UNLABELLED PREMISE OF THE STUDY Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • METHODS AND RESULTS A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolera...

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of th...

For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. Here, we report the identification a...

Random amplified polymorphic DNA (RAPD) is a simple, fast and cost effective method for assessing genetic diversity of plant varieties. The reproducibility of the RAPD amplification was affected by several factors, therefore causing misinterpretation. In this study, we analyzed the effect of changing the concentration of magnesium chloride, template genomic DNA and Taq DNA polymerase with the o...

The effects of 5 factors (template DNA, Mg(2+), dNTPs, Taq DNA polymerase, and primer) on the polymerase chain reaction (PCR) were investigated to optimize the start codon targeted polymor-phism (SCoT)-PCR system of Dactylis glomerata L., using an orthogo-nal design L16 (4(5)). A suitable SCoT-PCR system for D. glomerata was established; the 20 μL reaction volume contained 3.0 mM Mg(2+), 0.2 mM...

Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Taq) DNA polymerases. Incorrectly synthesized sequences were separated from the wil...

As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to m...

Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, wh...