Amplicon

P. Mavingui, T. Laeremans, M. Flores, D. Romero, E. Martinez-Romero, and R. Palacios, Genes es.sential for nod factor production and nodulation are located on a symbiotic amplicon (AMP/ rrCEN299pc60) in Rhizobium tropici. J. Bacteriol. 180 2866-2874 (1998). 

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. 

Agarose gel electrophoresis can be used to determine whether the PCR amplicon is the expected size. The density of the gel should be chosen to ensure resolution of... 

Sequencing the amplicon is the most conclusive confirmatory technique. The main consideration is that the DNA must be appropriately purified to achieve unambiguous sequencing data. However, sequencing requires expensive laboratory equipment that may not be available in all labs. Sequencing does not depend upon the specificity of a probe, or restriction enzyme, but gives a direct identification of the amplicon of interest. 

Another PCR variation used for detecting bacteria is nested PCR, which uses two successive rounds of amplification with different sets of primers.16,96,97 After amplification of one DNA fragment in the first round, the amplicon is used as a template for a second round of amplification, together with another primer set that amplifies an internal segment of the first amplicon.96,97 Nested PCR is especially useful when the concentration of the target bacterium is too... 

Although PCR amplification begins at an exponential rate, it enters a stationary phase after approximately 30 cycles, and additional cycles do not increase the concentration of amplicons. For this reason it is not practical to use PCR for quantifying bacteria directly, and other methods, such as real-time PCR, are used for this purpose. 

Real-time PCR is a quantitative method for measuring amplicons as they are produced by measuring the increase in fluorescence of a dye added to the reaction mixture.12,104,105 Methods using fluorescent reporters, such as SYBR Green,104,106 TaqMan ,107,108 or molecular beacons,9 collect quantitative data at the time when DNA is in the exponential phase of amplification. 

Mies55 Breast cancer RNAzol (Biotecx, Huston, TX) ER gene (exons 1 and 2) 150 bp amplicon is feasible for clinical and research studies. 

Lehmann et al.58 Microdissected CD68 (+) cell from liver section N/A Real time, for oncogenes with amplicon size of 150-300 bp It may provide a powerful tool to study gene alterations in FFPE tissue. 

Lahr et al.59 Microdissected thyroid tissues PUREscript kit (BlOzym, Germany) Nested PCR, RET oncogene with amplicon size of 141-383 bp Expressed genes can be analyzed from routine FFPE tissue slides or pooled single cells. 

Tested Genes Primer Sequences Amplicon Sizes (bp)... 

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