**7.8 Colony PCR**

*Genetic Engineering - A Glimpse of Techniques and Applications*

This is a standard PCR in which a single-primer pair is used to bind to the two separated target strands. The primers also define the target sequences that will be copied. The PCR generates millions of copies of the target DNA

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 patho-

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

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,

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

**7.1 Conventional PCR**

sequences [2–4].

**7.2 Multiplex-PCR**

genic specimens.

**7.3 Nested-PCR**

primer-dimer formations [40].

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

**20**

etc. [42–44].

**7.5 Hot start/cold finish PCR**

(e.g., 95°C) before adding the polymerase.

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