**3.1 The evolution of PCR**

Since the development of the polymerase chain technique (PCR) in 1971 when Gobind Khorana described the technique by explaining the replication of a DNA fragment using two primers [37]. But it was in 1983 that Kary Mullis and his colleagues at the Californian company Cetus Corporation first carried it out while working on making oligonucleotides and using primers for DNA sequencing. Used two primers that aligned with each of the DNA strands, added *Escherichia coli* DNA polymerase I and triphosphated nucleotides. As a result, they obtained the exponential replication of the DNA fragment flanked by the primers [38]. This endpoint PCR technique has evolved thanks to two factors, the discovery of the Taq polymerase and the thermal cycler (**Figure 3**). Thus, today we can find variants such as: reverse transcription PCR (RT-PCR) [39, 40], Circularized RT-PCR (cRT-PCR) [41], inverse PCR [42], nested PCR [43], in situ PCR [44], multiplex PCR [45], multiplex-ready PCR [46], asymmetric PCR [47], Ligation-anchored PCR [48], long fragment PCR [49], Fluorescent Quantitative PCR (Real Time PCR; qPCR) [50], Immune PCR (IPCR) [51], and digital PCR (dPCR) (**Figure 3**). dPCR was first conceptualized in the 1990s. It is based on limiting dilutions, PCR and Poisson distribution [52]. dPCR is classified into digital droplet PCR (ddPCR) and digital chip PCR (cdPCR). cdPCR presents a challenge to achieve high throughput, however, ddPCR can overcome this impediment [53]. It is worth mentioning that isothermal amplification methods called LAMP (LOOP-mediated isothermal amplification) have been developed that, unlike PCR technology, amplify the target sequence at a constant temperature of 60–65°C. based on strong strand displacement DNA polymerase and 4−6 specifically designed primers can be used that can recognize and amplify six or eight regions of a given target

## **Figure 3.**

*Evolution of the PCR technique. PCR technology has undergone continual improvements. The applications of PCR are multiple, encompassing from the evolution to the clinic, passing through the genetics, molecular biology, and biotechnology; in addition to applications in agriculture and livestock. The applications of this new technique seem to have no limits.*

gene to achieve higher analytical specificity. Amplification signals can be detected by photometry of the turbidity of the reaction or by colorimetry of the fluorescent intensity of the intercalated dyes. This approach offers a fast (1 h), a low-cost, easierto-use, thermocycler-free alternative method for PCR [54, 55].
