Session: 2019-2021

History of PCR, Primer Designing and

use of different polymerases in PCR

Assignment of PCR (theory)

Submitted to : Dr Ambreen
Submitted by: Muhammad Mushtaq
Class: M.Phil (Regular)
Roll.No: Mphil-BT-05F19

Contents:
Table of Contents

History of PCR...........................................................................................................................................4
Events before PCR invention:..................................................................................................................4
Invention and development of PCR:........................................................................................................8
Evolution of PCR:.................................................................................................................................10
Primer designing for PCR:.....................................................................................................................10
1. Primer Length:...................................................................................................................................11
2. Primer Melting Temperature:............................................................................................................11
3. Primer Annealing Temperature:........................................................................................................12
4. GC Clamp:.........................................................................................................................................13
5. GC Content:.......................................................................................................................................13
6. Primer Secondary Structures:............................................................................................................13
7. Runs:..................................................................................................................................................14
8. Repeats:.............................................................................................................................................14
9. 3' End Stability:................................................................................................................................14
10. Avoid Cross Homology:..................................................................................................................15
11. Avoid Template Secondary Structure:.............................................................................................15
Parameters of Designing Primer Pair.....................................................................................................15
1.Amplicon Length:.......................................................................................................................15
2. Product Positi on:......................................................................................................................16
3. Tm of Product:..........................................................................................................................16
4. Opti mum Annealing Temperature (T a   Opt):........................................................................16
5. Primer Pair Tm Mismatch Calculati on:................................................................................16
Primer Design using Software...............................................................................................................16
DNA polymerases used in PCR:.............................................................................................................17
Structure and Mechanism of Action:.....................................................................................................17
Properties of DNA polymerases used in PCR........................................................................................18
Fidelity...............................................................................................................................................19
Processivity........................................................................................................................................19
Extension rate....................................................................................................................................19
Thermostability..................................................................................................................................19
 Examples of PCR DNA polymerases:...................................................................................................20
Taq polymerase.................................................................................................................................20
Pfu DNA Polymerase..........................................................................................................................20
KOD DNA Polymerase........................................................................................................................21
Long-Range DNA polymerase............................................................................................................21
Hot-start DNA Polymerase.................................................................................................................22
Tth polymerase..................................................................................................................................22
Pwo DNA polymerase........................................................................................................................22
References:...............................................................................................................................................23
 History of PCR

Events before PCR invention:

There are many important events that helped in the PCR invention and development regarding to

the history of PCR. These include

 finding of DNA structure,

 replication mechanism,

 genetic code elucidation,

 discovery of Taq polymerase

 Sanger sequencing.

 On April 25, 1953James D Watson and

Francis Crick published the model for

DNA structure and founded the

molecular genetics. According to them it

consists of the two complimentary base

paired strands running is opposite

direction as a double helix. They said in

report. ‘it has not escaped our notice that

the specific pairing we have postulated

immediately suggests a possible copying

mechanism for the genetic material.

They were awarded the noble prize in

1962.
 In mid-1950s, Kornberg

started to study the mechanism of

DNA replication. In 1957, he

identified the first DNA

polymerases. He was awarded

noble prize for exploring

complexities of replication i.e

requirement of enzyme to open Kornberg

double strand and keep it open,

primers creation and removing

and replication in one direction.

 In early 1960s Gobind Khorana

made significant advancement in

explanation of genetic code. He initiated

to totally synthesize functional human

gene and made different techniques to

make synthetic DNA oligonucleotides.

He was awarded noble prize for his work

on genetic code in 1968.

Gobind Khorana
 In 1969 Thomas D. Brock reported the isolation of new species of bacterium,

Thermus aquaticus (Taq) from a hot spring in Yellowstone National Park. This bacterium

became source of enzyme Taq polymerase that can tolerate high temperature i.e. 96oC.

Yellowstone National Park

 In 1970 Klenow reported modified version of DNA polymerase I from E.coli without
forward nucleases activity rendering it to only synthesize DNA instead of degradation.

 In 1971 researchers in Khorana’s project, began looking at, ‘repair synthesis’ i.e.

artificial system of primers and templates that allows DNA polymerase to copy segment

of gene, who were concerned over their DNA yield. Although similar to PCR using

repeated application of DNA polymerase but would not give exponential amplification as

in PCR because of single primer-template complex.

