4 Polymerase Chain Reaction

The polymerase chain reaction is very different from gene cloning. Rather than a series of manipulations involving living
cells, PCR is carried out in a single test tube simply by mixing DNA with a set of reagents and placing the tube in a thermal
cycler, a piece of equipment that enables the mixture to be incubated at a series of temperatures that are varied in a
preprogrammed manner.
Fundamentals of the PCR
The fundamentals of this relatively
simple process are outlined here using a
fragment of double-stranded DNA. PCR
amplifies a specific region of a DNA
strand (the DNA target). Most PCR
methods amplify DNA fragments of
between 0.1 and 10 kilo base pairs (kb),
although some techniques allow for
amplification of fragments up to 40 kb in
size. The amount of amplified product is
determined by the available substrates in
the reaction, which become limiting as
the reaction progresses.

A basic PCR set-up requires several components and reagents, including:

 DNA template that contains the DNA target region to amplify
 DNA polymerase, an enzyme that polymerizes new DNA strands; heat-resistant Taq DNA polymerase is especially
common, as it is more likely to remain intact during the high-temperature DNA denaturation process
 Two DNA primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strands of
the DNA target (DNA polymerase can only bind to and elongate from a double-stranded region of DNA; without
primers there is no double-stranded initiation site at which the polymerase can bind); specific primers that are
complementary to the DNA target region are selected beforehand, and are often custom-made in a laboratory or
purchased from commercial biochemical suppliers
 Deoxy nucleoside triphosphates, or dNTPs (sometimes called "deoxy nucleotide triphosphates"; nucleotides
containing triphosphate groups), the building blocks from which the DNA polymerase synthesizes a new DNA strand
 Buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
 Bivalent cations, typically magnesium (Mg) or manganese (Mn) ions; Mg2+ is the most common, but Mn2+ can be
used for PCR-mediated DNA mutagenesis, as a higher Mn2+ concentration increases the error rate during DNA
synthesis
 Monovalent cations, typically potassium (K) ions

The reaction is commonly carried out in a volume of 10–200μl in small

reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler
heats and cools the reaction tubes to achieve the temperatures required at
each step of the reaction. Many modern thermal cyclers make use of the
Peltier effect, which permits both heating and cooling of the block holding
the PCR tubes simply by reversing the electric current. Thin-walled
reaction tubes permit favorable thermal conductivity to allow for rapid
thermal equilibration. Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube.
Steps of a PCR
Typically, PCR consists of a series of 20–40 repeated temperature changes, called
cycles, with each cycle commonly consisting of two or three discrete temperature
steps. The cycling is often preceded by a single temperature step at a very high
temperature (>90 °C (194 °F)), and followed by one hold at the end for final
product extension or brief storage. The temperatures used and the length of time
they are applied in each cycle depend on a variety of parameters, including the
enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in
the reaction, and the melting temperature (Tm) of the primers.
The individual steps common to most PCR methods are as follows:
 Initialization:
This step is only required for DNA polymerases that require heat activation by
hot-start PCR. It consists of heating the reaction chamber to a temperature of
94–96 °C (201–205 °F), or 98 °C (208 °F) if extremely thermostable
polymerases are used, which is then held for 1–10 minutes.
 Denaturation:
This step is the first regular cycling event and consists of heating the reaction
chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA
melting, or denaturation, of the double-stranded DNA template by breaking the
hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules.
 Annealing:
In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F) for 20–40 seconds, allowing annealing
of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the
reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are
single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only
very short sequences at the 3' end of each strand.
It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly
affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer
to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly
complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. If
it is too high, the primer may not bind at all. A typical annealing temperature is about 3–5 °C below the Tm of the
primers used. Stable hydrogen bonds between complementary bases are formed only when the primer sequence very
closely matches the template sequence. During this step, the polymerase binds to the primer-template hybrid and begins
DNA formation.
 Extension/elongation:
The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for the
thermostable DNA polymerase of Taq (Thermus aquaticus) polymerase is approximately 75–80 °C (167–176 °F),
though a temperature of 72 °C (162 °F) is commonly used with this enzyme. In this step, the DNA polymerase
synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction
mixture that are complementary to the template in the 5'-to-3' direction, condensing the 5'-phosphate group of the
dNTPs with the 3'-hydroxy group at the end of the nascent (elongating) DNA strand. The precise time required for
elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify. As a
rule of thumb, at their optimal temperature, most DNA polymerases polymerize a thousand bases per minute. Under
optimal conditions (i.e., if there are no limitations due to limiting substrates or reagents), at each extension/elongation
step, the number of DNA target sequences is doubled. With each successive cycle, the original template strands plus
all newly generated strands become template strands for the next round of elongation, leading to exponential
(geometric) amplification of the specific DNA target region.
The processes of denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required to
amplify the DNA target to millions of copies. The formula used to calculate the number of DNA copies formed after a
given number of cycles is 2n, where n is the number of cycles. Thus, a reaction set for 30 cycle’s results in 230, or
1073741824, copies of the original double-stranded DNA target region.
 Final elongation:
This single step is optional, but is performed at a temperature of 70–74 °C (158–165 °F) (the temperature range required
for optimal activity of most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any
remaining single-stranded DNA is fully elongated.
 Final hold:
The final step cools the reaction chamber to 4–15 °C (39–59 °F) for an indefinite time, and may be employed for short-
term storage of the PCR products.

