Have you ever heard about PCR? Well, PCR or Polymerase Chain Reaction is an effectual technique that is vastly implemented by the experts these days to amplify DNA. Kary Mullis, the one who developed this procedure back in 1983, also received the prestigious Nobel Prize for his work. However, the procedure PCR has become one of the most common, affordable ways of DNA replication. The technique is performed by making use of DNA polymerase, two DNA primers, nucleotide triphosphates as well as buffer solutions.
The technique is mostly done in cycles that allow DNA replication. At first, the experts denature the DNA at 94 to 98 °C for at least 20-30 seconds. This allows the separation of DNA appropriately into double strands. Splitting up the DNA into single strands facilitates the succeeding step of annealing. When it comes to the annealing procedure, the temperature is further lowered for 20 to 40 seconds to 50-65 °C. Well, this enables the DNA primers to bind well to their complementary targets that are single-stranded. The primer (also single-stranded) is generally shorter as compared to the DNA which is supposed to be replicated.
Then, the temperature is further increased to 75-80 °C. Well, the temperature may vary a bit depending on the DNA polymerase that is being used. The DNA polymerase further synthesizes that DNA in 5′ -3′ direction exclusively by condensing 5′ phosphate of added nucleotide triphosphates along with 3′ hydroxyl group. The procedure continually adds the newly synthesized strands of DNA to the pool comprising template strands in order to increase the total amount of DNA that is being produced.
Once you have the DNA products, you can easily start the purification procedure by eliminating the DNA primers, salts, enzymes, as well as nucleotide triphosphates. PCR purification can easily be attained by selectively trapping or binding the DNA target strand. However, you need to know that the task can be accomplished in varied ways. However, different purification kits are required in different methods. At first, one needs to employ the size exclusion chromatography. The beads are further added to that solution that has large pores which don’t allow the impurities and smaller primer strands to stick in. However, these pores can trap the target DNA.
After the elimination of certain unwanted byproducts and impurities, the target strands are eluted. The spin columns can be added that use the silica membrane in order to bind the DNA in high salt buffer. This particular sample is micro centrifuged as well as the impurities are also discarded. Then, target DNA is eluted with low salt buffer.
The purification procedure can also be performed by using paramagnetic beads. These beads selectively bind the DNA of accurate 100 bp or larger by bypassing those DNA primer strands easily. Once added, the easy washing steps would further remove primer dimers, salts, primers, enzymes as well as nucleotides. If you’re keen to know more about the methods of PCR purification, do feel free to visit www.biochain.com/products/rna/total-rna-rna/.