Polymerase chain reaction efficiency

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction. The techniques for chemical synthesis of oligonucleotides are highly developed. Very efficient automated methodologies based on solid phase synthesis are used extensively in fields that depend on the availability of defined DNA sequences.52... 

Iqbal, S. Robinson, J. Deere, D. Saunders, J. R. Edwards, C. Porter, J. Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water. Lett. Appl. Microbiol. 1997,24,498-502. 

An especially intriguing pair of products obtained from marine organisms in recent years are Vent and Deep Vent DNA polymerase. These products are used in DNA research studies. Their special feature is that they are at least 10 times as efficient as other similar products in polymerase chain reactions because they can tolerate temperatures just below the boiling point of water, a characteristic that comparable research tools lack. Vent and Deep Vent DNA polymerases are obtained from the bacterium Thermococcus litoralis, which is found around deep-sea hydrothermal vents at the bottom of the ocean. 

Parent, J.L., C. Le Gouill, M. Rola-Pleszczynski, and J Stankova, A highly efficient technique for cloning of refractory DNA fragments and polymerase chain reaction products. Anal Biochem, 1994. 220(2) 426-8. 

Roeder, T., Simple and efficient cloning of small polymerase chain reaction-generated DNA products. Anal Biochem, 2000.285(2) 278-80. 

Naked plasmid DNA was not only trapped in the cytoplasm around the site of microinjection, as visualized by fluorescence in situ hybridization (FISH) or using FITC-labeled DNA, but was also eliminated rapidly at physiological temperature (Lechardeur et al., 1999). The disposal of the DNA was completely prevented when the cells were kept at 4°C (Lechardeur et al., 1999). A similar conclusion was reached by monitoring the amount of microinjected expression cassette by the polymerase chain reaction (PCR) (Pollard et al., 2001), suggesting that the metabolic instability of naked DNA contributes to the low efficiency of gene transfer (Lechardeur et al., 1999 Mirzayans et al, 1992 Neves etal., 1999 Pollard et al., 2001). 

Zhou et al. [175] described the determination of severe acute respiratory syndrome (SARS) coronavirus by a microfluidic chip system. The unit included an LIF microfluidic chip analyzer, a glass microchip for both PCR and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. According to the authors, the system allowed efficient DNA amplification of the SARS coronavirus followed by electrophoretic sizing and detection on the same chip. 

Evolution has been shown to be an efficient tool for the improvement of enzymes and pathways. Stochastic approaches to introduce point mutations in genes include error prone polymerase chain reaction (ePCR),11 the use of low-fidelity (mutator) strains,12 and chemical mutagens.13... 

Many different techniques, such as bacteriological culture, DNA staining using fluorochrome and immunological or biochemical methods, are available to detect mycoplasma contamination (see section 1.6). However, none seem to be fully efficient, so a combination of different methods is often necessary. Molecular tools such as hybridization using rDNA gene probes or polymerase chain reaction (PCR) have been developed over the past few years. Several studies using 16S rDNA-based PCR concluded that PCR seems to be a very convenient method for routine detection of cell culture contaminations (Spaepen, 1992 Teyssou, 1993 van Kuppeveld, 1994). 

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