The annealing temperature is the important one because, again, this can affect the
specificity of the reaction. DNA–DNA hybridization is a temperature-dependent
phenomenon. If the temperature is too high no hybridization takes place; instead
the primers and templates remain dissociated. However, if the temperature is too
low, mismatched hybrids—ones in which not all the correct base pairs have
formed—are stable. If this occurs the earlier calculations regarding the
appropriate lengths for the primers become irrelevant, as these calculations
assumed that only perfect primer–template hybrids are able to form. If
mismatches are tolerated, the number of potential hybridization sites for each
primer is greatly increased, and amplification is more likely to occur at non-target
sites in the template molecule.

The ideal annealing temperature must be low enough to enable hybridization

between primer and template, but high enough to prevent mismatched hybrids
from forming. This temperature can be estimated by determining the melting
temperature or Tm of the primer–template hybrid. The Tm is the temperature at
which the correctly base-paired hybrid dissociates (“melts”). A temperature 1–
2°C below this should be low enough to allow the correct primer–template hybrid
to form, but too high for a hybrid with a single mismatch to be stable. The Tm
can be determined experimentally but is more usually calculated from the simple
formula:
Tm = (4 × [G + C]) + (2 × [A + T]) °C
in which [G + C] is the number of G and C nucleotides in the primer sequence, and [A + T] is the number of A and T
nucleotides.
The annealing temperature for a PCR experiment is therefore determined by calculating the Tm for each primer and using
a temperature of 1–2°C below this figure. Note that this means the two primers should be designed so that they have
identical Tms. If this is not the case, the appropriate annealing temperature for one primer may be too high or too low for
the other member of the pair.

To check whether the PCR successfully generated the anticipated DNA target region (also sometimes referred to as the
amplimer or amplicon), Agarose gel electrophoresis may be 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.

The usefulness of the PCR lies in its three steps—denaturation, hybridization, and DNA synthesis—that can be repeated
many times simply by changing the temperature of the reaction mixture. Each newly synthesized strand of DNA can serve
as a template, so the target DNA concentration increases at an exponential rate. In a process consisting of 20 cycles, the
amplification for the DNA fragment is about a million-fold. Thirty cycles, which can be completed in 1–3 hours, provide
a billion-fold amplification. Theoretically, one could begin with a single molecule of target DNA and produce in 1 hour
enough DNA for the Sanger dideoxy chain-terminating sequence procedure. Another benefit of the PCR is its simplicity.
Commercially available instruments called thermocyclers allow the laboratory technician to mix the reagents and insert
the reaction mixture in the cycler, which then automatically repeats the reaction steps by changing temperatures.
DESIGN OF PRIMERS
The primers are the key to the success or failure of a PCR experiment. In primer design, there are several aspects that
have to be considered. If the primers are designed correctly the experiment results in amplification of a single DNA
fragment, corresponding to the target region of the template molecule. If the primers are incorrectly designed the
experiment will fail, possibly because no amplification occurs, or possibly because the wrong fragment, or more than one
fragment, is amplified. Clearly a great deal of thought must be put into the design of the primers. Working out appropriate
sequences for the primers is not a problem: they must correspond with the sequences flanking the target region on the
template molecule. Each primer must, of course, be complementary to its template strand in order for hybridization to
occur, and the 3′ ends of the hybridized primers should point toward one another. The DNA fragment to be amplified
should not be greater than about 3 kb in length and ideally less than 1 kb. Fragments up to 10 kb can be amplified by
standard PCR techniques, but the longer the fragment the less efficient the amplification and the more difficult it is to
obtain consistent results. Amplification of very long fragments—up to 40 kb—is possible, but requires special methods.