  In 1971 Kjell Kleppe foresaw a process

very similar to PCR. At the end of the paper

he described:

“one would hope to obtain two structures,

each containing the full length of the

template strand appropriately complexed

with the primer. DNA polymerase will be

added to complete the process of repair

replication. Two molecules of the original

duplex should result. The whole cycle could

be repeated, there being added every time a

fresh dose of the enzymes.”

 In 1971, Cetus Corporation was founded in Berkeley, California by Ronald

Cape, Peter Farley and Donald Glaser. Early the company screened microorganisms

which were capable of producing important components, later began projects involving

new biotechnology industry, basically the cloning and expression of human genes and

also development of diagnostic tests for genetic mutations.

Peter Farley & Donald Glaser

  In 1976, a DNA polymerase was isolated from T. aquaticus, which is names as Taq

polymerase. It was found that it can retain its activity at high temperature above 75 oC

without denaturation.

 In 1977, Frederick

Sanger reported method of

DNA sequencing and he was

awarded noble prize in 1980.

It is most important technique

F. Sanger
in primer designing in PCR.

By 1980 , the scientific community was familiar with all of the components needed to

perform PCR amplification.

Invention and development of PCR:

o By May 1983, Mullis synthesized oligonucleotides

probe at Cetus for analyzing a sickle cell anaemia

mutation. Suffering from a problem with their work,

Mullis proposed an alternative technique based on

Sanger’s method sequencing. After realizing the

difficulty in applying sanger method specific to single

Karry Mullis
location in the genome, Mullis modified the idea to add

a second primer on the opposite strand.

o Later in 1983, Mullis began to test his idea. His first experiment was without any thermal
cycling and he hoped that polymerase could perform continued replication on its own.
 However, later experiments were performed by repeated thermal cycling but he failed to

convince other researchers.

o In June 1984, annual meeting was held in Monterey, California. Mullis presented a poster
on oligonucleotide production and also some results of PCR experiment. No one except

Joshua Lederberg showed interest. Later Mullis was removed from the headship of Oligo

synthesis lab in Cetus on physical altercation with a researcher over Cetus

o In September 1984, Mullis spend the whole month in designing PCR experiment on the
enforcement of his close friend Tom White, VP of research at Cetus. But results were not

visible in agarose gel electrophoresis.

o In November 1984, results were confirmed by southern blotting which was the first
visible signal, later researchers began the optimization of reaction.

o On march 1985, an application was filed by whole developing group including Mullis on
the analysis of the sickle cell anaemia mutation via PCR.

o In the spring 1985, PCR techniques were applied to the other targets and probes were

designed of the Huma leukocyte antigen and the results were visible on agarose gel

electrophoresis. Later the work was done on the thermostable DNA polymerases.

o In April 1985, abstract was submitted on American Society of Human Genetics meeting

in salt lake city and first announcement of PCR was made in October. Two papers were

planned to be published.

o 1) ‘Idea’ by Mullis

o 2) ‘Application’ from entire group.

Evolution of PCR:

o Manuscript of Mullis was rejected due to lack of results.

o In May 1986, Mullis presented PCR at the Cold Spring Harbor Symposium.

o On September 20, 1986 The use of Taq polymerase was announced by Henry Erlich in

Berlin meeting.

o In December 1985, reagents and the instruments for PCR were being developed by the
joint venture of Cetus and Perkil-Elmer.

o Klenow fragment based amplification Thermal cycler were developed. Later Taq based

cycler were developed and marketed in 1987.

o In 1985, Multiplex PCR was developed.

o On December 22, 1989 Taq polymerase and PCR was awarded as ‘molecule of the

year” by the journal Science.

o On July 23, 1991 Cetus corporation sold rights to the PCR patents to Hoffman-La Roche
for USD $300 million.

o On October 13, 1993 Karry Mullis was awarded the Noble Prize in Chemistry on the
morning when he was nearly arrested by Swedish authorities for use of laser pointer.
 Primer designing for PCR:
Good primer designing is essential for successful reactions. The important design

considerations described below are a key to specific amplification with high product.

Possibly, the most critical parameter for successful PCR is the design of PCR primer because

if all of the components of a conventional PCR reaction and conditions are focused and can be

assumed to be optimal, the success of a PCR reaction will ultimately depend upon the primers.

There are following important variables that are necessary to be taken in account while designing

PCR.

1. Primer Length:

 It is commonly considered that the optimal length of primers for PCR is 18-22 bp. This length

is neither too long nor too short. This length is sufficient for specificity and for easily binding

with the template DNA at given template.

