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Apeh Daniel O.         TYPES OF POLYMERASE CHAIN REACTION
DNA Replication which forms the basis of biological evolution and inheritance [1] is
a "semi conservative" process in that each (one) strand of the original double-stranded
DNA molecule serves as template for the reproduction of the complementary strand.
Hence, following DNA replication, two identical DNA molecules are been produced
from a single double-stranded DNA molecule [2].
The need to amplify genes for various purposes among which are forensic application,
genome studies, medical applications          have   led to the development of various
techniques now known as polymerase chain reaction (PCR) as a more convenient
alternative of gene cloning via recombinant DNA technology.
The idea of Polymerase chain reaction came up in 1983 when Kary Mullis a scientist
working for Cletus cooperation was driving along US route 101 in Northern
California; it was then introduced into the scientific community in 1985 at a
conference in October where Cetus also rewarded kary Mullis with $10,000 bonus for
his invention. Later, during a corporate reorganization, Cetus sold the patent for the
PCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million [3].
PCR technique which is also called a DNA photocopier, is an in vitro technique that
uses a few basic everyday molecular biology reagents to make large numbers of
copies of a specific DNA fragment or a specific region of a DNA strand in a test-tube.
The process which is carried out in a PCR machine requires DNA template, primer(s),
Taq or other polymerase(s), deoxynucleoside triphosphates (dNTPs), buffer solution
and divalent cations (eg.Mg2+ ) to run [2].
The basic steps in conducting a conventional PCR involves; Denaturation achieved by
heating the reaction mixture to a temperature between 90-98° C such that the dsDNA
is denatured into single strands by disrupting the hydrogen bonds between
complementary bases, Annealing achieved by cooling the reaction mixture to a
temperature of 45-60° C such that the primers base pair with the complementary
sequence in the DNA and the hydrogen bonds reform and Elongation achieved by
adjusting the temperature to 72° C which is ideal for polymerase allowing primers

                                                 1
extension by joining the bases complementary to DNA strands, the polymerase
   continually adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are
   added complementary to the template. This completes a first cycle another cycle is
   continued. As PCR machine is automated thermocycler the same cycle is repeated
   upto 30-40 times [1].
   This review attempts to summarize as many types of PCR as possible including the
   principles on which they work, their applications and in some cases their advantages
   and disadvantages as well as experimental procedures where necessary. The emphasis
   is neither placed on the PCR machine level of sophiscation nor time of its use (old or
   new) but on technical difference basically brought about by different applications of
   PCR.


                    TYPES OF POLYMERASE CHAIN REACTION


   INVERSE PCR
   The inverse PCR method includes a series of digestions and self-ligations with the
   DNA being cut by a restriction endonuclease. This cut results in a known sequence at
   either end of unknown sequences [4].
   Inverse PCR Steps
1) Target DNA is lightly cut into smaller fragments of several kilobases by restriction
   endonuclease digestion.
2) Self-ligation is induced under low concentrations causing the phosphate backbone to
   reform. This gives a circular DNA ligation product.
3) Target DNA is then restriction digested with a known endonuclease. This generates a
   cut within the known internal sequence generating a linear product with known
   terminal sequences. This can now be used for PCR (polymerase chain reaction).
4) Standard PCR is conducted with primers complementary to the now known internal
   sequences.


                                                  2
Inverse PCR uses standard PCR (polymerase chain reaction), however it has the
primers oriented in the reverse direction of the usual orientation. The template for the
reverse primers is a restriction fragment that has been ligated upon itself to form a
circle [5].




Figure 1.0. Inverse PCR Protocol
Uses: It is commonly used to identify the flanking sequences around genomic inserts.
Inverse PCR has numerous applications in molecular biology including the
amplification and identification of sequences flanking transposable elements, and the
identification of genomic inserts [6].

                                               3
MULTIPLEX PCR

   Multiplex PCR is a widespread molecular biology technique for amplification of
   multiple targets in a single PCR experiment. In a multiplexing assay, more than one
   target sequence can be amplified by using multiple primer pairs in a reaction mixture.
   As an extension to the practical use of PCR, this technique has the potential to
   produce considerable savings in time and effort within the laboratory without
   compromising on the utility of the experiment. 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 [7].

   Uses : Its has been found useful in Pathogen Identification, High Throughput SNP
   Genotyping, Mutation Analysis, Gene Deletion Analysis, Template Quantification,
   Linkage Analysis, RNA Detection and Forensic Studies [8].

   Types of Multiplex PCR

   Multiplexing reactions can be broadly divided into two

1. Single template PCR reaction; this technique uses a single template which can be a
   genomic DNA along with several pairs of forward and reverse primers to amplify
   specific regions within a template
2. Multiple template PCR reaction; this technique uses multiple templates and several
   primer sets in the same reaction tube. Presence of multiple primer may lead to cross
   hybridization with each other and the possibility of mis-priming with other templates.




                                                  4
Figure 2.0 Primer Design Parameters for Multiplex PCR

   Design of specific primer sets is essential for a successful multiplex reaction. The
   important primer design considerations described below are a key to specific
   amplification with high yield [9].

1. Primer Length: Multiplex PCR assays involve designing of large number of primers,
   hence it is required that the designed primer should be of appropriate length. Usually,
   primers of short length, in the range of 18-22 bases are used.
2. Melting Temperature: Primers with similar Tm, preferably between 55°C-60°C are
   used. For sequences with high GC content, primers with a higher Tm (preferably
   75°C-80°C) are recommended. A Tm variation of between 3°-5° C is acceptable for
   primers used in a pool.



                                                  5
3. Specificity: It is important to consider the specificity of designed primers to the target
   sequences, while preparing a multiplex assay, especially since competition exists
   when multiple target sequences are in a single reaction vessel.
4. Avoid Primer Dimer Formation: The designed primers should be checked for
   formation of primer dimers, with all the primers present in the reaction mixture.
   Dimerization leads to unspecific amplification. All other parameters are similar to
   standard PCR primer design guidelines.