Sequence of Primer:
Oligonucleotide primers are available from many commercial sources and can be synthesized to order in a few days.
Perhaps most obvious is the sequence of the primer -- more specifically, where does the sequence information come from?
It may be derived from amino acid sequence data, in which case the genetic code degeneracy has to be considered. In
synthesizing the primer, two approaches can be taken.
1. By incorporating a mixture of bases at the wobble position, a mixed primer can be made, with the ‘correct’
sequence represented as a small proportion of the mixture.
2. Alternatively, the base inosine (which pairs equally well with any of the other bases) can be incorporated as the
third base in degenerate codons.
If the primer sequence is taken from an already-determined DNA sequence, it may be from the same gene from a different
organism or may be from a cloned DNA that has been sequenced during previous experimental work.

Uniqueness of the sequence of primer:

The primer must also be long enough to ensure that it is a unique sequence in the genome from which the target DNA is
taken. Primer lengths of around 20-30 nucleotides are usually sufficient for most applications. With regard to the base
composition and sequence of primers, repetitive sequences should be avoided, and also regions of single-base sequence.
Primers should obviously not contain regions of internal complementary sequence or regions of sequence overlap with
other primers.
Because extension of PCR products occurs from the 3’ termini of the primer, it is this region that is critical with respect to
fidelity and stability of pairing with the target sequence. Some ‘looseness’ of primer design can be accommodated at the
5’ end, and this can sometimes be used to incorporate design features such as restriction sites at the 5’ end of the primer.

Primer design:
In (a) the amino acid sequence and number of codons per
amino acid are shown. Those amino acids with 6 codons
(circled) are best avoided. The boxed sequence is therefore
selected.
In (b) a mixed probe is synthesized by including the appropriate
mixture of dNTPs for each degenerate position. Note that in
this example the final degenerate position for proline is not
included, giving an oligonucleotide with 20 bases (a 20-mer).
There are 128 possible permutations of sequence in the
mixture.
In (c) inosine (I) is used to replace the four-fold degenerate
bases, giving eight possible sequences.
1. Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp. This length is long enough
for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature. The first
important issue to address is the length of the primers. If the primers are too short they might hybridize to non-target sites
and give undesired amplification products. Eg. Total human DNA is used in a PCR experiment with a pair of primers
eight nucleotides in length (in PCR jargon, these are called “8-mers”). The likely result is that a number of different
fragments will be amplified. This is because attachment sites for these primers are expected to occur, on average, once
every 48 = 65,536 bp, giving approximately 49,000 possible sites in the 3,200,000 kb of nucleotide sequence that makes
up the human genome. This means that it would be very unlikely that a pair of 8-mer primers would give a single, specific
amplification product with human DNA. The expected frequency of a 17-mer sequence is once every 417 = 17,179,869,184
bp. This figure is over five times greater than the length of the human genome, so a 17-mer primer would be expected to
have just one hybridization site in total human DNA. A pair of 17-mer primers should therefore give a single, specific
amplification product.
Why not simply make the primers as long as possible? The length of the primer influences the rate at which it hybridizes
to the template DNA, long primers hybridizing at a slower rate. The efficiency of the PCR, measured by the number of
amplified molecules produced during the experiment, is therefore reduced if the primers are too long, as complete
hybridization to the template molecules cannot occur in the time allowed during the reaction cycle. In practice, primers
longer than 30-mer are rarely used.

2. Primer Melting Temperature: Primer Melting Temperature (Tm) by definition is the temperature at which one half of
the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting
temperatures in the range of 52-58 oC generally produce the best results. Primers with melting temperatures above 65oC
have a tendency for secondary annealing. The GC content of the sequence gives a fair indication of the primer T m. All our
products calculate it using the nearest neighbor thermodynamic theory, accepted as a much superior method for estimating
it, which is considered the most recent and best available.
Formula for primer Tm calculation:
∆𝐻
Melting Temperature Tm (K) = { + R ln[C]},
∆𝑆
∆𝐻
Melting Temperature Tm (oC) = { + R ln(C)} - 273.15
∆𝑆
Where
ΔH (kcal/mole): H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in
Enthalpy. In the above formula the ΔH is obtained by adding up all the di-nucleotide pair’s enthalpy values of each nearest
neighbor base pair.
ΔS (kcal/mole): S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained
by adding up all the di-nucleotide pair’s entropy values of each nearest neighbor base pair. An additional salt correction is
added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this
is the default condition used for all calculations.
ΔS (salt correction) = ΔS (1M NaCl) + 0.368 x N x ln ([Na+])
Where
N: Number of nucleotide pairs in the primer (primer length -1).
[Na+]: Salt equivalent in mM.
[Na+] calculation: [Na+] = Monovalent ion concentration +4 x free Mg2+.