2. Primer Melting Temperature:

Primer Melting Temperature (T m ) refers to the temperature at which one half of the DNA

double strand will separate to become single stranded.

It shows the duplex stability. Primers with melting temperatures in the range of 52-58 o C

usually produce the best outcomes.

Primers with melting temperatures above 65 o C have a tendency for secondary annealing. The

T m of the primers depends upon the GC content.

There are many methods to calculate it but commonly used is the nearest neighbor

thermodynamic theory .

T m of primer is calculated by given formula :  

Melting Temperature T m (K)={ΔH/ ΔS + R ln(C)}, Or Melting Temperature T m ( o C) = {ΔH/ ΔS +

R ln(C)} - 273.15 where as

ΔH (kcal/mole): Enthalpy is represented by H. The quantity of heat energy that

substances possessed is called Enthalpy. ΔH is the change in Enthalpy. In the

given equation, change in Enthalpy is determined by sum of all the above di-

nucleotide pair’s enthalpy values of each nearest neighbor base pair.

ΔS (kcal/mole): Entropy is represented by s. The disorderness present in system

is termed as entropy. ΔS are change in Entropy. In the given equation, it is

obtained by sum of all di-nucleotide pairs entropy values of each nearest

neighbor base pair. An supplementary salt amendment is added as the Nearest

Neighbor parameters were obtained from DNA melting studies conducted in 1M

Na+ buffer and this is the default state used for all calculations.

ΔS (salt correction) = ΔS (1M NaCl) + 0.368 x N x ln ([Na+])

Where

N is the number of nucleotide pairs in the primer (primer length -1).

[Na+] is salt corresponding in mM.

[Na+] computation:

[Na+] = Monovalent ion concentration +4 x free Mg2+.

3. Primer Annealing Temperature: 

The temperature at which primer bind to the template is called annealing temperature. The

most critical point in annealing temperature T m because it shows hybrid stability. Improper

primer-template hybridization will occur when annealing temperature is very high that cause

low PCR product yield. On the other hand, too low T a  non-specific annealing. PCR specificity

strongly depends on the mismatch tolerance.

 T a  = 0.3 x T m (primer) + 0.7 T m  (product) – 14.9

where,

T m (primer) = Melting Temperature of the primers

T m (product) = Melting temperature of the product

4. GC Clamp:

 The G or C bases present in the last five bases from the 3' end of primers (GC clamp) helps to

promote the specific binding at the 3' end because of the stronger bonding of G and C bases.

More than 3 G's or C's are avoided in the last 5 bases at the 3' end of the primer.

5. GC Content: 

40-60% of the GC content (the number of G's and C's in the primer as a percentage of the total

bases) should be present in the primer.

6. Primer Secondary Structures

 Secondary structures present in the primers which are produced by intermolecular or

intramolecular interactions may result in the poor or no yield of the product. They affect the

annealing of primer template and then the amplification. The availability of primers to the

reaction may be reduced severely by these secondary structures.

i) Hairpins: They are produced by intramolecular interaction within the primer and must be

avoided. Favorably, a 3' end hairpin with a ΔG of -2 kcal/mol and an inner hairpin with a ΔG

of -3 kcal/mol is permitted generally.

ΔG definition: The Gibbs Free Energy G is defined as the measurement of the amount of work

that may be calculated from a process which is operating at a constant pressure. It is the

measure of the spontaneity of the reaction. The hairpin’s stability is commonly denoted by its

ΔG value, the energy needed to break the secondary structure. The larger the negative value

for ΔG, the more stable and unnecessary hairpins is indicated. Presence of hairpins at the 3'

end affects the reaction in an adverse manner.

ΔG = ΔH – TΔS

ii) Self Dimer: A primer self-dimer is produced by intermolecular interactions between the

two (same sense) primers, where the primer is similar to itself. In general, a large amount of

primers is required for PCR in comparison to the amount of target gene. When primers

produce intermolecular dimers more easily than hybridizing to target DNA, they decrease the

amount of product. Optimally a 3' end self-dimer with a ΔG of -5 kcal/mol and an internal self

-dimer with a ΔG of -6 kcal/mol is permitted generally.

iii) Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense

and antisense primers, where they are homologous. Optimally a 3' end cross dimer with a ΔG

of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.

