YISHUN INNOVA JUNIOR COLLEGE

9744 H2 BIOLOGY
LECTURE NOTES H2
CORE IDEA 2: GENETICS AND INHERITANCE
TOPIC 2.7: DNA MOLECULAR TECHNIQUES

Learning Outcomes:

(k) Describe the principles and procedures of these molecular techniques:

i. polymerase chain reaction (including advantages and limitations)
ii. gel electrophoresis
iii. Southern blotting and nucleic acid hybridisation

References:
Reece, J. B., et al. (2011). Campbell Biology (9th Ed), Chapter 20: Biotechnology, pp449 – 453

Note: This textbook is available in our library. You may wish to borrow them to supplement your reading when
necessary.

1) Introduction

2) Polymerase Chain Reaction

 Reagents
 Process
 Advantages
 Limitations

3) Gel Electrophoresis
 Agarose Gel Electrophoresis
 Polyacrylamide Gel Electrophoresis (PAGE)

4) Visualisation of DNA
 Southern Blotting
 Nucleic Acid Hybridisation
1 INTRODUCTION

 Biotechnology has led to recent advancements in genetic engineering, especially in agriculture and
medicine. Molecular techniques involving DNA manipulation are often employed to achieve these
goals.

 Techniques in DNA technology can be used in processes such as:

– genomic mapping (e.g. linkage mapping),
– disease detection (e.g. sickle-cell anaemia) and
– DNA fingerprinting to identify individuals and establish relationship.

2 POLYMERASE CHAIN REACTION

Learning Outcome 2(k)(i):

Describe the principles and procedure of polymerase chain reaction (including advantages and
limitations).

– PCR is an in vitro (outside the cell) method of replicating (amplifying) relatively short DNA
sequences into millions of copies over a short period of time. It is an automated process used when
a target DNA sequence needs to be amplified.

2.1 Reagents

– A PCR reaction requires a pair of primers, Taq polymerase, free deoxyribonucleoside triphosphates
(dNTPs), the target DNA sequence to be amplified and a buffer solution containing Mg2+.

(i) Target DNA Sequence

– The target sequence to be amplified is usually ~1 to 2 kb in length.

(ii) Primers
– Primers are short DNA sequences (single-stranded oligonucleotides) approximately 20 – 30 bp
in length.

– Primers are synthesised in the laboratory and are designed to be specifically complementary
to DNA sequences flanking / at the ends of the target sequence to be amplified. Thus, the
nucleotide sequence flanking the target DNA sequence must be known for primer design.

– Two different primers, forward and reverse primers, are required for PCR.

Primers serve to:

1. mark out the section of DNA to be amplified by attaching to complementary bases on the
3’ ends of the target DNA sequence via formation of hydrogen bonds between
complementary base pairs;

2. provide a free 3’-OH end for DNA polymerase to replicate the target sequence by elongating
the DNA strand in the 5’ to 3’ direction. This allows the replication of the double-stranded
DNA molecule.
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 Fig. 2.1.1: Use of primer to initiate PCR process

– Primers are present in large excess in reaction mixture to increase likelihood of primers
binding to target DNA (decrease likelihood of template DNA strands reannealing to each other
again).

– The primers become part of the amplified segments.

CHECKPOINT (1)

1. Deduce the sequence of two 10-nucleotide long primers that can be used to amplify the complete
target DNA segment.

Forward primer:

Reverse primer:

2. What is the difference between the primers used in DNA replication in cells and that used in PCR?

(iii) DNA Polymerase

– A DNA polymerase derived from a thermophilic bacterium, Thermus aquaticus (Taq polymerase)
is used in PCR. Taq polymerase is thermostable (i.e. resistant to denaturation at high
temperatures), with an optimum temperature of ~72C. This is important as the PCR process
requires repeated cycles of heating and cooling.

– Taq polymerase binds to the 3’ OH end of the primer, adding deoxyribonucleotides

complementary to the target sequence, to elongate the DNA strand.

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 CHALLENGE YOURSELF!

With reference to your knowledge of protein structure, what structural features do you expect to find in
Taq polymerase?

(iv) Free deoxyribonucleoside triphosphates (dNTP) / DNA nucleotides

– The four types of deoxyribonucleoside triphosphates, dATP, dTTP, dGTP and dGTP, are added
in approximately equal proportions in excess. They served as substrates for Taq polymerase
in the polymerase chain reaction.

(v) Buffer containing Mg2+

– Mg2+ is a cofactor for proper Taq polymerase function.
– The buffer solution maintains pH and ionic strength of the reaction solution suitable for the
activity of Taq polymerase.

2.2 Process

 The process involves a 3-stage cycle of denaturation, annealing and elongation which results in an
exponential increase in the number of copies of the target DNA molecule of identical sequences.

