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

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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.

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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].

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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.

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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.

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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.

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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].

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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.

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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].

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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

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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.

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Figure 5.0 Ligation-Mediated PCR flow setup

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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].

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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.

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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

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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] .

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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.

18Figure 7.0 Hot-Start PCR flow

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].

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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].

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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

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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)

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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.

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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.

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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

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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

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(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 Tm

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

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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.

.

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