DNA polymerases and buffers
Polymerase chain reaction (PCR) is powerful technique used for in vitro exponential amplification of specific DNA region (target region) that lies between two regions of known DNA sequence (oligonucleotide primer binding sites).
There are three major steps in PCR performed at different temperatures. The first is denaturation of template DNA at 94oC in which double-stranded DNA melts to single-stranded DNA, and all enzymatic reactions stop. Next is annealing step (at 50 - 68oC, depending on primers composition) in which oligonucleotide primers bind to the template. Final step is extension in which DNA complementary to template DNA is synthesized at direction 5´->3´ at 72oC. These three steps are repeated (usually 20 - 40 times) and in each step the amount of the amplified DNA fragment is doubled.
Reaction mixture for PCR contains the following components: thermostable DNA polymerase, reaction buffer with Mg2+, deoxynucleoside triphosphates set (dATP, dGTP, dCTP and dTTP), template DNA and a pair of oligonucleotide primers (usually 20 - 30 nucleotides in specific sequence). Top-Bio offers six products containing thermostable DNA polymerases extensively tested for their suitability for PCR. These enzyme preparations differ in concentration, composition of storage buffers and presence of additives.
- This product contains Taq DNA polymerase at a concentration of 5 U/μl in storage buffer and is widely used for routine diagnosis purposes.
- This product contains Taq DNA polymerase at a concentration of 5 U/μl in optimized storage buffer which increases stability of the enzyme and robustness of PCR
- In this product, concentration of Taq DNA polymerase is reduced to 1 U/μl and storage buffer is supplemented with inert Dye for easy loading and control of the presence of the enzyme in PCR.
- This product contains Taq DNA polymerase at a concentration 1 U/μl for easier manipulation. Furthermore, it is supplied with modified buffer (Blue buffer) which is more tolerant to elevated Mg2+ concentrations and decreases production of nonspecific products.
- This product contains two DNA polymerases at a concentration of 5 U/μl. One of them, Taq DNA polymerase, facilitates rapid and robust synthesis of DNA, whereas the second one with 3´->5´ exonuclease activity enhances PCR fidelity (~5 times higher when compared with Taq DNA polymerase). LA DNA polymerases Mix is suitable for amplification of long DNA fragments (up to 40 kbp) and also for amplification of DNA fragments aimed for DNA cloning.
- PCR Blue Buffer contains ammonium sulfate instead of potassium chloride and thereby exhibits higher tolerance towards changing concentrations of MgCl2.