2.7 Polymerase Chain Reaction (PCR)

2.7 Polymerase Chain Reaction (PCR)

In 1971, Dr. Har Gobind Khorana’s group described the idea to amplify a DNA (Templeton, 1992). Later on in 1985, Kary B. Mullis invented the Polymerase Chain Reaction (PCR) which is used today in different fields, such as scientific research, clinical diagnostics and criminal investigations. PCR is a molecular biology tool that is used to amplify a fragment of DNA to generate thousands to millions of copies. This technique is based on an enzymatic reaction which is controlled by thermal cycling, where every cycle consists of heating and cooling steps. The generated DNA fragments after every cycle are used as templates for the next cycle. This reaction consists of 5 major components: DNA template, two primers that are complementary to the 3’ ends of each strand of the DNA template, DNA polymerase, desoxynucleoside

Cells were disrupted using RLT buffer and the lysate was homogenized by centrifugation through a QIAshradder spin column for 2 min at full speed. The flowthrough was mixed with 70% ethanol (equal volume as the RLT buffer used), transferred into an RNeasy spin column and centrifuged at 17000 g for 1 min. after having the flowthrough discard, RW1 buffer was added to the column, centrifuged at 17000 g for 1 min and the flowthrough was discard. The spin column was washed twice with RPE buffer under at 17000 g centrifugation for 2 min. The column was centrifuged empty at full speed for 1 min to remove any residual ethanol. The RNA was eluted by adding nuclease-free water into the column and centrifugation of 1 min at full speed. The yielded RNA was checked by nanodrop (Thermo) and the integrity of the RNA was assessed in the Experion automated electrophoresis station