   Advantages of Multiplex PCR

1. Internal Controls: Potential problems in a simple PCR include false negatives due to
   reaction failure or false positives due to contamination. False negatives are often
   revealed in multiplex assays because each amplicon provides an internal control for
   the other amplified fragments.
2. Efficiency: The expense of reagents and preparation time is less in multiplex PCR
   than in systems where several tubes of uniplex PCRs are used. A multiplex reaction is
   ideal for conserving costly polymerase and templates in short supply.
3. Indication of Template Quality: The quality of the template may be determined
   more effectively in multiplex than in a simple PCR reaction.
4. Indication of Template Quantity: The exponential amplification and internal
   standards of multiplex PCR can be used to assess the amount of a particular template
   in a sample. To quantitate templates accurately by multiplex PCR, the amount of
   reference template, the number of reaction cycles, and the minimum inhibition of the
   theoretical doubling of product for each cycle must be accounted.




                                                   6
NESTED PCR

This PCR increases the specificity of DNA amplification, by reducing background
due to non-specific amplification of DNA [10]. Two sets (instead of one pair) of
primers are used in two successive PCRs. In the first reaction, on pair of primers
“outer pair” is used to generate DNA products, which besides the intended target, may
still consist of non-specifically amplified DNA fragments. The product(s) are then
used in a second PCR after the reaction is diluted with a set of second set “nested or
internal” primers whose binding sites are completely or partially different from and
located 3' of each of the primers used in the first reaction. The specificity of PCR is
determined by the specificity of the PCR primers. For example, if your primers bind
to more than one locus (e.g. paralog or common domain), then more than one segment
of DNA will be amplified. To control for these possibilities, investigators often
employ nested primers to ensure specificity [11].
Nested PCR means that two pairs of PCR primers were used for a single locus (figure
1). The first pair amplified the locus as seen in any PCR experiment. The second pair
of primers (nested primers) bind within the first PCR product (figure 4) and produce
a second PCR product that will be shorter than the first one (figure 5). The logic
behind this strategy is that if the wrong locus were amplified by mistake, the
probability is very low that it would also be amplified a second time by a second pair
of primers [12].




                                              7
Figure 3.0 Nested PCR pattern




Figure 3.1. Nested PCR strategy. Segment of DNA with dots representing nondiscript
DNA sequence of unspecified length. The double lines represent a large distance
between the portion of DNA illustrated in this figure. The portions of DNA shown
with four bases in a row represent PCR primer binding sites, though real primers
would be longer.




Figure 3.2. The first pair of PCR primers (blue with arrows) bind to the outer pair of
primer binding sites and amplify all the DNA in between these two sites.

                                              8
Figure 3.3. PCR product after the first round of amplificaiton. Notice that the bases
outside the PCR primer pair are not present in the product.




Figure 3.4. Second pair of nested primers (red with arrows) bind to the first PCR
product. The binding sites for the second pair of primers are a few bases "internal" to
the first primer binding sites.




Figure 3.5. Final PCR product after second round of PCR. The length of the product
is defined by the location of the internal primer binding sites.

When a complete genome sequence is known, it is easier to be sure you will not
amplify the wrong locus but since very few of the world's genomes have been
sequenced completely, nested primers will continue to be an important control for
many experiments.

A drawback of this technique is that the addition of new primers after the first
amplification round increases the chances of nonspecific contamination; many clinical
labs avoid this technique for this reason [11].




                                                  9
MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA)

MLPA is used to establish the copy number of up to 45 nucleic acid sequences in one
single multiplex reaction. The method can be used for genomic DNA (including both
copy number detection and methylation quantification) as well as for mRNA
profiling, it permits multiple targets to be amplified with only a single primer pair,
thus avoiding the resolution limitations of multiplex PCR [13].
The principle of MLPA is based on the identification of target sequences by
hybridization of pairs of MLPA probes that bind to adjacent sequences and can then
be joined by a ligation reaction. In order to make one copy of each target sequence,
specific MLPA probes are added to a nucleic acid sample for each of the sequences of
interest.
The sequences are then simultaneously amplified with the use of only one primer pair,
resulting in a mixture of amplification products, in which each PCR product of each
MLPA probe has a unique length [13].
One PCR primer is fluorescently or isotopically labelled so that the MLPA reaction
products can be visualized when electrophoresed on a capillary sequencer or a gel.
Resulting chromatograms show size-separated fragments ranging from 130 to 490 bp.
The peak area or peak height of each amplification product reflects the relative copy
number of that target sequence. Comparison of the electrophoresis profile of the
tested sample to that obtained with a control sample enables the detection of deletions
or duplications of genomic regions of interest [14].
Figure 4.0 summarizes the MLPA workflow for common devices and tools for data
analysis




                                              10
11
Figure 4.0. MLPA workflow for common devices and tools for data analysis
LIGATION-MEDIATED PCR
Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' (or adaptors)
that are first ligated to fragments of the target DNA. PCR primers that anneal
to the linker sequences are then used to amplify the target fragments. This
method is deployed for DNA sequencing, genome walking, and DNA
footprinting A related technique is Amplified fragment length polymorphism,
which generates diagnostic fragments of a genome [15].
Primer-extension step (Step 3): a gene-specific primer (Primer 1) was annealed
at 48°C and the primer was extended with Sequenase enzyme at 48°C. Ligation
step (Step 4): all extended DNA fragments with a blunt-end and 5'-phosphate
group were ligated to an unphosphorylated synthetic asymmetric double-strand
linker. Linear amplification step (Step 5): a second gene-specific primer
(Primer 2) was annealed to DNA fragments for a one-cycle extension using
Taq DNA polymerase. Exponential amplification step (Step 6): the primer 2
and the linker primer (the longest of the two oligonucleotides of the linker)
were used to exponentially and specifically amplify DNA fragments.
Sequencing gel electrophoresis and electroblotting (Step 7): amplified DNA
fragments were size-separated on a denaturing 8% polyacrylamide gel and
transferred onto a nylon membrane by electroblotting. Hybridization (Step 8)
the nylon membrane was hybridized overnight with a gene-specific probe.