 PCR reagents are added in a reaction tube and PCR is carried out in a thermal reactor, known as
a thermal cycler, which automates the timing of each step in the amplification cycle.

Stage 1: DNA denaturation

– The target DNA sample and required
reagents are heated to ~ 95°C, to
denature and separate the double-
stranded (ds) DNA into single-stranded
(ss) DNA.

– Heating increases kinetic energy of DNA

molecules, resulting in breaking of
hydrogen bonds between
complementary base pairs.

Stage 2: Annealing
– The mixture is then cooled to ~ 55°C in the
presence of excess primers.

– Forward and reverse primers would

bind to complementary sequences
flanking the target DNA sequence via
hydrogen bonds.

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– Due to presence of excess primers, the
probability of primer annealing is higher
than the DNA target sequence renaturing.

Stage 3: Elongation
– The temperature is raised to 72°C.

– Taq polymerase synthesizes the

complementary DNA strand in the 5’ to 3’
direction by adding free dNTPs / DNA
nucleotides to the free 3' OH end of the
primers, using the target DNA sequence
which acts as template.

– Taq polymerase catalyses the formation

of a phosphodiester bond between the
adjacent deoxyribonucleotides. The
hydrolysis of phosphate bonds in the
5' phosphate groups of incoming dNTPs
releases energy required for this reaction.

Fig. 2.2.1: The PCR cycle

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 After the first cycle, the same process (denaturation, annealing, elongation) is repeated 20 to 30
times in an automated thermal cycler.

TRY THIS
OUT!
Use the QR
code to access
the PCR Virtual
Lab Simulation

Fig. 2.2.2: PCR process

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 Each cycle results in the doubling of the target DNA sequences. Thus, number of copies of
dsDNA is 2n where, n is the number of cycles.

Fig. 2.2.3: Exponential increase in DNA copies

CHALLENGE YOURSELF!

With reference to Fig. 2.2.3, why are there longer than normal PCR products (i.e. not the desired PCR
product) present in the first few PCR cycles?

CHECKPOINT (2)
Complete the table to show the number of DNA molecules (double-stranded) and DNA strands (single-
stranded) after each PCR cycle.

Number of target Number of DNA

Cycle
DNA molecules (ds) strands (ss)
0 1 2

30

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2.3 Advantages

 Rapid and efficient

– Each cycle takes only 3 - 5 minutes. Thus, a PCR reaction involving 30 cycles of denaturation,
annealing and elongation can be completed in a few hours, yielding a large number of target
DNA molecules.

 Ease of use and automation

– PCR can be performed using relatively simple equipment, a thermal cycler. Reagents are
added to PCR reaction tubes in appropriate amounts and conditions (e.g temperature, duration,
number of cycles) are set. The use of thermostable Taq polymerase which is able to withstand
high temperatures without being denatured allows the reaction process to be fully automated
and the cycles can run unattended. There is no need to replace the enzyme after every cycle.

 Sensitive and robust

– The process is sensitive and can amplify minute amounts of target DNA. This is especially
useful in forensic work where DNA may not be present in large quanties.

– PCR is a robust process which can amplify DNA sequences from badly degraded material or
even DNA embedded in material that is difficult to extract, as long as a few molecules contain
the complete target sequence. DNA fingerprints can thus be prepared from cells in a drop of
blood or from the root and shaft of a single human hair.

 Specific
– As primers base pair via hydrogen bonds only to complementary sequences that flank the target
DNA sequence, PCR is a specific process which amplifies only target sequence

 Relatively high fidelity

– The amplification is relatively accurate with error rates ranging between 1 in 10,000 bases to
1 in 100,000 bases. Error rates vary with the choice of DNA polymerase used.

2.4 Limitations

 Primer design
– The specificity of the amplification process is dependent on specificity of the primers. Thus, the
knowledge of the base sequence flanking the target sequence is required in order to
synthesize specific primers.

– Primers that are short are easier to synthesize but tend to bind to multiple sites along the DNA
molecule. Long primers are more specific but their synthesis is more difficult and costly.

 Limited length of target sequence

– The length of target DNA restricted to 0.1 to 5 kb with an optimum length of 2 to 3 kb. A further
increase in length of the target sequence decreases efficiency of amplification because
polymerase tends to detach from the DNA template before chain elongation is complete.

 Error in replication
– Taq polymerase lacks proofreading activity. This results in an error rate of approximately 1 in
10,000 bases. If the error occurs early in the PCR cycle, the erroneous sequence will be
amplified together with the target sequence, resulting in all daughter molecules resulting from
this early error being exponentially affected.

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3 GEL ELECTROPHORESIS

Learning Outcome 2(k)(ii):

Describe the principles and procedure of gel electrophoresis.

