**4. Procedure**

Each cycle or round of PCR comprises three major steps, viz., denaturation, annealing, and extension, repeated for 30 or 40 cycles on a thermocycler (**Figure 2**) [2, 4, 7–10]. A number of parameters determine the range of temperature and the duration of each cycle step (**Figure 3**), e.g., the polymerase used for DNA synthesis; melting temperature (Tm) of the primers; and the concentration of reagents used, i.e., divalent ions and dNTPs. The melting temperature depends on the length and specific nucleotide sequence of a primer. At Tm, half of the DNA molecules are in the single-stranded form.

## **4.1 Denaturation**

It is the first cycling step that involves heating the reaction mixture to 94–98°C for 20–30 seconds. Such higher temperature disrupts the hydrogen bonding of the two complementary strands to produce the single-stranded DNA templates. Thus, denaturation prepares the DNA template for the binding of primers.

**Figure 2.** *A basic PCR protocol—DNA synthesis cycle.*

**Figure 3.** *PCR—a simple thermocycling protocol.*

## **4.2 Annealing**

After denaturation, the next cycling step is annealing, in which the temperature of the PCR reaction is decreased to 50–65°C and kept for 20–60 seconds. This promotes hybridization between primers and single-stranded templates. Optimal annealing occurs at temperatures that are 3–5°C less than the primer Tm. The primers should have sufficient length and GC content to strongly bind to their target template during annealing.

### **4.3 Extension**

DNA polymerase synthesizes (polymerizes) new DNA molecule by adding dNTPs complementary to the template bases in a 5′–3′ direction. The temperature and extension time depend on the type of DNA polymerase used: Taq polymerase performs optimally between 75 and 80°C. However, the enzyme is routinely used at 72°C. The extension time also depends on the length of the template.

A single cycle of PCR comprises the entire processes of denaturation, annealing, and extension/elongation. Under ideal conditions (optimal temperature ramping, presence of substrates/reagents, absence of inhibitors), the quantity of target DNA is doubled at the end of each cycle, resulting in exponential amplification of the specific DNA segment.

Final elongation: This single step is optional and performed at 70–74°C. The final elongation may take for 5–15 minutes after the last PCR cycle and allows any remaining single-stranded DNA to be fully extended.

**19**

*Polymerase Chain Reaction*

*DOI: http://dx.doi.org/10.5772/intechopen.81924*

**5. Magnitude of amplification**

be calculated using the formula "2n

ium bromide is 0.5–5.0 ng/band.

amplicon indirectly [29].

agricultural, and environmental fields [6].

**7. Types of PCR**

**6. Validating PCR**

Final hold: The final step may be employed for short-term storage of the reaction

Multiple cycling steps of a PCR can exponentially increase the copies of target DNA template to millions. The number of DNA copies produced by a PCR can

example, a PCR set for 36 cycles results in 68 billion copies of the template. Under optimal conditions, even with minimal efficiency, a PCR in 50 ml volume may produce 0.2 mg of 150 bp DNA from 100 template molecules after 35–40 cycles,

The degree of a PCR success can be determined in many ways [3, 29–36]:

1.Ethidium bromide (EtBr) can be used for the staining of amplified DNA product [31, 32]. It has UV absorbance maxima at 300 and 360 nm, and an emission maximum at 590 nm, and being a DNA intercalator, EtBr inserts itself between the base pairs in the double helix. The detection limit of DNA bound to ethid-

2.A three primer combination approach can provide a more cost-effective endlabeling of PCR products: (i) fluorescently labeled universal primer, (ii) modified locus-specific primers, and (iii) 5′ universal primer sequence tails [34].

3.Agarose gel electrophoresis: The most commonly employed validating method, gel electrophoresis, makes use of electric current to separate charged molecules like DNA using gel as molecular sieve. Gelling agents can be agarose (for DNA >500 bp) or polyacrylamide (<500 bp). Different DNA sequences are separated out based on their sizes. DNA staining dyes (like EtBr) are applied to the gel to help visualize the DNA bands using UV transilluminator [34, 36]. The presence of a correct size DNA band (confirmed using a DNA ladder) indicates that the target sequence was present and that the PCR has amplified a correct product. Absence of any DNA band indicates that the target DNA was absent, while the presence of

incorrect size DNA band indicates production of a spurious product [37].

4.The direct sequencing is often not practiced due to in accessibility or cost of DNA sequencer or even the time needed to undertake such an analysis. However, restriction enzyme digestion can also be used to assess the sequence of an

Owing to ever-growing applications, a wide variety of PCR techniques have emerged over the past few decades [2–4]. Some of the variants are mere optimization close to the basic PCR to fulfill the specific needs. Others have undergone massive modifications to suit novel applications in different biological, biomedical,

", where n is the number of cycles [27, 28]. For

by cooling the reaction chamber to 4–15°C for an indefinite time.

with the molar weight of the fragment equal to 99,000 Da.

Final hold: The final step may be employed for short-term storage of the reaction by cooling the reaction chamber to 4–15°C for an indefinite time.
