuals. Human genome is estimated to have 3 billion base pairs. In which, less then 5%
codes for functional proteins (the functional genes). Most of the remaining DNA has no clear
function and is called ‘junk DNA’. ‘Junk DNA’ contains many repeat sequences. One kind of
repeat sequence, VNTR (variable number of tandem repeats), has its repetitive sequence
elements arranged in tandem only at certain sites. They are a rich source of genetic
polymorphism: the number of repeats in a given VNTR site varies widely in the population.
We can consider the VNTR site a locus with multiple ‘alleles’ (various number of repeats) and
use it to analyze pedigree.
A common example of VNTR is a tandem repeat family called D1S80, which located
on chromosomal 1 with a repeating sequence of 16 base pairs (GAG GAC CAC CGG CAA G).
Below shows an example of a D1S80 locus. There are 21 and 15 repeating units on the
fraternal and maternal chromosomes, respectively. Those repeating sequences are usually
bracketed by regions of conserved sequences, called flanking sequences.
Repeating sequence
Fraternal
Flanking sequence
Maternal
PCR primer
16 bp
bp
If we examine a particular VNTR locus of a family, say father has repeat numbers 12
and 18 (12, 18) and mother (15, 30), the offspring should have one of the following pattern: (12,
15), (12, 30), (15, 18) or (18, 30). However, there is chance that an unrelated individual bears
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This manual is available for download at http://www.bch.cuhk.edu.hk/teaching/manual.doc
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the same offspring VNTR repeat pattern in the population. To reduce the occurrence of false
positive, we always examine several VNTR loci in paternity testing. Below is an example of
studying three VNTR loci: X, Y, Z of a family. Do you know who is the adopted child?
Locus X Locus Y
Chromosome A
Locus Z
Chromosome B
X Y Z
Father 10, 14 18, 24 8, 9
Mother 12, 20 22, 22 8, 15
Son 10, 20 22, 24 8, 8
Daughter 12, 14 22, 22 26, 9
Polymerase chain reaction
To detect the difference of the tandem repeat length at a given locus between
individuals, we need to go through a series of procedures:
1) DNA sample collection;
2) undergo Polymerase Chain Reaction (PCR) to amplify the specific tandem repeat
sequences;
3) perform Agarose Gel Electrophoresis to separate the amplified DNA sequences based
on their size; and
4) Visualize the separated DNA pattern by staining dye.
Cheek cells, hair root follicle cells and blood cells are common sources for DNA
extraction. The DNA obtained from these tissue samples is in very minute quantity. The DNA
regions of interests are then selectively amplified by PCR, a revolutionary technique developed
by Dr. Kary B. Mullis at 1985. Dr. Mullis was awarded the Nobel Prize for this invention. The
idea of PCR is simple. It starts with a reaction tube containing (1) the template DNA, (2)
primers (short synthetic oligonucleotides that are complementary to the two ends of the DNA
sequences to be amplified), (3) thermostable DNA polymerase and (4) nucleotides.
The reaction only comprises of three steps:
Double stranded target DNA
Step 1 : Denaturation
Two DNA targets available for PCR
95oC DNA denatured
PCR
amplification
cycle
Step 2 : Primer Annealing Step 3 : DNA Extension
~55oC Primers bind to target DNA 72oC Double stranded DNA duplicated
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These three steps together are called a cycle, which is repeated 20 to 40 times. DNA
amount is doubled in each cycle. The total amount of DNA you will obtain after a PCR
reaction is described by the formula
2n x C
where n = number of PCR cycles; and
C = the initial number of copies of DNA template present in the tube.
So, you will get 1,048,576 copies of DNA after 20 cycles of PCR reaction even you start with
only one copy of DNA template initially. What a powerful reaction!
Agarose Gel electrophoresis
DNA is negative in charge due to its sugar-phosphate backbone. Therefore, DNA will
move towards the positive cathode in the presence of an electric field. Agarose is a chain of
sugar moleucles extracted from seaweed. If we dissolves agarose in boiling water and let it
cools down, the sugar chains crosslink with each other (a process called polymerization),
creating ‘pores’ in between, and finally forms a semi-solid gel-like matrix. Those ‘pores’
present in agarose gel are very tiny and comparable in size to DNA molecules. Agarose gel can
confer a sieving effect when DNA is passing through. Agarose gel electrophoresis is a process
that separate DNA fragments by applying electricity to cause DNA passing through an agarose
gel. DNA fragments shorter in size will move faster and longer DNA fragments will lag
behind. The distances moved by linear DNA molecules are inversely proportional to the log10
of their molecular size.
DNA molecule is colourless. A staining step is needed so that it can be visualized on
an agarose gel. Ethidium bromide is a routine chemical used to stain DNA. Once it
intercalated into the DNA molecule, it emits orange fluorescent when irradiated by ultraviolet
light. Ethidium bromide is carcinogenic, however. We will use methylene blue, a safer
staining dye, in this experiment.
Paternity Test
In this experiment, you are given six DNA samples and one DNA marker to perform
agarose gel electrophoresis. Each sample contains artificially generated DNA fragments that
simulate DNA tandem repeat fragments amplified from three VNTR loci by PCR method. The
DNA fragments resolved on the gel can be viewed as a fingerprint pattern of a particular
individual. Assuming all the samples came from a family with adopted child and stepchild, you
are required to find out the true biologic child/children from the DNA fingerprint patterns on
your gel.
