PCR, DNA (cDNA). In many cases this method

PCR,
abbreviation of polymerase chain reaction, is an in vitro technique to
synthesize large quantities of a given DNA molecule. PCR separates the DNA into
their two complementary strands, synthesizes new DNA molecules using DNA
polymerase and repeats this process very quickly. PCR makes logarithmic
amplification of short DNA sequences (100 to 600 bp) within a longer double
stranded DNA molecule. PCR was invented in 1985 by Kary Banks Mullis in Cetus
Corporation (Berkeley, California, USA). Cetus Corporation was the orginal
owner of PCR patent and the patent was sold to Hoffmann-La Roche Inc. in 1991.
Dr. Kary Banks Mullis awarded Norbel Prize of chemistry for the invention of
PCR in 1993, only 8 years after the invention of PCR. As a fast gene detection,
PCR technique has revolutionized many aspects of life sciences, such as the
diagnosis of genetic defects, the detection of the AIDS virus in human cells,
criminologist applications and genetic researches.

PCR uses a pair of primers
(about 20 bp each), that are complementary to a specific sequence on each of
the two strands of the target DNA. These primers are extended by a DNA

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Polymerase and the
sequence of the new DNA pieces matches the sequence of the template followed
the primer. After the new DNA synthesized, the same primers will be released
and used again. This let the DNA make a logarithmic amplification. Since DNA
amplification must be processed under the single strand condition, it needs
high temperature to separate the double strand DNA in each round of the
amplification process. The milestone of DNA amplification exploring is the
discovery of a thermo-stable DNA polymerase that is isolated from Thermus
aquaticus (Taq), a bacterium growing in hot pools near volcanic vent. The
thermo-stable DNA polymerase comes from Taq and is called Taq polymerase, which
composes the core component of the PCR technique. For PCR, it is not necessary
to add new polymerase in every round of amplification. After some rounds of
amplification (about 40), the PCR product is abundant enough to be detected
with ethidium bromide stain and it can be analyzed on an agarose gel. In order
to measure messenger RNA (mRNA), the PCR is extended to use reverse
transcriptase to convert mRNA into complementary DNA (cDNA). In many cases this
method has been used to measure the levels of a particular mRNA (quantitative)
under different condition. Reverse transcriptase PCR analysis of mRNA is often
abbreviated as “RT-PCR”, which is unfortunate as it can be confused
with “real-time PCR” that also abbreviated as RT-PCR (Abdul-Careem, 2006).
In this paper the RT-PCR represents real-time PCR.

Traditionally,
PCR uses a peltier heat pump to quickly heat and cool the DNA strands and uses
the Taq polymerase for the synthesis of DNA molecules. Taq, the abbreviation of
Thermus aquaticus, is a bacterium that lives in volcanic sulfer jets at the
bottom of the ocean where the temperature is very high. They can withstand
extremely high temperatures, and that is why they are so valuable in PCR. For
reverse transcription PCR, primers are short strands of RNA that bind to the
target site of DNA molecule. DNA polymerases need to have RNA primers for the
beginning of DNA replication. Four dNTPs (deoxyribonucleotide triphosphates)
(dGTP, dCTP, dATP and dTTP) are letters of the DNA alphabet and the Taq
polymerase uses the dNTPs to build the new DNA molecular chains.

PCR needs to
place a very small amount of DNA molecules that contains the target gene into a
PCR test tube. A large amount of primer, which matches the certain sequence of
the target gene, is also added for the DNA synthesis tubes. These primers find
the right sequence in the DNA, and play starting points for DNA synthesis. When
the Taq enzyme is added, the loose nucleotides lock into a DNA sequence
dictated by the sequence of that target gene located between the two primers.

The test tube
is heated, and the DNA’s double helix separates into two strands at the high
temperature. The DNA sequence of each strand of the helix is opened and as the
temperature is lowered the primers automatically bind to their complementary
sequences of the DNA molecules. At the same time, the Taq enzyme links the
loose nucleotides to the primer and to each of the separated DNA strands in the
appropriate sequence. The complete reaction results in two double helices
containing the desired portion of the original sequence. The heating and
cooling is repeated many times (normally around 40), doubling the number of DNA
copies each heating cycle. After 30 to 50 heating cycles are completed a single
copy of a piece of DNA can be multiplied to hundreds of millions.

PCR has made a
revolution for the life science. As Dr. Kary Banks Mullis wrote in Scientific
American, “Beginning with a single molecule of the genetic material DNA,
the PCR can generate 100 billion similar molecules in an afternoon. The reaction
is easy to execute. It requires no more than a test tube, a few simple reagents
and a source of heat. The DNA sample that one wishes to copy can be pure, or it
can be a minute part of an extremely complex mixture of biological materials.
The DNA may come from a hospital tissue specimen, from a single human hair,
from a drop of dried blood at the scene of a crime, from the tissues of a
mummified brain or from a 40,000-year-old wooly mammoth frozen in a
glacier” (Mullis, 1990).

PCR History:

Dr. Kary Banks
Mullis invented PCR technique in 1985 while he worked as a chemist at Cetus
Corporation, a biotechnology company established in Berkeley, California, USA
in 1972. Cetus Corporation had the orginal ownership of PCR patent. Kary Mullis
awarded Norbel Prize of chemistry for the invention of PCR in 1993.