3. Primer Annealing Temperature: The primer melting temperature is the estimate of the DNA-DNA hybrid stability
and critical in determining the annealing temperature. Too high Ta will produce insufficient primer-template hybridization
resulting in low PCR product yield. Too low T a may possibly lead to non-specific products caused by a high number of
base pair mismatches, Mismatch tolerance is found to have the strongest influence on PCR specificity.
Ta = 0.3 x Tm (primer) + 0.7 Tm (product) – 14.9
Where,
Tm(primer) = Melting Temperature of the primers
Tm (product) = Melting temperature of the product

4. GC Content: The GC content (the number of G's and C's in the primer as a percentage of the total bases) of primer
should be 40-60%.
5. GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote
specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in
the last 5 bases at the 3' end of the primer.

6. Primer Secondary Structures: Presence of the primer secondary structures produced by intermolecular or
intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing
and thus the amplification. They greatly reduce the availability of primers to the reaction.

 Hairpins: It is formed by intramolecular interaction within the primer and should be avoided.
Optimally a 3' end hairpin with a ΔG of -2 kcal/mol and an internal hairpin with a ΔG of -3
kcal/mol is tolerated generally. The Gibbs free energy ΔG is the measure of the amount of
work that can be extracted from a process operating at a constant pressure. It is the measure of
the spontaneity of the reaction. The stability of hairpin is commonly represented by its ΔG
value, the energy required to break the secondary structure. Larger negative value for ΔG indicates stable,
undesirable hairpins. Presence of hairpins at the 3' end most adversely affects the reaction.
ΔG = ΔH – TΔS
 Self-Dimer: A primer self-dimer is formed by intermolecular interactions between the two (same sense) primers,
where the primer is homologous to itself. Generally a large amount of primers are used in PCR compared to the
amount of target gene. When primers form intermolecular dimers much more readily than hybridizing to target
DNA, they reduce the product yield. Optimally a 3' end self-dimer with a ΔG of -5 kcal/mol and an internal self-
dimer with a ΔG of -6 kcal/mol is tolerated generally.

 Cross Dimer: Primer cross dimers are formed by intermolecular

interaction between sense and antisense primers, where they are
homologous. Optimally a 3' end cross dimer with a ΔG of -5
kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is
tolerated generally.

7. Repeats: A repeat is a di-nucleotide occurring many times consecutively and should be avoided because they can
misprime. For example: ATATATAT. A maximum number of di-nucleotide repeats acceptable in an oligo is 4 di-
nucleotides.

8. Runs: Primers with long runs of a single base should generally be avoided as they can misprime. For example,
AGCGGGGGATGGGG has runs of base 'G' of value 5 and 4. A maximum number of runs accepted is 4bp.

9. 3' End Stability: It is the maximum ΔG value of the five bases from the 3' end. An unstable 3' end (less negative ΔG)
will result in less false priming.

10. Avoid Template Secondary Structure: A single stranded Nucleic acid sequences is highly unstable and fold into
conformations (secondary structures). The stability of these template secondary structures depends largely on their free
energy and melting temperatures(Tm). Consideration of template secondary structures is important in designing primers,
especially in qPCR. If primers are designed on a secondary structures which is stable even above the annealing
temperatures, the primers are unable to bind to the template and the yield of PCR product is significantly affected. Hence,
it is important to design primers in the regions of the templates that do not form stable secondary structures during the PCR
reaction. Our products determine the secondary structures of the template and design primers avoiding them.

