**3. The components of PCR**

Setting up a basic PCR requires many ingredients, reagents, and conditions which are described below (**Figure 1**) [2, 4, 7–10].

**15**

*Polymerase Chain Reaction*

ers, and polymerization.

by using degenerate primers [2].

**3.2 Primers**

**3.1 DNA template**

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

The double-stranded DNA molecule amplified by PCR is called the target or template DNA. The template defines the sequence in which new nucleotides are added during the PCR process [11]. This process is carried out in vitro by cyclically varying temperature, enabling separation of DNA strands, hybridization of prim-

DNA isolated from any source can be used as a template for PCR provided that it contains the target sequence. The DNA used in PCR can be isolated from blood, tissue, forensics specimens, paleontological samples, or microbial/tissue cells grown in the lab. Whatever the source, we need to have some information of the target DNA sequence, so that primers for PCR can be designed [2, 12]. The PCR primers can be designed very easily nowadays owing to the plethora of software tools that only requires target sequence information. If the sequence information is not known, the designing of primers becomes very challenging. This problem can be circumvented

Primers are single-stranded DNA molecules usually synthesized commercially, i.e., polynucleotides of variable sizes [2, 7–10]. These short polynucleotide DNA strands have a free 3′ hydroxyl group, also called as 3′ end. The free 3′ hydroxyl group on the primer is needed by the DNA polymerase to add new nucleotides during the polymerization process, thereby synthesizing a new complementary strand [12, 13]. The binding of DNA primer to the target requires the separation of two complementary DNA strands (Denaturation) which is generally achieved by heating process. To perform PCR, two primers are needed to enhance both the strands of the template: a primer for one strand (or sense strand), called the "forward primer," which is the beginning of the template, and another primer for the complementary strand (or the antisense strand) called the "reverse primer." Thus, both the

The length of primers plays an important role in correctly identifying their designated target complementary regions. Increasing the length of primers improves their chances of matching the target (specificity). These primers are usually commercially synthesized with their size ranging between 18 and 25 nucleotides.

Primers should bind (anneal or hybridize) to the template with good specificity and strength to ensure amplification of the correct sequence. The specific temperature that is needed for primer annealing also depends on the primer sequences, e.g., the longer the primer, the higher the annealing temperature. Therefore, the maximum specificity and efficiency of PCR depends on optimal primer sequences and appropriate primer concentrations [2, 7, 8]. This in return depends on the way primers are designed and used. Improper primers may amplify undesired DNA segments (nonspecific products), lower the yield of specific products, or completely fail the results of PCR. These undesired outcomes can be circumvented by designing and validating primers that preferentially bind to their target sequences. The online IDT Sci Tools Software Oligo Analyzer 3.1 and Primer Quest are invaluable aids both in primer design and validation [14]. These software tools also ensure that the two primers do not contain sequences that are complementary to each other. If primers contain selfcomplementary sequences, then hybridization will occur to each other, and they form "primer-dimmers." Consequently, the primers will fail to bind to their target template, leading to a compromised PCR efficiency. In addition, presence of complementary sequences within a primer leads to the formation of hairpin loop structures [15].

primers bind to 5′ ends of the sense and antisense strand.

**Figure 1.** *The component of PCR.*