                                         12
Figure 5.0 Ligation-Mediated PCR flow setup


                                     13
The principle of Ligation Mediated PCR (LM-PCR). 1-Ligation with excess of
primers, 2-Polymerase chain reaction of individual fragments. In LM-PCR,
each fragment is amplified independently so that due to intrinsic differences
among individual fragments, some fragments are amplified less efficiently than
others. This results in non-uniform representation of original genetic material
in the resultant amplicon, which consequently leads to loss of genetic
information and inaccurate results [16].
Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive
sequencing technique available to map single-stranded DNA breaks at the
nucleotide level of resolution using genomic DNA. LMPCR has been adapted
to map DNA damage and reveal DNA–protein interactions inside living cells.
However, the sequence context (GC content), the global break frequency and
the current combination of DNA polymerases used in LMPCR affect the
quality of the results. In this study, we developed and optimized an LMPCR
protocol adapted for Pyrococcus furiosus exo– DNA polymerase (Pfu exo–).
The relative efficiency of    Pfu exo– was compared to T7-modified DNA
polymerase (Sequenase 2.0) at the primer extension step and to Thermus
aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR.
At all break frequencies tested, Pfu exo– proved to be more efficient than
Sequenase 2.0. During both primer extension and PCR amplification steps, the
ratio of DNA molecules per unit of DNA polymerase was the main
determinant of the efficiency of Pfu exo–, while the efficiency of Taq was less
affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer
of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo–
was clearly more efficient than Taq to specifically amplify extremely GC-rich
genomic DNA sequences. Our results show that a combination of Pfu exo– at
the primer extension step and Taq at the PCR amplification step is ideal for in
vivo DNA analysis and DNA damage mapping using LMPCR [17].


                                           14
METHYLATION-SPECIFIC PCR (MSP)
Methylation-specific PCR (MSP) is used to identify patterns of DNA
methylation at cytosine-guanine (CpG) islands in genomic DNA [18]. Target
DNA is first treated with sodium bisulphite, which converts unmethylated
cytosine bases to uracil, which is complementary to adenosine in PCR primers.
Two amplifications are then carried out on the bisulphite-treated DNA: One
primer set anneals to DNA with cytosines (corresponding to methylated
cytosine), and the other set anneals to DNA with uracil (corresponding to
unmethylated cytosine). MSP used in Q-PCR provides quantitative information
about the methylation state of a given CpG island [18].
Bisulphite sequencing (also known as bisulphite sequencing) is the use of
bisulfite treatment of DNA to determine its pattern of methylation. DNA
methylation was the first discovered epigenetic mark, and remains the most
studied. In animals it predominantly involves the addition of a methyl group to
the carbon-5 position of cytosine residues of the dinucleotide CpG, and is
implicated in repression of transcriptional activity.
Treatment of DNA with bisulphite converts cytosine residues to uracil, but
leaves 5-methylcytosine residues unaffected. Thus, bisulphite treatment
introduces specific changes in the DNA sequence that depend on the
methylation status of individual cytosine residues, yielding single- nucleotide
resolution information about the methylation status of a segment of DNA.
Various analyses can be performed on the altered sequence to retrieve this
information. The objective of this analysis is therefore reduced to
differentiating between single nucleotide polymorphisms (cytosines and
thymidine) resulting from bisulphite conversion.




                                            15
Figure 6.0 Outline of the chemical reaction that underlies the bisulphite-
mediated conversion of cytosine to uracil.




Figure 6.1 Methylation-specific PCR flow
Methylation-specific PCR is a sensitive method to discriminately amplify and
detect a methylated region of interest using methylated-specific primers on
bisulfite-converted genomic DNA. Such primers will anneal only to sequences
that are methylated, and thus containing 5-methylcytosines that are resistant to
conversion by bisulfite. In alternative fashion, unmethylated-specific primers
can be used. This alternative method of methylation analysis also uses
bisulfite-treated DNA but avoids the need to sequence the area of interest.
Instead, primer pairs are designed themselves to be "methylated-specific" by

                                          16
including sequences complementing only unconverted 5-methylcytosines, or,
on the converse, "unmethylated-specific", complementing thymines converted
from unmethylated cytosines.
Methylation is determined by the ability of the specific primer to achieve
amplification. This method is particularly useful to interrogate CpG islands
with possibly high methylation density, as increased numbers of CpG pairs in
the primer increase the specificity of the assay. Placing the CpG pair at the 3'-
end of the primer also improves the sensitivity. The initial report using MSP
described sufficient sensitivity to detect methylation of 0.1% of alleles. In
general, MSP and its related protocols are considered to be the most sensitive
when interrogating the methylation status at a specific locus.
The MethyLight method is based on MSP, but provides a quantitative analysis
using real-time PCR. Methylated-specific primers are used, and a methylated-
specific fluorescence reporter probe is also used that anneals to the amplified
region. In alternative fashion, the primers or probe can be designed without
methylation specificity if discrimination is needed between the CpG pairs
within the involved sequences. Quantitation is made in reference to a
methylated reference DNA. A modification to this protocol to increase the
specificity of the PCR for successfully bisulphite-converted DNA (ConLight-
MSP) uses an additional probe to bisulphite-unconverted DNA to quantify this
non-specific amplification [19].
Further methodology using MSP-amplified DNA analyzes the products using
melting curve analysis (Mc-MSP).This method amplifies bisulphite-converted
DNA with both methylated-specific and unmethylated-specific primers, and
determines the quantitative ratio of the two products by comparing the
differential peaks generated in a melting curve analysis. A high-resolution
melting analysis method that uses both real-time quantification and melting
analysis has been introduced, in particular, for sensitive detection of low-level
methylation [20] .

                                           17
HOT-START PCR
A technique that reduces non-specific amplification during the initial set up
stages of the PCR. It may be performed manually by heating the reaction
components to the melting temperature (e.g., 95°C) before adding the
polymerase. Specialized enzyme systems have been developed that inhibit the
polymerase's activity at ambient temperature, either by the binding of an
antibody or by the presence of covalently bound inhibitors that only dissociate
after a high-temperature activation step. Hot-start/cold-finish PCR is achieved
with new hybrid polymerases that are inactive at ambient temperature and are
instantly activated at elongation temperature [21].
Mechanical hot start PCR: all components of PCR are added to the PCR vial
except for the DNA polymerase enzyme which will be added just at the first
denaturation step.
Non mechanical hot start PCR: The use of a form of Taq DNA polymerase,
for example, Amplitaq Gold which is activated only if the reaction mixture is
heated at about 94°C (the first denaturation step). Other method depends on
covalent linking of the polymerase enzyme to certain inhibitors. The enzyme
becomes dissociated from these inhibitors at the first denaturation step.




