DNA PCR
In 1983, the Cetus scientist Kary Mullis developed an ingenious “in vitro”
nucleic acid amplification technique termed the polymerase chain reaction (PCR). Since its
conception in the mid-1980s, it has rapidly become a routine procedure in every molecular
biology laboratory for identifying and manipulating genetic material, from cloning,
sequencing, mutagenesis, to diagnostic research and genetic analysis. What’s astounding
about this invention is that new and innovative applications of PCR have been generated with
stunning regularity; its potential has shown no signs of leveling off. New applications for
PCR are literally transforming molecular biology. In the postgenomic era, PCR has especially
become the method of choice to clone existing genes and generate a wide array of new genes
by mutagenesis and/or recombination within the genes of interest. The fast and easy
availability of these genes is essential for the study of functional genomics, gene expression,
protein structure-function relationships, protein–protein interactions, protein engineering, and
molecular evolution. Reverse-transcriptase-polymerase chain reaction has become one of the
most widely applied techniques in biomedical research. The ease with which the technique
permits specific mRNA to be detected and quantified has been a major asset in the molecular
investigation of disease pathogenesis. Disease related imbalances in the expression of specific
mRNAs can be sensitively and quantitatively determined by real-time (RT)-PCR. RT-PCR
also offers many opportunities in diagnostics, allowing sensitive detection of RNA viruses
such as Human imunodeficiency Virus (HIV) and Hepatitis C Virus (HCV).
This technique involves the use of a pair of short (usually 20 bp long) pieces of
synthesized DNA called primers and a thermostable DNA polymerase to achieve nearexponential enzymatic amplification of target DNA. The starting material for PCR may be
DNA from a variety of sources such as blood, tissues, paraffin-embedded material, ancient
archaeological samples, or forensic material. The PCR may also be used to amplify RNA,
which must first be converted into cDNA by the enzyme reverse transcriptase (RT-PCR). In
contrast to DNA, great care must be taken in the preparation and handling of RNA because of
its instability and susceptibility to degradation. Polymerase chain reaction proceeds in three
stages:
1. Denaturation of double-stranded DNA. (This initial denaturation step is not
necessary when amplifying RNA because it is a smaller target molecule.)
2. Primer annealing.
3. Extension of the annealed primers.
The target DNA is suspended in a reaction mixture consisting of distilled water, buffer
(containing MgCl2, which is necessary for the polymerase to efficiently), the thermostable
Taq polymerase, and each of the four deoxynucleotide triphosphates (dNTPs). Also present
are a pair of primers whose sequences are complementary to that of the DNA flanking the
target region. The reaction mixture is first heated to denature the double-stranded DNA into
single strands, then cooled to an optimum temperature to facilitate primer annealing. The
primer pair consists of a forward “sense” primer that binds to its complementary sequence
upstream of the region to be amplified and a reverse “antisense” primer that binds
downstream, both with their 3' ends facing inward. During primer extension the DNA
polymerase progressively adds dNTP’s, complementary to the target, to the 3' end of each
primer so that the target sequence is copied. The 5' end of the primers defines the length of the
PCR product. These three steps constitute a PCR cycle. Usually, 30 cycles are performed in a
programmable thermal cycler, with each cycle theoretically doubling the quantity of target
sequence.
PCR protocol
Note: Always wear, and frequently change, gloves to prevent contamination by DNA
from human skin cells. As likely sources of contamination include other samples and previous
amplification products, separate rooms, away from sites of DNA/RNA extraction,
manipulation, and recovery of PCR products, should ideally be allocated for the setup of PCR.
Designate separate sets of pipets for DNA and RT-PCR and use filter tips whenever possible.
Solutions and buffers must be stored in sterile containers and always prepared with fresh
distilled water.
1. Into a sterile tube, prepare the following master mix for 2 samples to be analyzed
(positive sample and blank control):
Deionized water – 73 µL,
10x reaction buffer -10 µL,
1.5 mM MgCl2 – 6 µL,
200 µM of dNTPs – 2 µL,
10 pmol forward primer (F) - 2 µL,
10 pmol reverse primer (R) - 2 µL,
0.5 µL (2.5 U) Taq polymerase - 1 µL.
2. Aliquot 48 µL of master mix into a PCR reaction tube for positive sample. Add 2
µL cDNA to give a final volume of 50 µL. PCR may be done in much smaller volumes down
to 10 µL, especially for analytical PCR, by reducing the reaction components proportionately.
3. Perform a blank control alongside the sample by replacing the genomic DNA with 2
µL of dH2O. This will check for contamination.
4. Overlay the reaction mixture with 1 drop of mineral oil to prevent evaporation,
unless using an “oil-free” thermal cycler that has a heated lid to prevent evaporation.
