Contamination polymerase chain reaction

Molecular methods used to uncover mutations are subject to several variables. The anticoagulants used for blood collection can affect digestion with restriction enzymes and amplification reactions. The type of detergent used in cell lysis can affect amplification of DNA by inhibiting the DNA-amplifying enzyme such as the taq polymerase used in the polymerase chain reaction (116). The control of contamination is crucial in ensuring the quality of results obtained by molecular analysis (117). 

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. 

Patino B, Medina A, Domenech M, Gonzalez-Jaen MT, Jimenez M and Vazquez C. 2007. Polymerase chain reaction (PCR) identification of Penicillium brevicompactum, a grape contaminant and mycophenolic acid producer. Food Addit Contam 24(2) 165-172. 

L4. Longo, M. C., Beminger, M. S., and Hartley, J. L., Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93, 125-128 (1990). 

Fig. 9.2 Representative examples of the methylation-specific polymerase chain reaction (MSP) analyses for gene promoter regions. Lanes Lf and M indicate the presence of unmethylated and methylated template, respectively. Placental DNA (PDNA) and in vitro methylated DNA (IMD) served as negative and positive controls, respectively. Water (H) was used to detect contamination. Samples 1, 3, 4, and 7 indicate the presence of a methylated promoter DNA with various degrees of methylation, and samples 2, 5, and 6 represent an unmethylated promoter...

Dean, T. R., Roop, B., Betancourt, D., and Menetrez, M. Y. (2005). A simple multiplex polymerase chain reaction assay for the identification of four environmentally relevant fungal contaminants. /. Microbiol. Methods 61, 9-16. 

Zur, G., Shimoni, E., Hallerman, E., and Kashi, Y. (2002). Detection of Alternaria fungal contamination in cereal grains by a polymerase chain reaction-based assay. /. Food Prot. 65, 1433-1440. 

M. G. Roper, C. J. Easley, and J. P. Landers, Advances in Polymerase Chain Reaction on Microfluidic Chips, Anal. Chem. 2005, 77, 3887 K. D. Dorfman, M. Chabert, J.-H. Codarbox, G. Rousseau, P. de Cremoux, and J.-L. Vtovy, Contamination-Free Continuous Flow Microfluidic Polymerase Chain Reaction for Quantitative and Clinical Applications, Anal. Chem 2005,... 

It should be emphasized that the polymerase chain reaction requires specific primers for synthesis therefore, the sequence flanking the sequence to be amplified must be known. PCR reactions are also very susceptible to contamination by other DNA. Precautions against contamination need to be... 

There are various methods for detecting Mycoplasma contamination of cell culture. A sensitive polymerase chain reaction test with broad specificity for Mycoplasma species is our method of choice (8). There are several products available for the eradication of Mycoplasma species from cell lines. The effectiveness of the treatment will depend on the cells and involves trail and error. This is because some cell lines are very sensitive to the chemicals used to eradicate Mycoplasma and may become static or die during treatment. 

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). 

Spaepen M, Angulo AF, Marynen P Cassiman JJ (1992) Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction. FEES Microbiology Letters 99 89-94. 

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