3.1 Principles of Gel Electrophoresis

 Electrophoresis is the movement of charged molecules in an electric field.

 Biological molecules such as DNA, RNA and proteins exist in solution as electrically-charged
particles at a given pH. DNA, for example, is negatively-charged due to phosphate groups of sugar-
phosphate backbone.

 When placed in an electric field, DNA molecules which are negatively-charged will move from
the negative electrode (cathode) and towards the positive electrode (anode).

 A meshwork of polymer fibers that makes up agarose gel impedes movement of the DNA fragments,
affecting the longer fragments more than shorter ones. Therefore, shorter DNA fragments will
move towards the positive electrode at a higher rate than longer fragments.

 Because all DNA molecules are negatively charged, the rate of DNA migration and separation
through an agarose gel depends on the size (molecular length) of a DNA molecule. Migration
distance is inversely proportional to the molecular size of a DNA fragment.

3.2 Agarose Gel Electrophoresis

 Agarose gel electrophoresis is used in the separation of DNA fragments.

Process:

1. Agarose powder is dissolved in Tris-Borate-EDTA (TBE) buffer and poured into the gel casting
tray. A comb is inserted to form wells.

2. Agarose is left to cool and harden forming an agarose gel (of 0.8% to 2%).

3. The comb is removed.

4. The gel is placed into a gel chamber and submerged in TBE buffer. The buffer maintains
appropriate pH and contains ions that conduct a direct current across the gel.

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 Fig. 3.2.1: Preparation of gel

5. DNA molecules are mixed with loading dye and tracking dye, and loaded in wells of the
agarose gel near the cathode (negative electrode) using a micropipette.

– The loading dye contains glycerol that helps weigh the DNA fragments into the wells.

– The tracking dye contains a low and a high molecular weight coloured compound,
acting as front and back markers of migration as the migrating DNA fragments are not
visible to the naked eye. The high molecular weight compound migrates behind the largest
DNA fragment while the low molecular weight compound migrates ahead of the smallest
DNA fragment.

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 Fig. 3.2.2: Gel electrophoresis

Fig. 3.2.3: Side view of gel chamber

6. A DNA ladder is loaded usually in the first well of the gel. The DNA ladder is a mixture of DNA
fragments of known sizes which acts as a standard for comparison with DNA fragment of
unknown sizes (fragment length) in the sample.

Fig. 3.2.4: Use of DNA ladder for estimation of DNA fragment length
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7. A voltage (between 90V to 150V) is applied across the gel.

When the current is turned on, the negatively-charged DNA fragments migrate out of the well
into the agarose. DNA is attracted towards the positive electrode (anode) due to negative
charges on its sugar phosphate backbone.

The gel acts as a molecular sieve that separates the DNA fragments by size (molecular weight)
and shape. Shorter DNA fragments are less impeded by the gel and migrate faster than longer
DNA fragments.

Fig. 3.2.5: Agarose gel acts as molecular sieve to separate DNA fragments based on size

8. After gel electrophoresis, DNA fragments of the same sizes are localised in the same region
of the gel forming a discrete band. However, DNA molecules are not visible to the naked eye.
Thus, the gels need to be stained for the DNA bands to be seen.

The electrophoresed gel is submerged in stains such as:

 methlyene blue, a safe to use but low sensitivity dye
 ethidium bromide (EtBr), a higher sensitivity dye but is a carcinogenic chemical which binds
to DNA and fluorescence under UV light.

However, such methylene blue and EtBr stains all DNA present in the gel. If only specific DNA
fragments are needed, Southern blot and nucleic acid hybridisation can be used instead.

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 CHECKPOINT (3)

(a) Label the cathode and anode.

(b) Indicate the direction of movement of DNA fragments.
(c) Label the longest and shortest DNA fragments.
(d) If well #1 contained DNA found at a crime scene, and wells #2 – 4 contained DNA from suspects,
which suspect was at the crime scene?

3.3 Polyacrylamide Gel Electrophoresis (PAGE) – For enrichment only

 Agarose is frequently used in gel electrophoresis as it is easy to use, relatively cheap, and non-toxic.
However, it has relatively low resolving power i.e. DNA fragments of very similar sizes cannot be
separated in an agarose gel.

 Polyacrylamide, on the other hand, has higher resolution. It is capable of separating DNA fragments
differing by one base pair. However, polyacrylamide is more difficult to use, relatively more
expensive than agarose and polyacrylamide solution is a neurotoxin. Thus, PAGE is usually used
to separate DNA molecules for purposes such as DNA sequencing which requires resolution up to
1 base pair while agarose gel electrophoresis is used for most other applications.

 PAGE is also commonly used for separating proteins of different molecular weight.