  Figure 7.0 Hot-Start PCR flow
                                           18
ALLELE-SPECIFIC PCR
A diagnostic or cloning technique which is based on single-nucleotide
polymorphisms (SNPs) (single-base differences in DNA). It requires prior
knowledge of a DNA sequence, including differences between alleles, and uses
primers whose 3' ends encompass the SNP. PCR amplification under stringent
conditions is much less efficient in the presence of a mismatch between
template and primer, so successful amplification with an SNP-specific primer
signals presence of the specific SNP in a sequence [22].




Figure 8.0 Allele-Specific PCR flow

HELICASE-DEPENDENT AMPLIFICATION
This PCR is similar to traditional PCR, but uses a constant temperature rather
than cycling through denaturation and annealing/extension cycles. DNA
helicase, an enzyme that unwinds DNA, is used in place of thermal
denaturation [27].


                                          19
REVERSE TRANSCRIPTION PCR (RT-PCR)

A PCR designed for amplifying DNA from RNA. Reverse transcriptase
reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR
is widely used in expression profiling, to determine the expression of a gene or
to identify the sequence of an RNA transcript, including transcription start and
termination sites. If the genomic DNA sequence of a gene is known, RT-PCR
can be used to map the location of exons and introns in the gene. The 5' end of
a gene (corresponding to the transcription start site) is typically identified by
RACE-PCR (Rapid Amplification of cDNA Ends) [23].




Figure 9.0 Reverse Transcription PCR flow

IN SITU PCR (ISH)

A polymerase chain reaction that actually takes place inside the cell on a slide.
In situ PCR amplification can be performed on fixed tissue or cells [24].

                                           20
ASSEMBLY PCR OR POLYMERASE CYCLING ASSEMBLY (PCA)

This entails the artificial synthesis of long DNA sequences by performing PCR
on a pool of long oligonucleotides with short overlapping segments. The
oligonucleotides alternate between sense and antisense directions, and the
overlapping segments determine the order of the PCR fragments, thereby
selectively producing the final long DNA product [25].

ASYMMETRIC PCR

This reaction preferentially amplifies one DNA strand in a double-stranded
DNA template. It is used in sequencing and hybridization probing where
amplification of only one of the two complementary strands is required. PCR is
carried out as usual, but with a great excess of the primer for the strand
targeted for amplification. Because of the slow (arithmetic) amplification later
in the reaction after the limiting primer has been used up, extra cycles of PCR
are required. A recent modification on this process, known as Linear-After-
The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher
melting temperature (Tm) than the excess primer to maintain reaction
efficiency as the limiting primer concentration decreases mid-reaction [26].

THERMAL ASYMMETRIC INTERLACED PCR (TAIL-PCR)

This reaction is applied in the isolation of an unknown sequence flanking a
known sequence. Within the known sequence, TAIL-PCR uses a nested pair of
primers with differing annealing temperatures; a degenerate primer is used to
amplify in the other direction from the unknown sequence [27].

Uses: TAIL-PCR as a powerful tool for amplifying insert end segments from
P1, BAC and YAC clones, the amplified products were highly specific and
suitable as probes for library screening and as templates for direct sequencing

                                          21
while the recover insert ends can also be used for chromosome walking and
mapping

Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is a
fast and efficient method to amplify unknown sequences adjacent to known
insertion sites in Arabidopsis. Nested, insertion-specific primers are used
together with arbitrary degenerate primers (AD primers), which are designed to
differ in their annealing temperatures. Alternating cycles of high and low
annealing temperature yield specific products bordered by an insertion-specific
primer on one side and an AD primer on the other. Further specificity is
obtained through subsequent rounds of TAIL-PCR, using nested insertion-
specific primers. The increasing availability of whole genome sequences
renders TAIL-PCR an attractive tool to easily identify insertion sites in large
genome tagging populations through the direct sequencing of TAIL-PCR
products. For large-scale functional genomics approaches, it is desirable to
obtain flanking sequences for each individual in the population in a fast and
cost-effective manner.
Experimental Details
      Primary reaction.
In the primary reaction, one low stringency PCR cycle is conducted to create
one or more annealing sites for the AD primer in the targeted sequence.
Specific products are then amplified over non-specific ones by interspersion of
two high-stringency PCR cycles with one reduced-stringency PCR cycle.
Set up 4 reactions as follows (one with each AD primer):
2 μl 10 X PCR buffer
1.2 μl 25 mM MgCl2
0.2 μl 10 mMdNTP’s
0.2 μl 100 ngμl-1 specific primer 1 (furthest away from AD) (0.15μM final)
2 μl20 μM AD primer (2 μM final)

                                          22
0.2 μlTaq DNA polymerase
0.4 μl DMSO
1 μl DNA (1-20ngμl-1)
12.8 μlH2O
There is no need to run out this primary reaction. It should contain a medium
yield of specific products, a high yield of non-targeted products, and a low
yield of non-specific products. The nested primers used in the secondary and
tertiary reactions result in very low yields of non-specific products, very high
yields of specific products and no amplification of non-targeted products.


      Secondary reaction
For the secondary reaction, a 1/40 dilution of the primary PCR product is used
as template, and the specific primer is the middle one of the three specific
primers.
Set up reaction as follows:
2.5 μl 10 X PCR buffer
1.5 μl 25 mM MgCl2
0.25 μl 10 mMdNTP’s
0.3 μl 100 ngμl-1 specific primer 2 (middle nested) (0.2 μM final)
2.5 μl 20 μM AD primer (2 μM final)
0.2 μl Taq DNA polymerase
0.5 μl DMSO
1 μl DNA (1/40 dilution of primary PCR products)
16.25 μl H2O
      Tertiary reaction
For the tertiary reaction, the SAME template (i.e primary PCR product) is used
but thistime in a 1/10 dilution. I usually simply add 4 X of the 1/40 dilution
used for thesecondary reaction. This removes the possibility of getting false
positives. The specificprimer used is the primer nearest the unknown sequence.

                                          23
Set up reaction as follows:
5 μl10 X PCR buffer
3 μl25 mM MgCl2
0.5 μl10 mMdNTP’s
0.6 μl 100ngμl-1 specific primer 3 (closest to AD) (0.2 μM final)
5 μl20 μM any one AD primer (2 μM final)
0.4 μlTaq DNA polymerase
1 μl DMSO
4 μlDNA (1/40 dilution of primary PCR products)
31 μl H2O




Agarose gel analysis
The secondary and tertiary products are run in adjacent lanes on a 1.2%
agarose gel. The specificity of the products is confirmed by the expected size
change between the secondary and tertiary products.