11. Avoid Cross Homology: To improve specificity of the primers it is necessary to avoid regions of homology. Primers
designed for a sequence must not amplify other genes in the mixture. Commonly, primers are designed and then BLASTed
to test the specificity. Our products offer a better alternative. You can avoid regions of cross homology while designing
primers. You can BLAST the templates against the appropriate non-redundant database and the software will interpret the
results. It will identify regions significant cross homologies in each template and avoid them during primer search.
Parameters for Primer Pair Design

1. Amplicon Length: The amplicon length is dictated by the experimental goals. For qPCR, the target length is closer to
100 bp and for standard PCR, it is near 500 bp. If you know the positions of each primer with respect to the template, the
product is calculated as: Product length = (Position of antisense primer-Position of sense primer) + 1.

2. Product Position: Primer can be located near the 5' end, the 3' end or anywhere within specified length. Generally, the
sequence close to the 3' end is known with greater confidence and hence preferred most frequently.

3. Tm of Product: Melting Temperature (Tm) is the temperature at which one half of the DNA duplex will dissociate and
become single stranded. The stability of the primer-template DNA duplex can be measured by the melting temperature
(Tm).

4. Optimum Annealing Temperature (Ta Opt): The formula of Rychlik is most respected. Our products use this formula
to calculate it and thousands of our customers have reported good results using it for the annealing step of the PCR cycle.
It usually results in good PCR product yield with minimum false product production.
Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 14.9
Where
Tm of primer is the melting temperature of the less stable primer-template pair
Tm of product is the melting temperature of the PCR product.

5. Primer Pair Tm Mismatch Calculation: The two primers of a primer pair should have closely matched melting
temperatures for maximizing PCR product yield. The difference of 5oC or more can lead no amplification

DIFFERENT TYPES OF PCR

1. NESTED PCR
Nested polymerase chain reaction (Nested PCR) is a modification of polymerase chain reaction intended to reduce non-
specific binding in products due to the amplification of unexpected primer binding sites.
Polymerase chain reaction itself is the process used to amplify DNA samples, via a temperature-mediated DNA
polymerase. The products can be used for sequencing or analysis, and this process is a key part of many genetics research
laboratories, along with uses in DNA fingerprinting for forensics and other human genetic cases. Conventional PCR
requires primers complementary to the termini of the target DNA. The amount of product from the PCR increases with the
number of temperature cycles that the reaction is subjected to. A commonly occurring problem is primers binding to
incorrect regions of the DNA, giving unexpected products. This problem becomes more likely with an increased number
of cycles of PCR.
Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction,
the second set intended to amplify a secondary target within the first run product. This allows amplification for a low
number of runs in the first round, limiting non-specific products. The second nested primer set should only amplify the
intended product from the first round of amplification and not non-specific product. This allows running more total cycles
while minimizing non-specific products. This is useful for very rare templates or PCR with high background.
Processes
1. The target DNA undergoes the first run of polymerase chain reaction with the first set of primers, shown in green. The
selection of alternative and similar primer binding sites gives a selection of products, only one containing the intended
sequence.
2. The product from the first reaction undergoes a second run with the second set of primers, shown in red. It is very
unlikely that any of the unwanted PCR products contain binding sites for both the new primers, ensuring the product
from the second PCR has little contamination from unwanted products of primer dimers, hairpins, and alternative
primer target sequences.
2. RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR), a variant of polymerase chain reaction (PCR), is a technique
commonly used in molecular biology to detect RNA expression. RT-PCR is often confused with real-time polymerase chain
reaction (qPCR) by students and scientists alike, but they are separate and distinct techniques. While RT-PCR is used to
qualitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA, qPCR is used
to quantitatively measure the amplification of DNA using fluorescent dyes.
Although RT-PCR and the traditional PCR both produce multiple copies of particular DNA isolates through amplification,
the applications of the two techniques are fundamentally different. Traditional PCR is used to exponentially amplify target
DNA sequences. RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA
complement through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amplified using
traditional PCR.
In addition to the qualitative study of gene expression, quantitative PCR can be utilized for quantification of RNA, in both
relative and absolute terms, by incorporating qPCR into the technique. The combined technique, described as quantitative
RT-PCR or real-time RT-PCR (sometimes even quantitative real-time RT-PCR), is often abbreviated as qRT-PCR, [9] RT-
qPCR, or RRT-PCR. Compared to other RNA quantification methods, such as northern blot, qRT-PCR is considered to be
the most powerful, sensitive, and quantitative assay for the detection of RNA levels. It is frequently used in the expression
analysis of single or multiple genes, and expression patterns for identifying infections and diseases.
In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse
transcriptase. The cDNA is then used as a template for exponential amplification using PCR. QT-NASBA is
currently the most sensitive method of RNA detection available. The use of RT-PCR for the detection of RNA
transcript has revolutionized the study of gene expression in the following important ways:
 Made it theoretically possible to detect the transcripts of practically any gene
 Enabled sample amplification and eliminated the need for abundant starting material that one faces when
using northern blot analysis
 Provided tolerance for RNA degradation as long as the RNA spanning the primer is intact.