Dr. Kary Banks Mullis,
male,  was born on December 28 of 1944 in
Lenoir North, North Carolina, USA, He obtained his bachelor’s degree in
Chemistry in 1966 from the Georgia Institute of Technology and received a PhD
in Biochemistry from the University of California at Berkeley in 1972. He then
spent seven years of post-doctoral research on Pediatric Cardiology and
Pharmaceutical Chemistry at the University of Kansas Medical School. After his
period at Kansas Medical School he got a technician position at the Cetus
Corporation of Emeryville (in 1978). It was during the time here that he
created the idea for PCR. In 1983, while driving along the Pacific Coast
Highway 128 of California in his Honda Civic from San Francisco to his home in
La Jolla, California, USA, Kary Mullis was thinking about a simple method of
determining a specific nucleotide from along a stretch of DNA. He then, like
many great scientists, claimed having a sudden flash of inspirational vision.
He had conceived a way to start and stop DNA polymerase action and repeating
numerously, a way of exponentially amplifying a DNA sequence in a test tube.
Mullis then took his concept to his colleagues at Cetus Company and together
they made it work in an experimental system.

This technique was first
opened to the world at a conference in 1985 and was widely accepted by the
scientific community after then. The enzyme molecule used in PCR was named as
Taq Polymerase in 1989. In 1989, Cetus got the patent for the PCR technique. By
1991 the use of PCR in laboratories across the world was extremely widespread.

Traditional PCR

 As a molecular biology technique, PCR
replicates DNA enzymatically in vitro. Like DNA amplification in living
organisms, PCR makes a small amount of the DNA molecule to be amplified
exponentially. However, because it is an in vitro technique, it can be
performed without restrictions on the form of DNA and it can be extensively
modified to perform a wide array of genetic manipulations (Kaldosh, 2006).

PCR is
commonly used in life science researches for a variety of tasks, such as
detection of hereditary diseases, identification of genetic fingerprints,
clinical diagnosis of infectious diseases, cloning of genes, paternity testing,
and DNA computing, etc. PCR is used to amplify a short, well-defined part of a
DNA strand. This can be a single gene, or just a part of a gene. As opposed to
living organisms, PCR process can copy only short DNA fragments, usually up to
10 kb. Certain methods can copy fragments up to 47 kb in size, which is still
much less than the chromosomal DNA of a eukaryotic cell. A human chromosome
contains about 3×106 kp.

Two synthetic
oligonucleotide primers, which are complementary to two regions of the target
DNA (one for each strand) to be amplified, are added to the target DNA, in the
presence of excess deoxynucleotides and Taq polymerase, a heat stable DNA
polymerase. In a series of temperature cycles, the target DNA is repeatedly
denatured (at around 95oC), annealed to the primers (at around 55oC) and a new
strand extended from the primers (at around 72oC). As the new strands
themselves act as templates for subsequent cycles, DNA fragments matching both
primers are amplified exponentially, rather than linearly.

1) Normally, PCR requires
several basic components:

(1)        DNA template, which contains the target DNA fragment

(2)        Two primers (sense and anti-sense), which determine the
beginning and end of the region to be amplified

(3)        Taq polymerase (a thermal DNA polymerase), which synthesize
DNA for the amplification and can stand for high temperature

(4)        Four deoxynucleotides-triphosphates (dNTP, i.e. dATP, dTTP,
dGTP, dCTP), from which the DNA polymerase builds the new DNA molecules

(5)        Buffer, which provides a suitable chemical environment for
the DNA amplification

(6)        The PCR process is carried out in a thermal cycler. This is a
machine that heats and cools the reaction tubes within it to the precise
temperature required for each step of the reaction. These machines cost about
US$2,000 – US$20,000.

2) Materials for normal
running of PCR

(1)        Template DNA (genomic, plasmid, cosmid, bacterial/yeast
colony, etc.).

(2)        Primers (resuspended to a known concentration with sterile
TE).

(3)        Buffer (usually 10×, normally supplied with Taq polymerase).

(4)        MgCl2 (25 mM).

(5)        Taq polymerase.

(6)        dNTPs (2 mM for each dNTP stock). Store at -20°C.

(7)        Sterile distilled water.

(8)        Gloves.

(9)        PCR machine (cycler).

(10)      Aerosol tips.

(11)      Ice.

3) The final
concentrations of reagents in PCR reactions

(1)        Buffer: 1×, usually comes as 10× stock.

(2)        dNTPs: For most general PCR, the final concentration is 0.2
mM.

(3)        Primers: Normally the primer concentration is 50-100 pmol of
each primer per reaction.

(4)        Template: It needs experiences to estimate the amount of
template added to a reaction.

(5)        MgCl2: MgCl2 is variable in PCR, and it could be from 1 to 6
mM. It is important for the MgCl2 amount added in PCR reaction.

4) The cycling reactions

There are three major
steps in a PCR, which are repeated for 30 to 50 cycles. This is done on an
automated cycler, which can heat and cool the tubes with the reaction mixture
in a very short time.

(1)Denaturation at 94°C:
During the denaturation, the double strand melts open to single stranded DNA
and all enzymatic reactions stop.

(2)Annealing at 54°C: The
primers are moving around by Brownian motion and ionic bonds are constantly
formed and broken between the single stranded primer and the single stranded
template.

(3)Extension at 72°C: The polymerase
synthesizes DNA molecules.

The bases that
are complementary to the DNA template are coupled to the primer on the 3′ side
(the polymerase adds dNTP’s from 5′ to 3′, reading the template from 3′ to 5′
side). Because both strands are copied during PCR, there is an exponential
increase of the number of copies of the gene.