Maximizing PCR and RT-PCR Success - Template quality

PCR Brochure 10/2016 15 Template quality Because PCR consists of multiple cycles of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol, chloroform, salts, ethanol, EDTA and other chemical solvents than single-step, enzyme-catalyzed processes. These impurities, which are commonplace in home-made template preparations, can reduce the sensitivity and efficiency of PCR amplification. Template preparation based on phenol extraction, ethanol precipitation, or salting-out procedures may not remove all contaminants and, thus, adversely affect PCR reliability (Table 3). We recommend QIAGEN sample prep technologies using silica membrane-based kits (e.g., QIAamp® and QIAprep® Kits) for reliable PCR results. Additionally, whole genome amplification kits, which amplify very small amounts of genomic DNA while introducing no sequence bias, can be used if the starting amount of DNA is limiting (e.g., REPLI-g® Kits). Visit www.qiagen.com to see our complete range of DNA purification products, all of which provide pure DNA from a wide variety of sample types and ensure accurate PCR results. In addition to DNA quality, the amount of template used for PCR may also influence PCR success – especially too much template, which often results in smearing or nonspecific amplification products. Impurity Inhibitory concentration SDS >0.005% (w/v) Phenol >0.2% (v/v) Ethanol >1% (v/v) Isopropanol >1% (v/v) Sodium acetate ≥5 mM Sodium chloride ≥25 mM EDTA ≥0.5 mM Hemoglobin ≥1 mg/ml Heparin ≥0.15 i.U./ml Urea >20 mM RT reaction mixture ≥15% (v/v) Table 3. Impurities exhibiting inhibitory effects on PCR Challenging PCR methods and new technologies Standard PCR applications are routinely used in numerous research areas, such as biomarker discovery, genotyping, gene regulation and cancer research. New PCR applications have been developed to address the need for increased sophistication and accuracy. These developments have been driven by demands for increased throughput while reducing costs, increasing assay sensitivity and ensuring reliable data normalization (Table 4, next page). Specialized PCR buffer systems and associated additives are often necessary to meet these challenges and ensure successful results.

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