7. Runs:

 Primers with long runs of a single base should generally be avoided as they can cause

mispriming. For example, AGCGGGGGATGGGG has runs of base 'G' of value 5 and 4. A

maximum number of runs accepted is 4bp.

8. Repeats: 

A repeat is a di-nucleotide occurring many times consecutively and should be avoided because

they can cause mispriming. For example: ATATATAT. A maximum number of di-nucleotide

repeats acceptable in an oligo is 4 di-nucleotides.

9. 3' End Stability: 

It is the maximum ΔG value of the five bases from the 3' end. An unstable 3' end (less

negative ΔG) will result in less false priming.

10. Avoid Cross Homology: 

To improve specificity of the primers it is necessary to avoid regions of homology. Primers

designed for a sequence must not amplify other genes in the mixture. Commonly, primers are

designed and then BLASTed to test the specificity. Our products offer a better alternative.

You can avoid regions of cross homology while designing primers. You can BLAST the

templates against the appropriate non-redundant database and the software will interpret the

results. It will identify regions significant cross homologies in each template and avoid them

during primer search.

11. Avoid Template Secondary Structure: 

A single stranded Nucleic acid sequences is highly unstable and fold into conformations

(secondary structures). The stability of these template secondary structures depends largely on

their free energy and melting temperatures(T m ). Consideration of template secondary

structures is important in designing primers, especially in qPCR. If primers are designed on a

secondary structures which is stable even above the annealing temperatures, the primers are

unable to bind to the template and the yield of PCR product is significantly affected. Hence, it

is important to design primers in the regions of the templates that do not form stable

secondary structures during the PCR reaction. Our products determine the secondary

structures of the template and design primers avoiding them.

Parameters for Designing of Primer Pair

Amplicon Length:

The amplicon length depends upon the experimental objectives. Its length for qPCR is up to

100 base pairs and for standard PCR, it is near 500 base pairs. If the position of each primer is

known with respect to the template, the product is calculated by following formula:

o Product length = (Position of antisense primer-Position of sense primer) + 1.

Product Position: 

Primer can be positioned near the 5' end, the 3' end or anywhere within specified length.

Commonly, the sequence near to the 3' end is known with more confidence and hence chosen

most frequently.

Tm of Product: 

Melting Temperature (T m ) is the temperature at which one half of the DNA duplex

will detach and become single stranded. The stability of the primer-template DNA

duplex can be determined by the melting temperature (T m ).

Optimum Annealing Temperature: 

The formula of Rychlik is most appreciated for the calculation annealing

temperature. It generally results in good PCR product yield with minimum false

product production.

o T a  opt = 0.3 x(T m  of primer) + 0.7 x(T m  of product) - 14.9

where

T m  of primer is the melting temperature of the less stable primer-template pair

T m  of product is the melting temperature of the PCR product.

Primer Pair Tm Mismatch Calculation:

 The two primers in primer pair should have nearly equaled melting temperatures for

maximizing PCR product yield. The difference of 5 o C or more can cause failure in

amplification.

Primer Design using Software

A number of primer design software have been developed that can help out in PCR primer

design for new and experienced users. These tools reduce the cost as well as time consumed in

experimentation because they lower the chances of failure.

 Primer Premier is a primer designing software that  follows all the guidelines

specified for PCR primer design. Primer Premier can be used to design primers for

single templates, alignments, degenerate primer design, restriction enzyme analysis.

Contig analysis and design of sequencing primers. However the guidelines

for qPCR primer design vary somewhat as compared to others.

 Primer Plex is also a software that is used to design primers for Multiplex PCR.

 Some software for example Allele ID and Beacon Designer are used in  species

specific primer design and reduce the cost of experimentation failure. They can design

primers and oligonucleotide probes for complex detection assays such as cross species

primer design,

DNA polymerases used in PCR:

DNA polymerases are enzymes responsible for assembling nucleotides to create new DNA molecules. During

DNA replication, the polymerase reads the existing DNA strands and semi-conservatively creates new

complementary DNA strands.

Structure and Mechanism of Action:

All DNA polymerases are similar in shape in that they resemble a “right hand” with common structural

features of a “thumb,” “palm,” and “fingers” domains.

o The “palm” area is the most similar among the polymerase families, and is associated with

catalysis of the phosphoryl transfer reaction (Steitz,1999).

o The “fingers” domain is involved in the interactions between the nucleoside triphosphate being

inserted and the existing template base, and

o The “thumb” is suggested to assist in aligning the double-stranded DNA.