                                          24
QUANTITATIVE PCR (Q-PCR)
Used to measure the quantity of a PCR product (commonly in real-time). It
quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is
commonly used to determine whether a DNA sequence is present in a sample
and the number of its copies in the sample. Quantitative real-time PCR has a
very high degree of precision. QRT-PCR methods use fluorescent dyes, such
as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as
TaqMan, to measure the amount of amplified product in real time. It is also
sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR
or RTQ-PCR are more appropriate contractions, since RT-PCR commonly


                                        25
refers to reverse transcription PCR (see below), often used in conjunction with
Q-PCR [27].

OTHER TYPES OF PCR

LONG PCR
Long PCR is a PCR is which extended or longer than standard PCR, meaning
over 5 kilobases (frequently over 10 kb). Long PCR is usually only useful if it
is accurate. Thus, special mixtures of proficient polymerases along with
accurate polymerases such as Pfu are often mixed together. Applications of
Long PCR Long PCR is often used to clone larger genes or large segments of
DNA which standard PCR cannot [27].
COLONY PCR
The screening of bacterial (E.Coli) or yeast clones for correct ligation or
plasmid products [27]. Selected colonies of bacteria or yeast are picked with a
sterile toothpick or pipette tip from a growth (agarose) plate. This is then
inserted into the PCR master mix or pre-inserted into autoclaved water. PCR
is then conducted to determine if the colony contains the DNA fragment or
plasmid of interest [28].
THE DIGITAL PCR
The Digital polymerase chain reaction simultaneously amplifies thousands of
samples, each in a separate droplet within an emulsion [29].
OVERLAP-EXTENSION PCR
A genetic engineering technique allowing the construction of a DNA sequence
with an alteration inserted beyond the limit of the longest practical primer
length [30].
SOLID PHASE PCR
encompasses multiple meanings, including Colony Amplification (where PCR
colonies are derived in a gel matrix, for example), 'Bridge PCR' (primers are
covalently linked to a solid-support surface), conventional Solid Phase PCR
                                          26
(where Asymmetric PCR is applied in the presence of solid support bearing
primer with sequence matching one of the aqueous primers) and Enhanced
Solid Phase PCR (where conventional Solid Phase PCR can be improved by
employing high Tm and nested solid support primer with optional application
of a thermal 'step' to favour solid support priming) [31].
TOUCHDOWN PCR (STEP-DOWN PCR)
A variant of PCR that aims to reduce nonspecific background by gradually
lowering the annealing temperature as PCR cycling progresses. The annealing
temperature at the initial cycles is usually a few degrees (3-5°C) above the T m
of the primers used, while at the later cycles, it is a few degrees (3-5°C) below
the primer Tm. The higher temperatures give greater specificity for primer
binding, and the lower temperatures permit more efficient amplification from
the specific products formed during the initial cycles [32].
MINIPRIMER PCR
This reaction uses a thermostable polymerase (S-Tbr) that can extend from
short primers ("smalligos") as short as 9 or 10 nucleotides. This method
permits PCR targeting to smaller primer binding regions, and is used to
amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA
gene [27].
UNIVERSAL FAST WALKING PCR
Used for genome walking and genetic fingerprinting using a more specific
'two-sided' PCR than conventional 'one-sided' approaches (using only one
gene-specific primer and one general primer - which can lead to artefactual
'noise') by virtue of a mechanism involving lariat structure formation.
Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested
PCR for rapid amplification of genomic DNA ends), 5'RACE LaNe and
3'RACE LaNe [27].


VARIABLE NUMBER OF TANDEM REPEATS (VNTR) PCR

                                            27
This method targets areas of the genome that exhibit length variation. The
analysis of the genotypes of the sample usually involves sizing of the
amplification products by gel electrophoresis. Analysis of smaller VNTR
segments known as Short Tandem Repeats (or STRs) is the basis for DNA
Fingerprinting databases such as CODIS [27].

INTERSEQUENCE-SPECIFIC PCR (OR ISSR-PCR)

This is a method for DNA fingerprinting that uses primers selected from
segments repeated throughout a genome to produce a unique fingerprint of
amplified product lengths. The use of primers from a commonly repeated
segment is called Alu-PCR, and can help amplify sequences adjacent (or
between) these repeats [27].

CONCLUSION
Current variations of PCR in use are more in number than those highlighted in this
discussion, most of these PCRs have specific applications even in new areas of
science. This revive have brought out most types of PCR and detailed some even to
specific methodology, their principles of operation and their use and also successfully
shown that the possibilities that come with the manipulation of DNA are inexhaustible.


.




REFERENCES

                                          28
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17. Calvo B, Bilbao JR, Urrutia I, Eizaguirre J, Gaztambide S, Castano L
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Identification of a novel nonsense mutation and a missense substitution in the
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neurohypophyseal diabetes insipidus. J Clin Endocrinol Metab. 83(3):995-7

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                                         33

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Types of PCR ((APEH Daniel O.))