The quantification of mRNA using RT-PCR can be achieved as either a one-step or a two-step reaction. The
difference between the two approaches lies in the number of tubes used when performing the procedure.
 In the one-step approach, the entire reaction from cDNA synthesis to PCR amplification occurs in a single
tube. The one-step approach is thought to minimize experimental variation by containing all of the
enzymatic reactions in a single environment. However, the starting RNA templates are prone to
degradation in the one-step approach, and the use of this approach is not recommended when repeated
assays from the same sample is required. Additionally, one-step approach is reported to be less accurate
compared to the two-step approach. It is also the preferred method of analysis when using DNA binding
dyes such as SYBR Green since the elimination of primer-dimers can be achieved through a simple
change in the melting temperature.
 On the other hand, the two-step reaction requires that the reverse transcriptase reaction and PCR
amplification be performed in separate tubes. The disadvantage of the two-step approach is susceptibility
to contamination due to more frequent sample handling.

Types of RT-PCR
1. End-point RT-PCR
The measurement approaches of end-point RT-PCR requires the detection of gene expression levels by the use of
fluorescent dyes like ethidium bromide, P32 labeling of PCR products using phosphorimager, or by scintillation
counting. End-point RT-PCR is commonly achieved using three different methods: relative, competitive and
comparative.
2. Relative RT-PCR
Relative quantifications of RT-PCR involves the co-amplification of an internal control simultaneously with the gene
of interest. The internal control is used to normalize the samples. Once normalized, a direct comparison of relative
transcript abundances across multiple samples of mRNA can be made. One precaution to note is that the internal
control must be chosen so that it is not affected by the experimental treatment. The expression level should be constant
across all samples and with the mRNA of interest for the results to be accurate and meaningful. Because the
quantification of the results are analyzed by comparing the linear range of the target and control amplification, it is
crucial to take into consideration the starting target molecules concentration and their amplification rate prior to starting
 the analysis. The results of the analysis are expressed as the ratios of gene signal to internal control signal, which the
values can then be used for the comparison between the samples in the estimation of relative target RNA expression.
3. Competitive RT-PCR
Competitive RT-PCR technique is used for absolute quantification. It involves the use of a synthetic “competitor”
RNA that can be distinguished from the target RNA by a small difference in size or sequence. It is important for the
design of the synthetic RNA be identical in sequence but slightly shorter than the target RNA for accurate results. Once
designed and synthesized, a known amount of the competitor RNA is added to experimental samples and is co-
amplified with the target using RT-PCR. Then, a concentration curve of the competitor RNA is produced and it is used
to compare the RT-PCR signals produced from the endogenous transcripts to determine the amount of target present
in the sample.
4. Comparative RT-PCR
Comparative RT-PCR is similar to the competitive RT-PCR in that the target RNA competes for amplification reagents
within a single reaction with an internal standard of unrelated sequence. Once the reaction is complete, the results are
compared to an external standard curve to determine the target RNA concentration. In comparison to the relative and
competitive quantification methods, comparative RT-PCR is considered to be the more convenient method to use since
it does not require the investigator to perform a pilot experiment; in relative RT-PCR, the exponential amplification
range of the mRNA must be predetermined and in competitive RT-PCR, a synthetic competitor RNA must be
synthesized.
5. Real-time RT-PCR
The emergence of novel fluorescent DNA labeling techniques in the past few years have enabled the analysis and
detection of PCR products in real-time and has consequently led to the widespread adoption of real-time RT-PCR for
the analysis of gene expression. Not only is real-time RT-PCR now the method of choice for quantification of gene
expression, it is also the preferred method of obtaining results from array analyses and gene expressions on a global
scale. Currently, there are four different fluorescent DNA probes available for the real-time RT-PCR detection of PCR
products: SYBR Green, TaqMan, Molecular Beacons, and Scorpions. All of these probes allow the detection of PCR
products by generating a fluorescent signal. While the SYBR Green dye emits its fluorescent signal simply by binding
to the double-stranded DNA in solution, the TaqMan probes, Molecular Beacons and Scorpions generation of
fluorescence depend on Förster Resonance Energy Transfer (FRET) coupling of the dye molecule and a quencher
moiety to the oligonucleotide substrates