Primers are oligonucleotides that can bind to specific sequence of the DNA template to guide DNA

polymerase replication. When the DNA template strands dissociate, a primer with a free 3’ hydroxyl group

anneal to its specific template sequence. Researchers can selectively replicate any regions of interest on the

template DNA by flanking the region with specifically designed primers. Primer annealing initiates the DNA

polymerase to add free nucleotides onto the hydroxyl group via a phosphoryl transfer reaction to elongate the

new strand in a 5’-3’ direction. When a nucleoside triphosphate (NTP) binds to DNA polymerase, the DNA

polymerase undergoes a conformational change and generates a specific shape/pocket into which only the base

on the template strand and a properly shaped complementary nucleotide can fit (cytosine to guanine, thymine

to adenine). In this way, DNA polymerase is able to select the correct nucleotides for incorporation (Oshima

& Imahori,1974)
Properties of DNA polymerases used in PCR

 Fidelity

 Processivity

 Extension rate

 Thermostability

PCR buffer components and thermal cycling conditions can affect DNA polymerase fidelity,

Processivity thermostability and Extension rate.

Fidelity

A DNA polymerase’s fidelity refers to its accuracy during replication of the amplicon; high fidelity is achieved

by having a low mis-incorporation rate as well as a proofreading mechanism. Many polymerases have a 3’-5’

exonuclease domain independent of the polymerization process, allowing them to remove mis-incorporated

nucleotides from the 3' end while the DNA is being formed in the 5'-3' direction (Steitz,1999).

Processivity

In addition to fidelity, DNA polymerases can be evaluated by their processivity, which is the number of

nucleotides a polymerase is able to incorporate before dissociating (Losick et al, 2008). A polymerase with

high processivity binds to the template and extends far and possibly to the end, adding several nucleotides per

second. However, most DNA polymerases are intrinsically low in processivity, that they frequently bind to the

template and dissociates, adding one nucleotide per second and producing short DNA product per

association/dissociation event (Losick et al, 2008).

Extension rate

Extension rate refers to the speed at which the nucleotides are added per molecule of DNA polymerase

during extension, and is proportional to the processivity of the DNA polymerase. Extension temperature,

buffer conditions and template sequences can affect the extension rate, and a higher extension rate generally

leads to reduced thermal cycling time.

Thermostability

Finally, thermal stability is also essential for DNA polymerases to maintain its stabilities at high temperature in

PCR. It can be measured by the half-life of DNA polymerases to retain its activity under sustained temperature

as high as 95°C. It is intimately related to the fidelity and processivity attributes of the DNA polymerase. PCR

originally utilized the Klenow fragment, a proteolytic product of DNA polymerase I isolated from E. coli

Klenow, & Henningsen,1970). While it has high fidelity, having retained both polymerase activity and 3’-5’

exonuclease activity, it is irreversibly denatured from the high temperatures used to separate the new strands of

DNA (Holland,1991). New enzyme must be manually added after every cycle – for the typical 30-40 cycles

in PCR, this poses a contamination risk and is labour- and time- consuming. The Klenow fragment has since

been replaced by thermostable enzymes from thermophilic bacteria species, which do not require addition of

new polymerase between cycles (Holland,1991).

Examples of PCR DNA polymerases:

Taq polymerase

Taq DNA polymerase (commonly abbreviated to “Taq”) is a thermostable DNA polymerase used in PCR,

originally isolated from Thermus aquaticus, a thermophilic bacteria found in hot springs and hydrothermal

vents. PCR takes advantage of Taq’s ability to withstand the high temperatures required during the

denaturation step for strand separation (Saiki et al, 1988). With an optimum activity temperature of 75-80°C,

Taq polymerase can be reused through several cycles of PCR without being denatured by the heat itself . Taq

polymerase also displays high processivity replicating 1 kb of DNA within 30-60 seconds during PCR (Chien

et al, 1976). Taq DNA polymerases also display 5'-3' exonuclease activity, allowing for the excision of

nucleotides from the 5' end of the DNA strand in a nick translation reaction (Holland,1991). This activity is

important as it cleaves labelled oligonucleotide probes from the 5' end of the DNA to generate a detectable

signal in real-time PCR application. (Heid et al, 1996).