  • 1. Apeh Daniel O. TYPES OF POLYMERASE CHAIN REACTION DNA Replication which forms the basis of biological evolution and inheritance [1] is a "semi conservative" process in that each (one) strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules are been produced from a single double-stranded DNA molecule [2]. The need to amplify genes for various purposes among which are forensic application, genome studies, medical applications have led to the development of various techniques now known as polymerase chain reaction (PCR) as a more convenient alternative of gene cloning via recombinant DNA technology. The idea of Polymerase chain reaction came up in 1983 when Kary Mullis a scientist working for Cletus cooperation was driving along US route 101 in Northern California; it was then introduced into the scientific community in 1985 at a conference in October where Cetus also rewarded kary Mullis with $10,000 bonus for his invention. Later, during a corporate reorganization, Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million [3]. PCR technique which is also called a DNA photocopier, is an in vitro technique that uses a few basic everyday molecular biology reagents to make large numbers of copies of a specific DNA fragment or a specific region of a DNA strand in a test-tube. The process which is carried out in a PCR machine requires DNA template, primer(s), Taq or other polymerase(s), deoxynucleoside triphosphates (dNTPs), buffer solution and divalent cations (eg.Mg2+ ) to run [2]. The basic steps in conducting a conventional PCR involves; Denaturation achieved by heating the reaction mixture to a temperature between 90-98° C such that the dsDNA is denatured into single strands by disrupting the hydrogen bonds between complementary bases, Annealing achieved by cooling the reaction mixture to a temperature of 45-60° C such that the primers base pair with the complementary sequence in the DNA and the hydrogen bonds reform and Elongation achieved by adjusting the temperature to 72° C which is ideal for polymerase allowing primers 1
  • 2. extension by joining the bases complementary to DNA strands, the polymerase continually adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template. This completes a first cycle another cycle is continued. As PCR machine is automated thermocycler the same cycle is repeated upto 30-40 times [1]. This review attempts to summarize as many types of PCR as possible including the principles on which they work, their applications and in some cases their advantages and disadvantages as well as experimental procedures where necessary. The emphasis is neither placed on the PCR machine level of sophiscation nor time of its use (old or new) but on technical difference basically brought about by different applications of PCR. TYPES OF POLYMERASE CHAIN REACTION INVERSE PCR The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a restriction endonuclease. This cut results in a known sequence at either end of unknown sequences [4]. Inverse PCR Steps 1) Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion. 2) Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation product. 3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction). 4) Standard PCR is conducted with primers complementary to the now known internal sequences. 2
  • 3. Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle [5]. Figure 1.0. Inverse PCR Protocol Uses: It is commonly used to identify the flanking sequences around genomic inserts. Inverse PCR has numerous applications in molecular biology including the amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts [6]. 3
  • 4. MULTIPLEX PCR Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment. 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 [7]. Uses : Its has been found useful in Pathogen Identification, High Throughput SNP Genotyping, Mutation Analysis, Gene Deletion Analysis, Template Quantification, Linkage Analysis, RNA Detection and Forensic Studies [8]. Types of Multiplex PCR Multiplexing reactions can be broadly divided into two 1. Single template PCR reaction; this technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template 2. Multiple template PCR reaction; this technique uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primer may lead to cross hybridization with each other and the possibility of mis-priming with other templates. 4
  • 5. Figure 2.0 Primer Design Parameters for Multiplex PCR Design of specific primer sets is essential for a successful multiplex reaction. The important primer design considerations described below are a key to specific amplification with high yield [9]. 1. Primer Length: Multiplex PCR assays involve designing of large number of primers, hence it is required that the designed primer should be of appropriate length. Usually, primers of short length, in the range of 18-22 bases are used. 2. Melting Temperature: Primers with similar Tm, preferably between 55°C-60°C are used. For sequences with high GC content, primers with a higher Tm (preferably 75°C-80°C) are recommended. A Tm variation of between 3°-5° C is acceptable for primers used in a pool. 5
  • 6. 3. Specificity: It is important to consider the specificity of designed primers to the target sequences, while preparing a multiplex assay, especially since competition exists when multiple target sequences are in a single reaction vessel. 4. Avoid Primer Dimer Formation: The designed primers should be checked for formation of primer dimers, with all the primers present in the reaction mixture. Dimerization leads to unspecific amplification. All other parameters are similar to standard PCR primer design guidelines. Advantages of Multiplex PCR 1. Internal Controls: Potential problems in a simple PCR include false negatives due to reaction failure or false positives due to contamination. False negatives are often revealed in multiplex assays because each amplicon provides an internal control for the other amplified fragments. 2. Efficiency: The expense of reagents and preparation time is less in multiplex PCR than in systems where several tubes of uniplex PCRs are used. A multiplex reaction is ideal for conserving costly polymerase and templates in short supply. 3. Indication of Template Quality: The quality of the template may be determined more effectively in multiplex than in a simple PCR reaction. 4. Indication of Template Quantity: The exponential amplification and internal standards of multiplex PCR can be used to assess the amount of a particular template in a sample. To quantitate templates accurately by multiplex PCR, the amount of reference template, the number of reaction cycles, and the minimum inhibition of the theoretical doubling of product for each cycle must be accounted. 6
  • 7. NESTED PCR This PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA [10]. Two sets (instead of one pair) of primers are used in two successive PCRs. In the first reaction, on pair of primers “outer pair” is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR after the reaction is diluted with a set of second set “nested or internal” primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. The specificity of PCR is determined by the specificity of the PCR primers. For example, if your primers bind to more than one locus (e.g. paralog or common domain), then more than one segment of DNA will be amplified. To control for these possibilities, investigators often employ nested primers to ensure specificity [11]. Nested PCR means that two pairs of PCR primers were used for a single locus (figure 1). The first pair amplified the locus as seen in any PCR experiment. The second pair of primers (nested primers) bind within the first PCR product (figure 4) and produce a second PCR product that will be shorter than the first one (figure 5). The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers [12]. 7
  • 8. Figure 3.0 Nested PCR pattern Figure 3.1. Nested PCR strategy. Segment of DNA with dots representing nondiscript DNA sequence of unspecified length. The double lines represent a large distance between the portion of DNA illustrated in this figure. The portions of DNA shown with four bases in a row represent PCR primer binding sites, though real primers would be longer. Figure 3.2. The first pair of PCR primers (blue with arrows) bind to the outer pair of primer binding sites and amplify all the DNA in between these two sites. 8