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4 VISUALISATION OF DNA

Learning Outcome 2(k)(iii):

Describe the principles and procedure of Southern blotting and nucleic acid hybridisation.

 Staining the electrophoresed gel with methylene blue or EtBr allows us to visualize all DNA bands
in the gel. However, if we want to locate a particular DNA fragment, Southern blotting and nucleic
acid hybridisation is used.

(i) Southern blotting is a method of transferring denatured DNA from a gel after electrophoresis
to a membrane.

(ii) After immobolizing the DNA onto the membrane, nucleic acid hybridisation is performed to
detect specific sequences, where a labelled DNA probe binds to the target sequence via
hydrogen bonding between complementary base pairs.

Fig. 4.1: Nucleic acid hybridisation and Southern blotting

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4.1 Southern Blotting

Principle of Southern Blotting

 A replica of the DNA bands obtained using agarose gel electrophoresis is made by transferring (or
blotting) the DNA on the gel onto a membrane made of nitrocellulose or nylon.

– The double-stranded DNA molecules must be denatured before hybridisation with the probe.
This is done either before or during the blotting process by exposing the gel to alkaline
denaturing conditions.

Process:

1. A sheet of nitrocellulose or nylon membrane / paper is laid over the electrophoresed gel
containing separated DNA fragments.

2. The gel is supported on a layer of sponge in a bath of alkaline solution.

3. Paper towels are stacked on top of the nitrocellulose membrane to wick alkaline solution
through the gel and nitrocellulose membrane by capillary action.

4. As the alkaline solution passes through the gel, it denatures the DNA fragments from double-
stranded DNA to single-stranded DNA (ssDNA). It also transfers the ssDNA fragments from
the gel to the nitrocellulose membrane. The ssDNA fragments bind to the membrane in the
same exact position as they were in the gel.

5. The membrane is then baked in an oven at 80°C for 2 hours or exposed to UV to fix the DNA
fragments firmly onto nitrocellulose membrane.

4.2 Nucleic Acid Hybridisation

Principle of Nucleic Acid Hybridisation

 The nitrocellulose / nylon membrane is incubated in a solution containing labelled, single-

stranded DNA probes.

 Single-stranded DNA fragments that are complementary and thus able to specifically hybridise
to the labelled DNA probe by complementary base-pairing, are located by detecting the bound
probe using autoradiography or by fluorescence means.

 The size of the DNA fragment that binds to the labelled probe can be determined by referring to
the DNA molecular weight marker that was electrophoresed together with the DNA sample during
gel electrophoresis.

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Process:

1. The nitrocellulose membrane is peeled off the gel and placed in a sealed plastic bag of solution
containing radioactive DNA probes. DNA probes are short fragments of deoxyribonucleic acid
with base sequence complementary to the target DNA fragment.

2. The mixture is incubated to allow radioactive probes to hybridise with target DNA sequence via
complementary base pairing by forming hydrogen bonds.

Fig. 4.2.1: Nucleic acid hybridisation

3. The nitrocellulose membrane is removed from the bag. Excess, unbound probes are washed
away.

4. The nitrocellulose membrane is subjected to autoradiography by exposing it to photographic

film. The DNA band that has hybridised to the radioactive probe will show up as dark bands on
the autoradiograph.

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Fig. 4.2.2: Southern blotting and nucleic acid hybridisation after gel electrophoresis

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Example: Sickle-Cell Anaemia

 Sickle cell disease is a recessively inherited genetic disorder. Glu is replaced by val at the 6th
position of the -globin chain of the haemoglobin molecule due to a A  T substitution.

 The DNA triplet code (i.e. on the coding sequence / 5' to 3' DNA strand) GAG, coding for glu, is
changed to GTG, coding for val, resulting in single nucleotide polymorphism.

 This results in the loss of a MstII restriction site (CCTNAGG) in the sickle cell allele.

DNA A S A A
ladder Hb Hb Hb Hb HbSHbS

1.5 kb

1 kb

0.5 kb

Fig. 4.2.3: Banding pattern of individuals with sickle cell and normal allele

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 CHECKPOINT (4)

The positions of the restriction sites for a restriction enzyme R in the normal and the mutated alleles of
a gene are shown in Fig. 1 below:

(a) Draw the location of the bands in the gel below when the DNA samples of two individuals:
1. a homozygote having 2 copies of the normal allele and
2. a homozygote having 2 copies of the mutant allele of this gene
was digested with restriction enzyme R and separated by gel electrophoresis.

(b) Label the band(s) to which the probe 1 will hybridise to with ‘probe 1’.

(c) Label the band(s) to which the probe 2 will hybridise to with ‘probe 2’.

(d) On Fig. 1, draw the location of a third probe (‘Probe 3’) which would give the same banding
pattern for both normal and mutant samples and hence will not able to differentiate between
both samples.
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