3. REAL TIME PCR

A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative polymerase chain reaction (qPCR),
is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification
of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR. Real-time PCR
can be used quantitatively (quantitative real-time PCR), and semi-quantitatively, i.e. above/below a certain amount of DNA
molecules (semi quantitative real-time PCR). Two common methods for the detection of PCR products in real-time PCR
are:
 Non-specific fluorescent dyes that intercalate with any double-stranded DNA, and
 Sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which
permits detection only after hybridization of the probe with its complementary sequence.
The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines propose that
the abbreviation qPCR be used for quantitative real-time PCR and that RT-qPCR be used for reverse transcription–qPCR.
The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but
not all authors adhere to this convention.

Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least
one specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able
to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA
polymerase.
The PCR process generally consists of a series of temperature changes that are repeated 25 – 50 times. These cycles
normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acids double chain; the
second, at a temperature of around 50-60 °C, allows the binding of the primers with the DNA template; the third, at between
68 - 72 °C, facilitates the polymerization carried out by the DNA polymerase. Due to the small size of the fragments the
last step is usually omitted in this type of PCR as the enzyme is able to increase their number during the change between
the alignment stage and the denaturing stage. In addition, in four step PCR the fluorescence is measured during short
temperature phase lasting only a few seconds in each cycle, with a temperature of, for example, 80 °C, in order to reduce
the signal caused by the presence of primer dimers when a non-specific dye is used. The temperatures and the timings used
for each cycle depend on a wide variety of parameters, such as: the enzyme used to synthesize the DNA, the concentration
of divalent ions and deoxyribonucleotides (dNTPs) in the reaction and the bonding temperature of the primers.

Non-specific detection: Real-time PCR with double-stranded DNA-binding dyes as reporters

A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye. An increase in DNA
product during PCR therefore leads to an increase in fluorescence intensity measured at each cycle. However, dsDNA dyes
such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer).
This can potentially interfere with, or prevent, accurate monitoring of the intended target sequence.
In real-time PCR with dsDNA dyes the reaction is prepared as usual, with the addition of fluorescent dsDNA dye. Then
the reaction is run in a real-time PCR instrument, and after each cycle, the intensity of fluorescence is measured with a
detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). This method has the advantage of only
needing a pair of primers to carry out the amplification, which keeps costs down; however, only one target sequence can
be monitored in a tube.

Specific detection: fluorescent reporter probe method

Fluorescent reporter probes detect only the DNA containing the sequence complementary to the probe; therefore, use of
the reporter probe significantly increases specificity, and enables performing the technique even in the presence of other
dsDNA. Using different-coloured labels, fluorescent probes can be used in multiplex assays for monitoring several target
sequences in the same tube. The specificity of fluorescent reporter probes also prevents interference of measurements
caused by primer dimers, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do
not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the
reaction.
The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the
opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence;
breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity
and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in
the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due
to the breakdown of the probe and release of the reporter.
1. The PCR is prepared as usual, and the reporter probe is added.
2. As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.
3. Polymerization of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-
3'-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an
increase in fluorescence.
4. Fluorescence is detected and measured in a real-time PCR machine, and its geometric increase corresponding to
exponential increase of the product is used to determine the quantification cycle (Cq) in each reaction.
(1) In intact probes, reporter fluorescence is quenched.
(2) Probes and the complementary DNA strand are hybridized and reporter fluorescence is still quenched.
(3) During PCR, the probe is degraded by the Taq polymerase and the fluorescent reporter released.
4. INVERSE PCR
Inverse polymerase chain reaction (Inverse PCR) is a variant of the
polymerase chain reaction that is used to amplify DNA with only one
known sequence. One limitation of conventional PCR is that it requires
primers complementary to both termini of the target DNA, but this
method allows PCR to be carried out even if only one sequence is
available from which primers may be designed. Inverse PCR is
especially useful for the determination of insert locations. For example,
various retroviruses and transposons randomly integrate into genomic
DNA. To identify the sites where they have entered, the known,
"internal" viral or transposon sequences can be used to design primers
that will amplify a small portion of the flanking, "external" genomic
DNA. The amplified product can then be sequenced and compared
with DNA databases to locate the sequence which has been disrupted.
The inverse PCR method involves a series of restriction digests and
ligation, resulting in a looped fragment that can be primed for PCR
from a single section of known sequence. Then, like other polymerase
chain reaction processes, the DNA is amplified by the temperature-
sensitive DNA polymerase:
1. A target region with an internal section of known sequence and
unknown flanking regions is identified
2. Genomic DNA is digested into fragments of a few kilobases by a usually low-moderate frequency (6-8
base) cutting restriction enzyme.
3. Under low DNA concentrations, self-ligation is induced to give a circular DNA product.
4. PCR is carried out as usual, with primers complementary to sections of the known internal sequence.*
Finally the sequence is compared with the sequence available in the data base.