  • 9. Figure 3.3. PCR product after the first round of amplificaiton. Notice that the bases outside the PCR primer pair are not present in the product. Figure 3.4. Second pair of nested primers (red with arrows) bind to the first PCR product. The binding sites for the second pair of primers are a few bases "internal" to the first primer binding sites. Figure 3.5. Final PCR product after second round of PCR. The length of the product is defined by the location of the internal primer binding sites. When a complete genome sequence is known, it is easier to be sure you will not amplify the wrong locus but since very few of the world's genomes have been sequenced completely, nested primers will continue to be an important control for many experiments. A drawback of this technique is that the addition of new primers after the first amplification round increases the chances of nonspecific contamination; many clinical labs avoid this technique for this reason [11]. 9
  • 10. MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA) MLPA is used to establish the copy number of up to 45 nucleic acid sequences in one single multiplex reaction. The method can be used for genomic DNA (including both copy number detection and methylation quantification) as well as for mRNA profiling, it permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR [13]. The principle of MLPA is based on the identification of target sequences by hybridization of pairs of MLPA probes that bind to adjacent sequences and can then be joined by a ligation reaction. In order to make one copy of each target sequence, specific MLPA probes are added to a nucleic acid sample for each of the sequences of interest. The sequences are then simultaneously amplified with the use of only one primer pair, resulting in a mixture of amplification products, in which each PCR product of each MLPA probe has a unique length [13]. One PCR primer is fluorescently or isotopically labelled so that the MLPA reaction products can be visualized when electrophoresed on a capillary sequencer or a gel. Resulting chromatograms show size-separated fragments ranging from 130 to 490 bp. The peak area or peak height of each amplification product reflects the relative copy number of that target sequence. Comparison of the electrophoresis profile of the tested sample to that obtained with a control sample enables the detection of deletions or duplications of genomic regions of interest [14]. Figure 4.0 summarizes the MLPA workflow for common devices and tools for data analysis 10
  • 11. 11 Figure 4.0. MLPA workflow for common devices and tools for data analysis
  • 12. LIGATION-MEDIATED PCR Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' (or adaptors) that are first ligated to fragments of the target DNA. PCR primers that anneal to the linker sequences are then used to amplify the target fragments. This method is deployed for DNA sequencing, genome walking, and DNA footprinting A related technique is Amplified fragment length polymorphism, which generates diagnostic fragments of a genome [15]. Primer-extension step (Step 3): a gene-specific primer (Primer 1) was annealed at 48°C and the primer was extended with Sequenase enzyme at 48°C. Ligation step (Step 4): all extended DNA fragments with a blunt-end and 5'-phosphate group were ligated to an unphosphorylated synthetic asymmetric double-strand linker. Linear amplification step (Step 5): a second gene-specific primer (Primer 2) was annealed to DNA fragments for a one-cycle extension using Taq DNA polymerase. Exponential amplification step (Step 6): the primer 2 and the linker primer (the longest of the two oligonucleotides of the linker) were used to exponentially and specifically amplify DNA fragments. Sequencing gel electrophoresis and electroblotting (Step 7): amplified DNA fragments were size-separated on a denaturing 8% polyacrylamide gel and transferred onto a nylon membrane by electroblotting. Hybridization (Step 8) the nylon membrane was hybridized overnight with a gene-specific probe. 12
  • 13. Figure 5.0 Ligation-Mediated PCR flow setup 13
  • 14. The principle of Ligation Mediated PCR (LM-PCR). 1-Ligation with excess of primers, 2-Polymerase chain reaction of individual fragments. In LM-PCR, each fragment is amplified independently so that due to intrinsic differences among individual fragments, some fragments are amplified less efficiently than others. This results in non-uniform representation of original genetic material in the resultant amplicon, which consequently leads to loss of genetic information and inaccurate results [16]. Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA–protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo– DNA polymerase (Pfu exo–). The relative efficiency of Pfu exo– was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo– proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo–, while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo– was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo– at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR [17]. 14
  • 15. METHYLATION-SPECIFIC PCR (MSP) Methylation-specific PCR (MSP) is used to identify patterns of DNA methylation at cytosine-guanine (CpG) islands in genomic DNA [18]. Target DNA is first treated with sodium bisulphite, which converts unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR primers. Two amplifications are then carried out on the bisulphite-treated DNA: One primer set anneals to DNA with cytosines (corresponding to methylated cytosine), and the other set anneals to DNA with uracil (corresponding to unmethylated cytosine). MSP used in Q-PCR provides quantitative information about the methylation state of a given CpG island [18]. Bisulphite sequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA to determine its pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity. Treatment of DNA with bisulphite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulphite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single- nucleotide resolution information about the methylation status of a segment of DNA. Various analyses can be performed on the altered sequence to retrieve this information. The objective of this analysis is therefore reduced to differentiating between single nucleotide polymorphisms (cytosines and thymidine) resulting from bisulphite conversion. 15
  • 16. Figure 6.0 Outline of the chemical reaction that underlies the bisulphite- mediated conversion of cytosine to uracil. Figure 6.1 Methylation-specific PCR flow Methylation-specific PCR is a sensitive method to discriminately amplify and detect a methylated region of interest using methylated-specific primers on bisulfite-converted genomic DNA. Such primers will anneal only to sequences that are methylated, and thus containing 5-methylcytosines that are resistant to conversion by bisulfite. In alternative fashion, unmethylated-specific primers can be used. This alternative method of methylation analysis also uses bisulfite-treated DNA but avoids the need to sequence the area of interest. Instead, primer pairs are designed themselves to be "methylated-specific" by 16
  • 17. including sequences complementing only unconverted 5-methylcytosines, or, on the converse, "unmethylated-specific", complementing thymines converted from unmethylated cytosines. Methylation is determined by the ability of the specific primer to achieve amplification. This method is particularly useful to interrogate CpG islands with possibly high methylation density, as increased numbers of CpG pairs in the primer increase the specificity of the assay. Placing the CpG pair at the 3'- end of the primer also improves the sensitivity. The initial report using MSP described sufficient sensitivity to detect methylation of 0.1% of alleles. In general, MSP and its related protocols are considered to be the most sensitive when interrogating the methylation status at a specific locus. The MethyLight method is based on MSP, but provides a quantitative analysis using real-time PCR. Methylated-specific primers are used, and a methylated- specific fluorescence reporter probe is also used that anneals to the amplified region. In alternative fashion, the primers or probe can be designed without methylation specificity if discrimination is needed between the CpG pairs within the involved sequences. Quantitation is made in reference to a methylated reference DNA. A modification to this protocol to increase the specificity of the PCR for successfully bisulphite-converted DNA (ConLight- MSP) uses an additional probe to bisulphite-unconverted DNA to quantify this non-specific amplification [19]. Further methodology using MSP-amplified DNA analyzes the products using melting curve analysis (Mc-MSP).This method amplifies bisulphite-converted DNA with both methylated-specific and unmethylated-specific primers, and determines the quantitative ratio of the two products by comparing the differential peaks generated in a melting curve analysis. A high-resolution melting analysis method that uses both real-time quantification and melting analysis has been introduced, in particular, for sensitive detection of low-level methylation [20] . 17
