PCR Mix for difficult samples
When DNA sequences with a high GC content are amplified, the formation of PCR products can often be compromised by inadequate strand separation and by the propensity for the formation of complex secondary structures. The inability of the DNA polymerase to function through regions of strong secondary structure, such as "hairpins", often leads to the formation of truncated PCR products. In addition, poorly defined amplification products are prevalent when the primers are GC-rich. Despite the inherent challenges, the detection of GC-rich sequences is becoming increasingly important for the molecular diagnosis of hereditary diseases.
We propose DNA polymerases with robust performance in a wide range of models rich in GC and AT. These DNA polymerases have improved tolerance to common PCR inhibitors for crude samples and / or DNA extractions.
PCR Master Mix is a premixed, ready-to-use solution containing DNA polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR
PCR master mix contains allcomponents for PCR, except DNA template and primers :
A DNA polymerase that is heat resistant, so that it can remain intact during the DNA denaturation process and makes the apmlification of the target DNA sequence.
On distingue différents types d'ADN polymérases haute-fidélité dont la Pfu polymérase, une ADN polymérase naturellement fidèle grâce à son activité exonucléase 3'-5' ainsi que des ADN polymérases modifiées pour obtenir des capacités de fidélité supérieures.
Les modifications apportés aux ADN polymérases pour leur apporter une fidélité supérieure sont de différentes natures : modification génétique, modification chimique, mélange de plusieurs enzymes ...
Deoxynucleoside triphosphates (dNTPs)
dNTPs consist of four basic nucleotides : dATP, dCTP, dGTP, and dTTP. dNTPs are building blocks of new DNA strands. These four nucleotides are typically added to the PCR reaction in equimolar amounts for optimal base incorporation. In common PCR applications, the recommended final concentration of each dNTP is generally 0.2 mM.
Magnesium ion (Mg2+)
Magnesium ion (Mg2+) functions as a cofactor for activity of DNA polymerases by enabling incorporation of dNTPs during polymerization. The magnesium ions at the enzyme's active site catalyze phosphodiester bond formation between the 3′-OH of a primer and the phosphate group of a dNTP. ). In addition, Mg2+ facilitates formation of the complex between the primers and DNA templates by stabilizing negative charges on their phosphate backbones. Magnesium ion (Mg2+) are present in master mix as MgCl2 solution.
PCR is carried out in a buffer that provides a suitable chemical environment for activity of DNA polymerase. Generally a 10-50 mM Tris/HCl buffer with a pH above 8.0 (typically 8.3-8.8). KCl can be added to facilitate primer annealing, but shouldn't be higher than 50 mM as this may inhibit polymerase.
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