B) Materials
Apparatus
i. Electrophoresis unit
ii. Power pack
iii. 1 ml syringe fitted with plastic tip
iv. Microwave oven
v. 250 ml conical flask
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Direction for
DNA migration Anode (+)
Wells for sample
loading Warning: High Voltage!
Agarose slab gel
Cathode (-) submerged in buffer
Figure 1. Agarose gel electrophoresis for separation of DNA fragments. [Adapted
from D.W.S. Wong (1997) The ABCs of Gene Cloning. Chapman and Hall, pp 80]
Preparation 1X TBE buffer (500ml)
To prepare 500ml of 1X TBE buffer, mix 50 ml 10X TBE buffer with 450ml distilled
water and mix well.
Agarose Gel Preparation (1.5%, 60mL)
i. Agarose powder, electrophoresis grade, 0.9 g
ii. 1X Tris-borate (TBE) buffer, 60 mL
(Note: Working solution of TBE buffer contains 0.089M tris-borate, 0.89M boric acid
and 0.02M EDTA)
Gel Electrophoresis of DNA
i. 6X gel-loading buffer
(Note: This buffer contains 0.2% bromophenol blue, 0.2% xylene cyanol, 60% glycerol
in H2O and 60mM EDTA.. Bromophenol blue and xylene cyanol are tracking dyes.
Glycerol can increase the density of the loading samples.)
ii. DNA Samples
a. M (Mother)
b. F (Father)
c. D1
d. D2
e. S1
f. S2
g. MK
iii. 1X TBE buffer, 300 mL
C) Procedure
(I) Preparation of Agarose Gel for Electrophoresis of DNA
1. Prepare gel casting unit by sealing both ends with tape.
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2. Dissolve 0.9 g agarose powder in 60 mL 1X TBE buffer in a 250 ml conical
flask with a loose-fitting cap. The buffer should not occupy more than 50% of
the volume of the flask.
3. Heat the slurry in a microwave oven (or a hot plate) until the agarose dissolves.
(Caution: the agarose solution can become superheated and may boil violently if it has
been heated for too long.)
4. Cool the solution to 70oC. Pour the gel immediately into the gel casting unit
placed horizontally on the bench. Place the comb and check there are no air
bubbles under or between the teeth of the comb.
5. Allow the gel to set at room temperature for at least 30 minutes.
II)Gel Electrophoresis of DNA
6. After the gel is completely set (30-45 minutes at room temperature), carefully
remove the comb and sealing tape, and mount the gel in the electrophoresis
tank.
7. Add just enough electrophoresis buffer to cover the gel to a depth of about 1
mm. Care should be taken not disturbing the loaded DNA samples.
(Note: It is important to use the same batch of electrophoresis buffer in both the
electrophoresis tank and the gel.)
8. Slowly load the DNA samples into the wells of the gel using a 1 ml syringe
fitted with plastic tip. Wash the syringe with buffer between successive loading.
MK is the DNA marker containing DNA fragments of known size.
9. Close the lid of the gel tank and attach the electrical leads so that the DNA will
migrate toward the anode (red lead).
10. Apply a voltage of about 80V. If the leads have been attached correctly, bubbles
should be generated at the electrodes.
11. Run the gel until the dye front migrated to 1 cm from the gel end (~ 60 mins).
12. Turn off the electric current and remove the gel from the gel tank.
13. To visualize the DNA, stain the gel overnight with appropriate volume of 1X
methylene blue staining solution. (Note: To make 1X staining solution, mix 1 ml
of 100X methylene staining solution with 99 ml of distilled water. It is important
to make sure that the gel is completely immersed in the staining solution)
D) Results
MK M F D1 D2 S1 S2
Lane MK: DNA marker
Lane M: Mother
Lane F: Father
2100 bp
1700 bp
Lane D1: Daughter 1
Lane D2: Daughter 2
1100 bp Lane S1: Son 1
850 bp
Lane S2: Son 2
600 bp
250 bp
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Question 1 : Analysis of DNA fingerprint patterns
Analyse the fingerprint patterns on your gel and fill in the following blanks.
D1: _______________________ (Biologic child / adopted child / stepchild)
D2: _______________________ (Biologic child / adopted child / stepchild)
S1: _______________________ (Biologic child / adopted child / stepchild)
S2: _______________________ (Biologic child / adopted child / stepchild)
Question 2 : Construction of calibration curve
If the‘log10 nucleotide pairs’ is plotted against the ‘distance migrated’ based on the markers, a
calibration curve will be obtained and the size of the DNA fragments can be determined by this
curve. (Note: The ‘distance migrated’ is the distance measured between the well and the DNA
fragment)
log10 nucleotide pairs
Distance migrated (mm)
Measure the distances migrated by the MK and S2 DNA fragments. (Distance between the
well and the band.) Then, construct a calibration curve based on MK and estimate the DNA
fragment sizes of S2.
Sample Fragment no. Fragment size Distance migrated (mm)
MK 1 2100 bp _____________
2 1700 bp _____________
3 1100 bp _____________
4 850 bp _____________
5 600 bp _____________
6 250 bp _____________
S2 1 _________ _____________
2 _________ _____________
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3 _________ _____________
4 _________ _____________
A calibration curve of log10 nucleotide pairs against distance migrated (mm)
log10 nucleotide pairs
Distance migrated (mm)
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