5. Denature the template for 5 min at 94°C.
6. PCR at the following settings for 30 cycles:
a. Denaturation: 94°C for 1 min.
b. Annealing: 62°C for 1 min (adjust temperature according to the calculated Tm of the
primers).
c. Extension: 72°C for 1 min (optimum temperature for Taq polymerase activity).
d. Final extension: 72°C for 10 min.
7. Analyze 20 µL of the sample on a 2% agarose gel.
Agarose gel electrophoresis
Electrophoresis through agarose or polyacrylamide gels is a standard method used to
separate, identify, and purify nucleic acids. The technique is simple, rapid to perform and
capable of resolving fragments that differ by as little as 0.2% in size. Electrophoresis occurs
under the influence of an electric field: Charged molecules such as nucleic acids migrate in
the direction of the electrode having the opposite charge (anode). The electrophoretic mobility
of nucleic acids is determined by a number of parameters, but molecules of linear doublestranded DNA migrate through gel matrices at rates that are inversely proportional to the
log10 of the number of base pairs and therefore larger molecules migrate more slowly
because of the greater frictional drag. Other factors affecting electrophoretic mobility include
base composition, concentration of gel matrix, composition and ionic strength of the
electrophoresis buffer, temperature and the use of intercalating dyes such as ethidium bromide
or sybrgreen (GelStar).
Agarose gels are the most popular medium for the separation of moderate and largesized nucleic acids and have a wide range of separation (Table 1) but a relatively low
resolving power. Polyacrylamide gels are most effective for separating smaller fragments
(less than 100bp).
Two electrophoresis buffers are commonly used and contain EDTA and Tris-acetate
(TAE) or Tris-borate (TBE) at a concentration of approx 50 mM. For historical reasons, TAE
is the most commonly used buffer for agarose gel electrophoresis, but its buffering capacity is
low and may become exhausted during extended electrophoresis. TBE is slightly more
expensive, but it offers significantly higher buffering capacity. Although the resolving power
of the buffers is almost identical, double-stranded linear DNA migrates approx 10% faster in
TAE than in TBE. Electrophoresis buffers are routinely prepared as concentrated solutions
(50xTAE or 10xTBE) and stored at room temperature.
The most convenient methods for visualizing DNA in agarose and polyacrylamide
gels is by staining with the fluorescent dye (ethidium bromide, SybrGreen etc.) which
intercalates between the stacked bases of the DNA. The fixed position and the close proximity
to the bases causes the bound dye to display an increased fluorescent yield compared to that
of the free dye in solution. Ultraviolet (UV) radiation at a range of 260–360 nm is absorbed
by the DNA and transmitted to the dye, and the energy is re-emitted in the visible spectrum.
Table 1.
Assembly and Pouring of the Gel
1. Place the tray/gel caster onto a horizontal section of bench, using a glass leveling
plate if necessary, place the spacers and the comb(s) in the appropriate position(s) so that
wells are formed at the cathode end of the gel.
2. Add the 2g powdered agarose (2% gel) to a 100 ml of 1x electrophoresis buffer
(TBE). Heat the mixture in a microwave oven, swirling every 30 s until the agarose is visibly
seen to have dissolved. Any undissolved agarose appears as small translucent particles.
3. Allow the solution to cool to 50°C. Add 3 µL of GelStar to the 50 ml of melted
agarose and mix, allowing the progression of the electrophoresis to be analyzed during
electrophoresis by illuminating the gel with UV light.
4. Pour 50 ml of the agarose into the gel mold, ensuring that no air bubbles form
between the teeth of the comb, and allow the gel to set at 4°C for 15-20 min (or 30–40 min in
room temperature).
Running the Gel
1. Carefully remove the comb and spacers, add sufficient 1X electrophoresis buffer to
cover the gel to a depth of approx. 1 mm (or more).
2. Mix the DNA sample (20 µL) with gel loading buffer (5 µL) to produce a 1X
concentration of buffer and load into the well through the thin layer of running buffer. Placing
a black piece of paper behind the wells may facilitate in the loading process by making the
wells more visible.
3. Load an 1 µL of DNA size standard (100bp, „Fermentas“) to allow the
determination of the sizes of the DNA fragments, because although the tracking dyes in the
loading buffer give a rough estimate of the migration of the DNA, they do not give the exact
size. Size standards can be purchased commercially or prepared by restriction enzyme
digestion of plasmid DNA, producing DNA fragments of known sizes.
4. Place the lid onto the gel tank, being careful not to disturb the samples, set voltage
approx. 110V and begin electrophoresis.
5. When the dyes have migrated the appropriate distance on the gel (takes ~1h), turn
off the power supply and proceed with visualization of the DNA.
Visualization of the DNA
Place the gel on a UV transilluminator and photograph the gel using (CCD)-based
digital analysis system.
Based on: PCR Mutation Detection Protocols. Human Press, Totowa, New Jersey, 2002.