Real-time PCR (qPCR) Master Mixes
Quantitative polymerase chain reaction (qPCR), also called real-time PCR is one of the most valuable techniques, which is used to amplify and simultaneously quantify targeted DNA fragments. In combination with reverse transcription (RT) in which RNA is transcribed to cDNA, RT-qPCR is an important tool for analysis of expression of individual genes.
Real-time detection of PCR products is made possible by including in the reaction a fluorescent molecule that reports an increase in the amount of DNA with a proportional increase in fluorescence signal. The fluorescent chemistries used for this purpose include "nonspecific" DNA-binding dyes and fluorescently labeled sequence-specific probes. Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence during PCR. The intensity of measured fluorescence reflects the amount of amplified product in each cycle. Key factor for successful qPCR is availability of reaction mixes which allow DNA amplification in the presence of DNA-binding dye or fluorescent probe. Top-bio offers several extensively optimized reaction mixtures, qPCR Master Mixes, which are designated for qPCR.
An important component of the qPCR Master Mixes is anti-Taq monoclonal antibody, which inactivates enzymatic activity of thermostable DNA polymerase. The enzyme is re-activated after denaturation of the antibody during the first denaturation phase of PCR cycle. This so called "hot start" PCR decreases formation of nonspecific PCR products. qPCR Master Mixes possess all components (buffer, nucleotides, enzyme and antibody) 2x concentrated; before qPCR is only added template DNA, primers, water and, in some cases, fluorescent oligonucleotide probe.
qPCR Master Mixes containing fluorescent DNA dye:
- A product containing fluorescent DNA dye, SYBR Green I, which intercalates into double-stranded DNA. It is suitable for most of qPCR amplifications which do not require specific fluorescent probes.
- This product contains SYTO-9 fluorescent DNA dye, which is suitable for qPCR amplification of longer fragments (up to 1 kbp) and for high resolution melting (HRM) analysis of DNA amplicons.
- TP SYBR 2x Master Mix is dedicated for universal analysis of DNA samples using qPCR with quantification of DNA amplicons with DNA dye SYBR Green I. It is based on recent finding that addition of Trehalose or 1,2-Propanediol (abbriated TP) into reaction mixture is capable of susbstantialy increasing efficiency of PCR and enhance amplification of samples which are otherwise difficult to amplify, including DNA from whole blood, GC rich amplicons and samples containing PCR inhibitors (Horáková a spol., BMC Biotechnology, 11:41, 2011; Free PMC article).
qPCR Master Mixes
- In composition this product is similar to qPCR 2x SYBR Master Mix, but does not contain fluorescent DNA dye. It is recommended for detection of qPCR amplicons using sequence-specific oligonucleotide probes such as TaqMan, Molecular beacons, FRET and others.
- This product also does not contain fluorescent dye and therefore is recommended for detection of qPCR amplicons using specific probes. Different composition of the reaction buffer makes it more suitable for some qPCRs.
- LA (Long and Accurate). The Mix contains unique blend of two thermostable DNA polymerases. One of them is Taq DNA polymerase, which is highly processive but lacks a 3’->5’ exonuclease proofreading activity, which accounts for relatively high error rate during DNA amplification. Second polymerase is less processive but possesses 3’->5’ exonuclease activity necessary for proofreading. The polymerases mix decreases error rate during synthesis of complementary DNA strands and preserves high speed of DNA synthesis. The repair capability allows amplification of complex genomic DNA fragments (up to 15 kbps in size) and less complex viral DNA (up to 40 kbps in size) and significantly decreases error rate when compared to Taq DNA polymerase.