Polymerase chain reaction protocol

Ml 3 Repeat Probe Polymerase Chain Reaction Protocol... 

Helps et al. (2004) slaughtered two female cattle, followed by one male, and then four female cattle and collected swab samples from the split vertebral-column surfaces. Real-time polymerase chain reaction protocols were followed to determine the extent to which tissue from the male carcass accumulated in the splitting saw and was disseminated to subsequent female carcasses. Under simulated abattoir conditions (i.e., washing the saw for 5 s between carcasses and washing the carcasses before collecting samples), these researchers reported that 0.01% of the tissue recovered from the split vertebral-column surface of the final female carcass in the sequence was from the male carcass and that 10% of the tissue remaining in the housing of the saw was from the male carcass. It was concluded from that study that "should a BSE-positive carcass be... 

Leung KT, Watt A, Lee H, Trevors JT (1997) Quantification detection of penta-chlorophenol-degrading Sphingomonas sp. UG30 in soil by a most-probable-number polymerase chain reaction protocol. J Microbiol Method 31 59-66... 

Apart from the traditional organic and combinatorial/high-throughput synthesis protocols covered in this book, more recent applications of microwave chemistry include biochemical processes such as high-speed polymerase chain reaction (PCR) [2], rapid enzyme-mediated protein mapping [3], and general enzyme-mediated organic transformations (biocatalysis) [4], Furthermore, microwaves have been used in conjunction with electrochemical [5] and photochemical processes [6], and are also heavily employed in polymer chemistry [7] and material science applications [8], such as in the fabrication and modification of carbon nanotubes or nanowires [9]. 

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ahility to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1-10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13). 

Fig. 3. Comparison of different enzyme-linked immuno sorbent assay (ELISA) methods adapted for immuno-polymerase chain reaction (IPCR). Dependent on the purification grade of the sample to be analyzed and the availability of specific and functionalized antibodies, several typical ELISA protocols were adapted to IPCR. In the direct approach (A), the pure antigen is immobilized to the microplate surface and subsequently detected by a labeled specific antibody. If no labeled antibody is available (e.g., because of unpurified ascites fluid containing the antibody or loss in activity following labeling), a standardized labeled secondary species-specific antibody is used for detection of the primary antigen-specific antibody (B). For the detection of the antigen from matrices such as serum, plasma, tissue homogenate, and so on, a capture antibody immobilized to the microplate surface was used either in a direct (C) or indirect (D) sandwich approach, with the latter one additionally including a secondary species-specific detection antibody. For different methods of coupling antibody and DNA, abbreviated by in this figure, compare Fig. 2. Note that protein A chimeras (Fig. 2A) are not compatible with capture antibodies (Fig. 3C, D).

Adler M. Hapten labeling of nucleic acids for immuno-polymerase chain reaction applications, bioconjugation protocols Strategies and methods. In Niemeyer CM, editor. Methods in Molecular Biology. Humana Press, 2004 163-180. 

The exception to limited technical coverage concerns the trend toward direct sequencing of plasmids and polymerase chain reaction (PCR) products. This is a reflection of the increasing need for ease and rapidity in sequence determination. However, many published protocols lack consistency. As discussed at greater length below, there are few or no (as yet) tried-and-true favorites. Thus, the techniques presented in this chapter comprise detailed protocols for only one fairly robust rapid isolation and... 

Basic Protocol for Polymerase Chain Reaction of Ancient DNA... 

DHPLC is usually performed on a styrene-divinylbenzene-based polymeric stationary phase, with a mobile phase that contains triethylammonium acetate as the IPR to provide adequate reversed phase (RP) retention for the negatively charged nucleic acid molecules. The samples are usually amplified according to polymerase chain reaction (PCR) protocols and then injected into the chromatographic system. 

---