Download PCR (Polymerase Chain Reaction)

PCR (Polymerase Chain Reaction)
Learning objective
 To know what PCR is
 To know its applications
 To know how it works
What is PCR?
A PCR or polymerase chain reaction is a laboratory procedure
in which millions of copies of a specific piece of DNA are made. It
is essentially an amplification method, whereby the tiniest amounts
of DNA that may be present in blood, hair or tissues can be copied
so that there is enough for analysis.
Kary Mullis, who developed PCR in 3891, won the Nobel
prize in Chemistry in 3881 for his invention. Since then, PCR has
been widely used as a diagnostic and research tool. Its applications
are continually growing and are widespread over many scientific
disciplines, including molecular biology, microbiology, genetics,
clinical diagnostics, forensic science, environmental science,
hereditary studies and paternity testing.
Mullis summarized the procedure: "Beginning with a single molecule of the
genetic material DNA, the PCR can generate 311 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." PCR has replaced previous methods of
DNA replication that used bacteria and could take several weeks to complete. PCR
can be done within a few hours, making it a very rapid assay.
The name of this method is derived from the key enzyme involved that
carries out the replication of the DNA, the DNA polymerase. This is an enzyme
that exists in nature. The most commonly used polymerase is Taq polymerase,
which is obtained from the bacterium Thermus aquaticus. This enzyme works
optimally at about 01oC. It can create a new DNA strand, using the original DNA
as a template, and using DNA oligonucleotides (also known as primers). The
primers used in PCR are synthesized, short sequences of DNA that are made to
match exactly the ends of the DNA region to be copied.
1
Applications of PCR
Detecting infectious agents
PCR is extensively used in analysing clinical specimens for the presence of
infectious agents, including HIV, hepatitis, human papillomavirus (the causative
agent of genital warts and cervical cancer), Epstein-Barr virus (glandular fever),
malaria, anthrax, etc….
PCR is particularly invaluable in the early detection of viral infections as it
can identify the DNA of the virus immediately following infection, as opposed to
the antibodies that are produced weeks or months after infection. PCR can also be
used to determine the viral load (i.e. how much virus is circulating around the
body), which is a useful measure of prognosis.
The role of PCR in cancer diagnostics
PCR is an invaluable tool as it can provide information on a patient’s
prognosis, and predict response or resistance to therapy. Many cancers are
characterised by small mutations in certain genes, and this is what PCR is
employed to identify.
For example, PCR can be applied in monitoring leukaemia patients
following treatment, by counting the number of cancerous cells that are still
circulating in their bodies.
Genetic diseases and paternity testing
Another important application of PCR is in the analysis of mutations that
occur in many genetic diseases. Because of the sensitivity of PCR, this can be done
from a single cell taken from an embryo before birth.
The paternity test is essentially carried out by PCR. A cheek swab is taken
from inside the mouth of both parents and the child. The DNA is extracted from
the cells obtained and is analysed by PCR. Everyone’s DNA is the same in every
cell in the body. A child’s DNA should have part of the mother's and father's DNA.
Several locations on the child's DNA are examined, and the sequences of these loci
are compared to the mother and father to see if there are matches from both
parents.
2
Others
Because PCR amplifies the regions of DNA that it targets, PCR can be used
to analyze extremely small amounts of sample. This is often critical for forensic
analysis, when only a trace amount of DNA is available as evidence. PCR may
also be used in the analysis of ancient DNA that is tens of thousands of years old.
These PCR-based techniques have been successfully used on animals, such as a
forty-thousand-year-old mammoth, and also on human DNA, in applications
ranging from the analysis of Egyptian mummies to the identification of a Russian
Tsar.
How does PCR work?
There are three basic steps involved in performing a PCR. The steps are
repeated 11-01 times in cycles of heating and cooling, with each step taking place
at a different temperature.
Components required to carry out a PCR
3.
A DNA template: The DNA to be copied,
usually extracted and purified from blood or other
tissue.
2.
Primers: Single stranded oligonucleotides
that match exactly the beginning and end of the
DNA template. Primers range from 31 to 11
nucleotides, and are used for the complementary
building blocks of the target sequence. They are
generated synthetically.
A primer for each target sequence on the end of
your DNA is needed. This allows both strands to
be copied simultaneously in both directions.
1.
A DNA polymerase (i.e. Taq polymerase): To
synthesise the DNA.
Taq stands for Thermus aquaticus, which is a
microbe found in 918C hot springs. Taq an DNA
polymerase, that amplifies the DNA from the primers by
the polymerase chain reaction.
Taq Polymerase
3
0.
dNTPs (Deoxyribonucleotide triphosphates): The
building blocks from which the Taq polymerase can
synthesise new DNA. These are added in excess amount.
Steps of PCR
All of the components are mixed together in one tube in very tiny volumes.
The reaction is carried out in an automated machine, known as a Thermal Cycler,
which is capable of rapidly increasing and decreasing the temperature.
Thermal
Cycler
3. The first step is known as
the denaturation step and is
carried out at around °49- °59C.
Since DNA exists in nature as a
double stranded molecule linked
together by weak hydrogen
bonds, to be able to copy it, the
DNA needs to be separated into
single strands (denatured). This
can be done by heating it to over
81oC.
2. The second step is the
annealing step and is carried out at
about 11-01oC.
Annealing is the process of
allowing two sequences of DNA to
form hydrogen bonds.
