**7.2 Multiplex-PCR**

It is a special type of PCR for the detection of pathogenic microorganisms by using several pairs of primers annealing to different target sequences in a single sample [2–4, 38]. The multiplex-PCR is mainly used to identify exonic or intronic sequences to detect mutations, deletions, insertions, and rearrangements in pathogenic specimens.

### **7.3 Nested-PCR**

It is used to increase the specificity of DNA amplification by reducing the nonspecific amplification [2–4, 39]. The two sets of primer pairs are used for a single locus point in two successive PCR reactions. The first round of PCR is performed with a primer pair that anneals to the sequence that flanks the target region. This generates a much larger DNA product that includes the target sequence. The second PCR is performed with a primer pair that precisely anneals to the target sequence, internal to the product of first PCR. This ensures that only the correct product is amplified in the second PCR [7]. Although Nested PCR improves specificity of amplification, it has disadvantage like primer-dimer formations [40].

### **7.4 Real-time PCR/quantitative PCR (qPCR)**

A qPCR is a technique used to quantify the amplification of a template DNA in real time during the PCR reaction. This type of PCR is commonly employed to estimate the number of DNA targets present in a sample or to study and compare the gene expression [7, 37]. When real-time PCR is used quantitatively (qPCR), the amount of amplification is measured either by using a nonspecific fluorescent dyes or sequence-specific DNA oligonucleotide fluorescent probes [4, 41]. When quantitative PCR is used above/below a certain amount of DNA molecules, it is called semi quantitative real-time PCR. Although the quantitative real-time PCR has many applications, it is more frequently used in basic research and diagnostic purposes. There is a growing industrial use of the technique, e.g., quantification of microbial load in processed foods, detection of GMOs, quantification of pathogenic viruses, etc. [42–44].

## **7.5 Hot start/cold finish PCR**

This technique reduces nonspecific amplification during the initial stages of a PCR [4, 7, 45]. To prevent nonspecific amplification at lower temperatures, hybrid polymerases are used which remain inactive at ambient temperature and is only activated at higher temperatures. Inhibition of the polymerase activity at ambient temperature is done by using an antibody or covalently bound inhibitors. Simply, in this technique the reaction components are heated to the DNA melting temperature (e.g., 95°C) before adding the polymerase.

**21**

*Polymerase Chain Reaction*

**7.8 Colony PCR**

**7.10 Inverse PCR**

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

**7.6 Touchdown PCR (step-down PCR)**

amplification later at the end [4, 7, 46].

**7.7 Assembly PCR or polymerase cycling assembly (PCA)**

template to generate the final full-length DNA structure [4, 7, 47].

initially extracting the bacterial genomic DNA [3, 4, 7, 48].

**7.9 Methylation-specific PCR (MSP)**

This type of PCR is designed to minimize nonspecific amplification by gradually decreasing the primer annealing temperature in the successive cycles. PCR is started with initial cycles having an annealing temperature 3–5°C higher than the primer Tm. The annealing temperature is then gradually decreased to 3–5°C lower below the Tm. The higher annealing temperature increases the specificity of the primers at initial stages of the reaction, while the lower temperature permits more efficient

This technique is used for the synthesis of long DNA molecules from long oligonucleotides with short overlapping segments, alternating between sense and antisense directions. The process begins with an initial PCR with primers that have an overlap, followed by a second PCR using the products of the first PCR as the

It is a convenient high-throughput technique used to confirm the addition of DNA insert in the recombinant clones and their uptake by the bacterial cell. A single set of insert specific primers are designed for the areas of the vector flanking the site where target DNA fragments are already inserted. This results in the amplification of the inserted sequences. The technique is used for the screening of bacterial colonies transformed with the recombinant vectors and to perform PCR without

It is a variant of PCR used to identify promoter hyper-methylation at CpG islands in cell lines and clinical samples, including fresh/frozen tissues. The target DNA is first treated with sodium bisulfite, which transforms the unmethylated cytosine bases in to uracil, which pair with adenosine of the PCR primers. The modified DNA is then amplified using two types of primers that only differ at their CpG islands. One primer set anneals to DNA with cytosine (corresponding to methylated cytosine), while the other anneals to DNA with uracil (corresponding to unmethylated cytosine). The MSP technique provides quantitative information

This type of PCR is used to detect the sequences that surround the target DNA (flanking sequences). It involves a series of restriction enzyme digestions and self-ligation. The primers amplify sequences at either end of the target by extending

In this technique, the PCR is preceded by a reaction converting RNA into cDNA using viral reverse transcriptase. The resulting cDNA is used as a template for a second conventional PCR. The technique is widely used in the detection of RNA viruses and to

about the methylation when used in quantitative PCR [3, 4, 7, 49, 50].

outward from the known DNA segment [4, 7, 51].

**7.11 Reverse transcription-PCR (RTP)**