One of the major drawbacks to Taq is its inability to proofread as it lacks 3'-5' exonuclease activity, therefore

giving low replication fidelity (1 error in 9000 base pairs) (Chien et al, 1976).
Pfu DNA Polymerase

Pfu DNA polymerase is a thermostable enzyme originally isolated from Pyrococcus furiosus, a

hyperthermophilic species of archaea (McTernan et al, 2014). Similarly to Taq, Pfu DNA polymerase can be

reused throughout several PCR cycles as it operates optimally at 90°C and is not denatured by the heating

steps. In addition, Pfu DNA polymerase displays 3’-5’ exonuclease activity, and therefore has the ability to

proofread by excising mis-incorporated nucleotides, giving it very high replication fidelity (1 error in 1.3

million base pairs).

One caveat to Pfu DNA polymerase’s superior fidelity is its slower speed, as it requires up to 2 minutes to

amplify 1 kb of DNA during a PCR cycle (Bustin, 2010). Pfu DNA polymerase also produces DNA products

with blunt ends, requiring the use of blunt-ended vectors for cloning applications (Chen et al, 2002).

KOD DNA Polymerase

KOD DNA polymerase is a recombinant form of DNA polymerase derived from the thermophilic solfatara

bacterium Thermococcus kodakaraensis KOD1 type strain. KOD DNA polymerase functions optimally at

85°C and displays 3'-5' exonuclease proofreading activity, producing blunt-ended DNA products ( Morikawa

et al, 1994)., ( Benson et al, 2003). While KOD DNA polymerase displays high fidelity and processivity for

small amplicons, long-distance amplification of amplicons over 5 kb tends to lower product yield due to its

strong 3’-5’ exonuclease activity. This can be avoided by mixing wildtype KOD polymerase with mutant

forms with lower 3’-5’ exonuclease activity, allowing for accurate amplification of amplicons up to 15 kb

(Nishioka et al, 2001).

Long-Range DNA polymerase

While conventional PCR can be used on amplicons up to 3-4 kb, Taq DNA polymerase is best optimized

for amplicons smaller than 2 kb. Taq DNA polymerase lacks 3’-5’ exonuclease activity renders it unable to

remove mis incorporated bases, causing it to stall and dissociate without completing the entire sequence

(Huang et al ,1992). On larger amplicons, accumulation of enough mismatches can inhibit PCR, leading to

truncated products.
Long-range DNA polymerase is optimized for DNA segments of up to 20 kb. These polymerases combine a

thermostable DNA polymerase, usually Taq polymerase for its high processivity, with a proofreading enzyme

containing 3'-5' exonuclease activity to increase fidelity. The proofreading polymerase is often derived from a

recombinant source and works to remove Taq polymerase’s 3’ mismatches during primer extension (Cheng et

al, 1994). 

Hot-start DNA Polymerase

Hot-start DNA polymerases are used to increase product yield by reducing nonspecific amplification

during PCR setup. Since most DNA polymerases can be active even at room temperature, the combination of

reaction components during PCR setup can lead to nonspecific primers annealing to each other or to the

template. These nonspecifically annealed primers compete for Taq polymerase binding and extension to create

undesirable PCR products.

Hot-start PCR is advantageous for amplifying low amounts of DNA template, highly complex DNA templates

or in multiplex PCR when multiple pairs of primers are used, as it can significantly improve the specificity and

yield of the product (Paul et al,2010).

Tth polymerase

Tth polymerase is derived from Thermus thermophilus, a thermophilic thermal vent bacterium (Oshima,

& Imahori, 1974). Tth DNA polymerase functions optimally at 75°C with high processivity, but lacks

proofreading 3'-5' exonuclease activity. Tth DNA polymerase displays efficient intrinsic reverse transcriptase

(RT) activity in the presence of manganese (Mn) ions, allowing it to assemble cDNA from RNA. Because of

this property, Tth polymerase can be used for RT-PCR, followed by subsequent amplification of the cDNA

product in the presence of magnesium (Mg) ions. Tth polymerase produces sticky-ended DNA products

(Mulhardt, 2010)).

Pwo DNA polymerase

Pwo DNA polymerase is derived from the ultra-thermophilic archaeon Pyrococcus woesei found in deep

marine environments (Zillig, 1987). Pwo polymerase functions optimally at 100-103°C, and displays high
proofreading 3'-5' exonuclease activity, giving it 18-fold higher fidelity than Taq polymerase. Pwo polymerase

creates blunt-ended products (Peter, 2014).

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(KOD) DNA Polymerase for PCR-mass spectrometry based analyses. J Am Soc Mass

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Humana Press.

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