Welcome to Applied Biological Materials’ PCR video series! In this video,

we will introduce the basics of PCR. Polymerase chain reaction, or PCR is a technology

indispensible to genetic and molecular biology. Many of its applications are widely used in

the world around us, including DNA sequencing, DNA fingerprinting, forensics, detection of

microorganisms, and diagnosis of hereditary disease. PCR allows fast and inexpensive amplification

of DNA fragments. Because it is able to generate large quantities of DNA from a small amount

of nucleic acid, PCR is also referred to as molecular photocopying.

PCR depends on a series of 20 to 40 repeated cycles of DNA replication by a DNA polymerase

enzyme. After each cycle, the number of DNA strands is doubled, and at the end of a 40

cycle reaction, more than 1 trillion copies are generated from a single copy of the DNA

molecule. The PCR process is separated into four steps:

initialization, denaturation, annealing, and elongation. In the first initialization step,

the reaction is heated to 94–96°C for 2 to 10 minutes to activate the DNA polymerase

and to denature other contaminates in the mixture. In the case of colony PCR screening,

this step also facilitates cell lysis to release DNA and denature other cellular proteins.

In the denaturation step, hydrogen bonds between the double-stranded DNA are broken by heating

the reaction to 94–98°C for 20–30 seconds. After the DNA strands are separated, primers

bind to a complementary sequence in the DNA template to guide DNA polymerase replication.

In this step, the reaction temperature is lowered to 50-65°C for 20–40 seconds to

allow for optimal primer annealing. After the primers establish a starting point for

the enzyme, DNA polymerase starts to incorporate dNTPs in a 5’ to 3’ direction to synthesize

a new DNA strand. The temperature and extension time for this elongation step depend on the

type of DNA polymerase enzyme and the target amplicon. Commonly used Taq Polymerase polymerizes

at a speed of 1-1.5 kilobases per minute and works ideally at 72-78°C. The cycle then

repeats from denaturation to elongation 20 to 40 times. At the end of the last cycle,

there is a final elongation step that keeps the reaction mixture at 72-78°C for 5-15

minutes. This ensures that any remaining single-stranded DNA is fully extended after the last PCR cycle.

A final holding step keeps the PCR at 4-15°C for an indefinite time, keeping the products

for short-term storage. The most common DNA polymerase enzyme used

in PCR is Taq polymerase, a thermostable DNA polymerase that allows multiple cycles of

amplification without the addition of new enzyme after each denaturation step. Since

the initial discovery of Taq DNA polymerase, many variations of the Taq enzyme have been

engineered with different attributes that can be utilized for specific PCR applications.

For example, we at Applied Biological Materials, also known as abm, carry a modified Taq enzyme

that has decreased error rates. Also, abm has engineered a robust Taq enzyme that amplifies

DNA directly from blood samples. There are many more engineered Taq enzymes and other

DNA polymerases available. To visualize the DNA fragments after amplification

by PCR, gel electrophoresis is used. By comparing the gel bands to a molecular weight marker,

researchers can estimate the size of the amplified products to know if their desired genes were

successfully amplified. Many factors can interfere with a PCR reaction.

Some are easy to eliminate while others are trickier to manipulate and would require experience

to tackle. In general, factors that are important in PCR include the nucleotide composition

of the DNA template, DNA polymerase enzyme choice, buffer components, primer design,

additives and inhibitors. Depending on the sequence of the target DNA,

different strategies may be needed for a successful PCR. For example, GC rich templates have more

hydrogen bonds compared to AT rich templates. This results in incomplete strand separation

which may cause the PCR to fail. In addition, high GC content templates tend to lead to

more secondary structures, which can arrest the polymerase leading to pre-mature termination.

In these cases, secondary structure destablizers such as DMSO can be added.

At the same time, AT rich templates also require special attention because of their low primer annealing

temperatures. Non-specific primer annealing can occur under low annealing temperature,

and primer specificity can be retained using a combination of lowered extension temperature

and the use of additives such as TMAC. Long templates are difficult to amplify because

of their higher likelihood of DNA template being broken or degraded by depurination.

Depurination refers to a chemical reaction during which the β-N-glycosidic bond in the

purine nucleoside is cleaved to release a nucletide base due to hydrolysis. As Taq DNA

polymerase will not extend through apurinic positions during replication, depurination

is an important limiting factor of long template amplification. This can be avoided by using

a higher pH reaction buffer and decreasing the denaturation time to limit depurination,

or by using a proofreading DNA polymerase. As mentioned earlier, there are many variations

of DNA polymerases commercially available, each with different features. Depending on

the DNA template, choosing the correct DNA polymerase, and pairing it with an appropriate

buffer can be critical for a successful reaction. Apart from DNA polymerase choice, having a

good pair of primers can also dictate the outcome of a PCR. Some considerations to keep

in mind include the length of the primer, the annealing temperature, and the sequence

of the primer. For example, the pair of primers should not have complementary sequences; otherwise

they can easily lead to self-dimer or primer dimer formation and compete with template-primer

annealing. Many naturally occurring substances, such

as polysaccharides, tannic acid and EDTA, can also inhibit PCR. Some inhibitors may

degrade or modify the DNA template, while others can disturb the annealing of primers

to DNA or alter the DNA polymerase activity. Inhibitors can be removed with sample-specific

nucleic acid isolation protocols, or sometimes the use of additives can help in reducing

the inhibition. For a complete list of PCR additives and their mechanisms of action,

please refer to our knowledge base. A combination of favorable factors contributes

to a successful PCR. Taking all these factors into consideration, abm provides a wide range

of DNA polymerases, and has formulated optimal PCR MasterMixes for each polymerase. We also

offer kits that make DNA amplification from plants, tissue and blood samples easy! Check

out our comprehensive list of PCR products in the link provided below. Thank you for

watching and make sure you follow our next video on the variations of DNA polymerases.

Don’t forget to leave your comments and questions below.