5. HOT START PCR

Hot start PCR is a modified form of polymerase chain reaction (PCR) which avoids a non-specific amplification of DNA
by inactivating the DNA polymerase at lower temperatures. In hot start PCR specific antibodies or reversible chemical
modifications of the protein (usually modifications of the lysine with organic acid anhydride) are used to block the activity
of the DNA polymerase at lower temperature. An initial activation step at 95℃ is required for activation of the protein.
This step will both denature antibodies linked to the active center of the enzyme, and also remove any lysine modifications
made with acid anhydride. The anti-Taq antibodies reduce the Taq polymerase activity below 72℃, the optimal
temperature at which the enzyme extends the primers. When the specific antibodies detach from Taq-polymerase, the
amplification proceeds with greater specificity.
In conventional PCR, the DNA polymerase is modestly active at room temperature and to a lesser degree, even on ice. In
some instances, when all the reaction components are put together, nonspecific primer annealing can occur due to these
low temperatures. This nonspecific annealed primer can then be extended by the DNA polymerase, generating nonspecific
products and lowering product yields.
Hot start PCR significantly reduces nonspecific priming, the formation of primer dimers, and often, increases product
yields. In Hot start long and accurate PCR, the impact on yield can be dramatic. Classic methods, while effective, involve
additional handling and increased risk of contamination.

6. MULTIPLEX PCR
Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of polymerase chain reaction to amplify several
different DNA sequences simultaneously (as if performing many separate PCR reactions all together in one reaction). This
process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal
cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing
temperature during PCR.
Multiplex-PCR was first described in 1988 as a method to detect deletions in the dystrophin gene. It has also been used
with the steroid sulfatase gene. In 2008, multiplex-PCR was used for analysis of microsatellites and SNPs.
Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that
are specific to different DNA sequences. By targeting multiple sequences at once, additional information may be gained
from a single test run that otherwise would require several times the reagents and more time to perform. Annealing
temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes,
i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
Alternatively, if amplicon sizes overlap, the different amplicons may be differentiated and visualized using primers that
have been dyed with different colour fluorescent dyes. Commercial multiplexing kits for PCR are available and used by
many forensic laboratories to amplify degraded DNA samples.
Applications
Some of the applications of multiplex PCR include:
1. Pathogen Identification
2. High Throughput SNP Genotyping
3. Mutation Analysis
4. Gene Deletion Analysis
5. Template Quantitation
6. Linkage Analysis
7. RNA Detection
8. Forensic Studies
9. Diet Analysis

7. TOUCHDOWN PCR
The touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase
chain reaction by which primers avoid amplifying nonspecific sequences. The annealing temperature during a polymerase
chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on
annealing temperature. At temperatures just above this point, only very specific base pairing between the primer and the
template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures
polymerase chain reaction results, as the nonspecific sequences to which primers anneal in early steps of amplification will
"swamp out" any specific sequences because of the exponential nature of polymerase amplification.

The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. The annealing
temperature is decreased in increments for every subsequent set of cycles (the number of individual cycles and increments
of temperature decrease is chosen by the experimenter). The primer will anneal at the highest temperature which is least-
permissive of nonspecific binding that it is able to tolerate. Thus, the first sequence amplified is the one between the regions
of greatest primer specificity; it is most likely that this is the sequence of interest. These fragments will be further amplified
during subsequent rounds at lower temperatures, and will outcompete the nonspecific sequences to which the primers may
bind at those lower temperatures. If the primer initially (during the higher-temperature phases) binds to the sequence of
interest, subsequent rounds of polymerase chain reaction can be performed upon the product to further amplify those
fragments. Touchdown increases specificity of the reaction at higher temperatures and increases the efficiency towards the
end by lowering the annealing temperature.