  • 18. HOT-START PCR A technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature [21]. Mechanical hot start PCR: all components of PCR are added to the PCR vial except for the DNA polymerase enzyme which will be added just at the first denaturation step. Non mechanical hot start PCR: The use of a form of Taq DNA polymerase, for example, Amplitaq Gold which is activated only if the reaction mixture is heated at about 94°C (the first denaturation step). Other method depends on covalent linking of the polymerase enzyme to certain inhibitors. The enzyme becomes dissociated from these inhibitors at the first denaturation step. Figure 7.0 Hot-Start PCR flow 18
  • 19. ALLELE-SPECIFIC PCR A diagnostic or cloning technique which is based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence [22]. Figure 8.0 Allele-Specific PCR flow HELICASE-DEPENDENT AMPLIFICATION This PCR is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation [27]. 19
  • 20. REVERSE TRANSCRIPTION PCR (RT-PCR) A PCR designed for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends) [23]. Figure 9.0 Reverse Transcription PCR flow IN SITU PCR (ISH) A polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can be performed on fixed tissue or cells [24]. 20
  • 21. ASSEMBLY PCR OR POLYMERASE CYCLING ASSEMBLY (PCA) This entails the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product [25]. ASYMMETRIC PCR This reaction preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After- The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction [26]. THERMAL ASYMMETRIC INTERLACED PCR (TAIL-PCR) This reaction is applied in the isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence [27]. Uses: TAIL-PCR as a powerful tool for amplifying insert end segments from P1, BAC and YAC clones, the amplified products were highly specific and suitable as probes for library screening and as templates for direct sequencing 21
  • 22. while the recover insert ends can also be used for chromosome walking and mapping Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is a fast and efficient method to amplify unknown sequences adjacent to known insertion sites in Arabidopsis. Nested, insertion-specific primers are used together with arbitrary degenerate primers (AD primers), which are designed to differ in their annealing temperatures. Alternating cycles of high and low annealing temperature yield specific products bordered by an insertion-specific primer on one side and an AD primer on the other. Further specificity is obtained through subsequent rounds of TAIL-PCR, using nested insertion- specific primers. The increasing availability of whole genome sequences renders TAIL-PCR an attractive tool to easily identify insertion sites in large genome tagging populations through the direct sequencing of TAIL-PCR products. For large-scale functional genomics approaches, it is desirable to obtain flanking sequences for each individual in the population in a fast and cost-effective manner. Experimental Details Primary reaction. In the primary reaction, one low stringency PCR cycle is conducted to create one or more annealing sites for the AD primer in the targeted sequence. Specific products are then amplified over non-specific ones by interspersion of two high-stringency PCR cycles with one reduced-stringency PCR cycle. Set up 4 reactions as follows (one with each AD primer): 2 μl 10 X PCR buffer 1.2 μl 25 mM MgCl2 0.2 μl 10 mMdNTP’s 0.2 μl 100 ngμl-1 specific primer 1 (furthest away from AD) (0.15μM final) 2 μl20 μM AD primer (2 μM final) 22
  • 23. 0.2 μlTaq DNA polymerase 0.4 μl DMSO 1 μl DNA (1-20ngμl-1) 12.8 μlH2O There is no need to run out this primary reaction. It should contain a medium yield of specific products, a high yield of non-targeted products, and a low yield of non-specific products. The nested primers used in the secondary and tertiary reactions result in very low yields of non-specific products, very high yields of specific products and no amplification of non-targeted products. Secondary reaction For the secondary reaction, a 1/40 dilution of the primary PCR product is used as template, and the specific primer is the middle one of the three specific primers. Set up reaction as follows: 2.5 μl 10 X PCR buffer 1.5 μl 25 mM MgCl2 0.25 μl 10 mMdNTP’s 0.3 μl 100 ngμl-1 specific primer 2 (middle nested) (0.2 μM final) 2.5 μl 20 μM AD primer (2 μM final) 0.2 μl Taq DNA polymerase 0.5 μl DMSO 1 μl DNA (1/40 dilution of primary PCR products) 16.25 μl H2O Tertiary reaction For the tertiary reaction, the SAME template (i.e primary PCR product) is used but thistime in a 1/10 dilution. I usually simply add 4 X of the 1/40 dilution used for thesecondary reaction. This removes the possibility of getting false positives. The specificprimer used is the primer nearest the unknown sequence. 23
  • 24. Set up reaction as follows: 5 μl10 X PCR buffer 3 μl25 mM MgCl2 0.5 μl10 mMdNTP’s 0.6 μl 100ngμl-1 specific primer 3 (closest to AD) (0.2 μM final) 5 μl20 μM any one AD primer (2 μM final) 0.4 μlTaq DNA polymerase 1 μl DMSO 4 μlDNA (1/40 dilution of primary PCR products) 31 μl H2O Agarose gel analysis The secondary and tertiary products are run in adjacent lanes on a 1.2% agarose gel. The specificity of the products is confirmed by the expected size change between the secondary and tertiary products. 24
  • 25. QUANTITATIVE PCR (Q-PCR) Used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly 25
  • 26. refers to reverse transcription PCR (see below), often used in conjunction with Q-PCR [27]. OTHER TYPES OF PCR LONG PCR Long PCR is a PCR is which extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb). Long PCR is usually only useful if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together. Applications of Long PCR Long PCR is often used to clone larger genes or large segments of DNA which standard PCR cannot [27]. COLONY PCR The screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products [27]. Selected colonies of bacteria or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of interest [28]. THE DIGITAL PCR The Digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion [29]. OVERLAP-EXTENSION PCR A genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length [30]. SOLID PHASE PCR encompasses multiple meanings, including Colony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR 26
  • 27. (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming) [31]. TOUCHDOWN PCR (STEP-DOWN PCR) A variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the T m of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles [32]. MINIPRIMER PCR This reaction uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene [27]. UNIVERSAL FAST WALKING PCR Used for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends), 5'RACE LaNe and 3'RACE LaNe [27]. VARIABLE NUMBER OF TANDEM REPEATS (VNTR) PCR 27
  • 28. This method targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting databases such as CODIS [27]. INTERSEQUENCE-SPECIFIC PCR (OR ISSR-PCR) This is a method for DNA fingerprinting that uses primers selected from segments repeated throughout a genome to produce a unique fingerprint of amplified product lengths. The use of primers from a commonly repeated segment is called Alu-PCR, and can help amplify sequences adjacent (or between) these repeats [27]. CONCLUSION Current variations of PCR in use are more in number than those highlighted in this discussion, most of these PCRs have specific applications even in new areas of science. This revive have brought out most types of PCR and detailed some even to specific methodology, their principles of operation and their use and also successfully shown that the possibilities that come with the manipulation of DNA are inexhaustible. . REFERENCES 28
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