PCR contamination

To minimize the risk of PCR contamination, PCR tubes and pipets can be exposed to ultraviolet (UV) irradiation (UV Stratalinker 2400, Stratagene) to inactivate contaminating DNA. 

Negative control DNA samples should be included for every amplicon being analyzed and positive control DNA samples should be included when available (see Note 5). A negative control is a sample with no sequence change in the amplicon and a positive control is a sample with a known sequence change in the amplicon being analyzed. A blank (H2O) control should also be included to check for PCR contamination. 

The PCR product can be stored at 4°C until needed. PCR trays should be briefly centrifuged as condensation may occur on the lid, which is a possible source of post-PCR contamination. 

Because the PCR exponentially copies the target molecule or molecules, amplicon contamination in the laboratory is a serious concern. It is recommended that the mastermix is prepared in an isolated area, such as a PCR station equipped with a UV light. This work area should be exposed to UV radiation after use to destroy any DNA contaminants. The use of dedicated pipets and Altered pipet tips is also recommended. The template DNA should be prepared and added to the reaction in an area that is isolated from the mastermix preparation hood. The thermal cycling and gel electrophoresis should be conducted in a third work area and care should be taken not to introduce amplified PCR products into the mastermix or template preparation work areas. 

Negative controls demonstrate the absence of laboratory contamination or sample cross-contamination. DNA extracts from nontransgenic plants, clean buffer and mastermix with no template DNA added are common negative controls that are run concurrently with the test samples in the PCR. 

Collecting samples of ancient nucleic acids is a delicate operation that requires what are basically surgical procedures. It is advantageous, whenever possible, that the samples be collected at excavation sites and precautions taken to ensure that they do not become contaminated with other, particularly more recent, nucleic acids. At high temperatures and humidity, nucleic acids decay quickly. Well-preserved ancient nucleic acids can, therefore, be expected in sites where low temperatures and a dry environment prevail, as, for example, in cold, desert areas of the world. Once collected, the samples need to be isolated from any other remaining materials until they can be amplified by PCR and their chemical composition and structure can then be studied. 

Since rolling circle amplification takes place at a constant temperature, there is no need for the target amplification process to take place in a thermal cycler, which is required to regulate the temperature for different parts of the reaction. The type of DNA polymerase to be used in RCA is not limited to thermostable enzymes, like the PCR-based diagnostics. On the other hand, the RCA method requires the environment to be free of contaminations as the RCA arrays are highly sensitive. Wiltshire [22]... 

Li ZJ, Xu JM, Tang C, Wu JJ, Akmal M, Wang HZ (2006) Application of 16S rDNA-PCR amplification and DGGE fingerprinting for detection of shift in microbial community diversity in Cu-, Zn-, and Cd- contaminated paddy soils. Chemosphere 62 1374-1380... 

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. 

The immobilized-hybridization methods provide simple detection systems that do not require separation of the target PCR product from contaminating DNA. These protocols are also applicable to the detection of an unmodified DNA target [5, 33, 34],... 

Current methods for DNA detection usually require enzymatic amplification of the target DNA sequence prior to analysis. For example, the PCR technique selectively increases the concentration of the target sequence relative to unrelated sequences. PCR methods, however, introduce ambiguities resulting from contamination by different DNA sequence. Therefore, a definitive method is required for the analysis of a single, original DNA sequence. To achieve this objective, the sensitivity and speed of the chemiluminescent enhancement techniques described in this chapter must be improved. 

PCR) and compared with sequences in other individuals and modern specimens. However, ancient DNA is severely damaged and fragmented. Contamination of aged samples and extracts with modern DNA is a serious problem and, whilst the study of DNA in archaeological samples will constitute a major area of future activity in the discipline, current research will continue to focus on the authentication of samples of ancient DNA advances have been so rapid that perusal of the appropriate scientific journals is essential. For somewhat more recent views of the state of ancient DNA research, see Willerslev and Cooper (2005). 

Exogenous sources such as a person s hair or skin, doorknobs, laboratory benches, dust, reagents, thermal cyclers, and pipet tips are some of the common sources of DNA contamination. Ideally, a laminar air flow bench with filtered air provides a clean, dust-free environment. Sample preparation should be done in a separate room or area. The addition of sample to the PCR reaction mixture in the... 

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