The primers anneal to their
matching sequence on the original
DNA strand.
4
1. The final extension step is
carried out at 02oC.
Taq DNA polymerase binds to the
annealed
primer.
Taq
polymerase works its way along the
DNA,
adding
complementary
nucleotides using the dNTPs
and other components in the
reaction mix. This completes the
replication process.
0. Once synthesis has been completed, the whole mixture is heated again to
81oC to melt the newly formed DNA complexes. This results in twice the amount
of template available for the next round of replication. Repeated heating and
cooling quickly amplifies the DNA segment of interest. Roughly one million
copies are made after 21 cycles. Number of cycles range from 21-01 cycles.
To summarize:
• Denaturalization: °49- °59C
• Primer Annealing: 559- 06°C
• Extension of DNA: °29
• Number of Cycles: 25-46
5
To check whether the PCR
generated
the
anticipated
DNA
fragment, agarose gel electrophoresis
is employed for size separation of the
PCR products. The size(s) of PCR
products is determined by comparison
with a DNA ladder (a molecular weight
marker), which contains DNA fragments
of known size, run on the gel alongside
the PCR products
PCR products after gel electrophoresis. Two sets of primers were used to
amplify a target sequence from three different tissue samples. No
amplification is present in sample 13; DNA bands in sample 12 and 11
indicate successful amplification of the target sequence. The gel also
shows a positive control, and a DNA ladder containing DNA fragments
of defined length for sizing the bands in the experimental PCRs
Variations of PCR
There are several variations of the PCR technique. One commonly used and
important variation is real-time PCR. Real-time PCR can be used to count the
amount of DNA, or number of copies of a gene, that is present in a sample.
It is employed to determine the viral load in viral infections, and also in
cancer diagnostics to count the number of cancerous cells remaining in a patient
undergoing treatment.
Limitations and benefits
As with many diagnostic tests in the laboratory, the possibility of false
positive and false negative results does exist when PCR is used for detecting
infectious agents. Therefore, follow up confirmation tests are always carried out.
6
Sanger Method for DNA Sequencing
DNA sequencing, first devised in 3801, has become a powerful technique in
molecular biology, allowing analysis of genes at the nucleotide level. For this
reason, this tool has been applied to many areas of research. For example, PCR
requires first knowing the flanking sequences of this piece. Another important use
of DNA sequencing is identifying restriction sites in plasmids. Before the advent of
DNA sequencing, molecular biologists had to sequence proteins directly; now
amino acid sequences can be determined more easily by sequencing a piece of
cDNA.
Dideoxynucleotide sequencing represents only
one method of sequencing DNA. It is
commonly called Sanger sequencing since
Sanger devised the method. This technique
utilizes 2',1'-dideoxynucleotide triphospates
(ddNTPs), molecules that differ from
deoxynucleotides by the having a hydrogen
atom attached to the 1' carbon rather than an
OH group. These molecules terminate DNA
chain elongation because they cannot form a
phosphodiester
bond
with
the
next
deoxynucleotide.
In order to perform the sequencing, one must first convert double stranded DNA
into single stranded DNA. This can be done by denaturing the double stranded
DNA with NaOH. A Sanger reaction consists of the following: a strand to be
sequenced (one of the single strands which was denatured using NaOH), DNA
primers (short pieces of DNA that are both complementary to the strand which is
to be sequenced and radioactively labelled at the 1' end), a mixture of a particular
ddNTP (such as ddATP) with its normal dNTP (dATP in this case), and the other
three dNTPs (dCTP, dGTP, and dTTP). The concentration of ddATP should be 31
of the concentration of dATP. The logic behind this ratio is that after DNA
polymerase is added, the polymerization will take place and will terminate
whenever a ddATP is incorporated into the growing strand. If the ddATP is only
31 of the total concentration of dATP, a whole series of labeled strands will result.
Note that the lengths of these strands are dependent on the location of the base
relative to the 1' end.
7
This reaction is performed four
times using a different ddNTP for
each reaction. When these reactions
are completed, gel electrophoresis
performed. One reaction is loaded
into one lane for a total of four
lanes. In electrophoresis, the
shortest fragments will migrate the
farthest. Therefore, the bottommost band indicates that its
particular dideoxynucleotide was
added first to the labeled primer.
Therefore, ddATP must have been
added first to the primer, and its
complementary base, thymine,
must have been the base present on
the 1' end of the sequenced strand.
One can continue reading in this
fashion. If one reads the bases from
the bottom up, one is reading the 1'
to 1' sequence of the strand
complementary to the sequenced
strand.
Ribosomal RNA sequencing
 60S rRNA is a part of the small subunit 11S
ribosomal RNA.
 It is highly conserved between different
species of bacteria and archaea.
 The new gold standard for the speciation of
bacteria.
8
Sources of DNA
There are three main sources of genes:
3. Gene libraries containing natural copies of genes
2. Gene libraries containing ‘complementary DNA’
copies of genes made from mRNA
1. Artificial synthesis of DNA.
Synthetic DNA
• Genes can be made in vitro with the help of DNA synthesis machines.
• A chain of over 321 nucleotides can be synthesized by this method, thus
several chains must be synthesized separately and linked together (by DNA
ligase) to form an entire gene.
• The difficulty of this approach is that the sequence of the gene must be
known.
• If the protein product of that gene is known and consequently the amino acid
sequence, is it possible to know the DNA sequence
9