PCR method

This acronym stands for Reverse Transcriptase Polymerase Chain Reaction, a method used to first copy a strand of RNA into cDNA, then amplify it through standard PCR methods. 

Short (2-6 bp), inherited, tandem repeat units of DNA occur about 50,000-100,000 times in the human genome (Chapter 36). Because they occur more frequently—and in view of the routine application of sensitive PCR methods—they are replacing RFLPs as the marker loci for various genome searches. 

SRM 2391 is designed to provide quality assurance to laboratories that perform DNA profiling using PCR methods. This SRM can be used to verify that each step of the analysis system is operating correctly and within the proper limits. 

PCR method for wheat, buckwheat, peanut, shrimp, and crab... 

Since the MHLW designated shrimp/prawn and crab for mandatory labeling in June 2008, respective PCR methods to discriminate between shrimp/prawn and crabs in processed foods have been developed. 

Tani, K. Kurokawa, K. Nasu, M. Development of a direct in situ PCR method for detection of specific bacteria in natural environments. Appl. Environ. Microbiol. 1998,64,1536-1540. 

Wittwer, C. Hahn, M. Kaul, K. (Eds.). Rapid Cycle Real-Time PCR Methods and Applications. Springer-Verlag Berlin, 2004. 

Hayashi, K. (1991) PCR-SSCP a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods and Applications 1, 34-38. 

Salles, F. J., and Strickland, S. (1995). Rapid and sensitive analysis of mRNA polyadenylation states by PCR. PCR Methods and Applications 4, 317-321. 

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. 

RyR3 has been reported to be the major isoform in some smooth muscle tissues (Neylon et al 1995), but RyR2 has been reported to be the primary isoform expressed in urinary bladder smooth muscle (Chambers et al 1999), in which CICR has been definitively established (Ganitkevich Isenberg 1992, Imaizumi et al 1998, Collier et al 2000). RyRl has also been reported in smooth muscle at the expression and functional levels (Neylon et al 1995). It should be noted that determination of isoform expression has largely been reported using non-quantitative RT-PCR methods, with associated interpretive difficulties. 

Fahey, E., Kwoh, D. Y. and Gingeras, T. R. Self-sustained sequence replication (3SR) an isothermal transcription-based amplification system alternative to PCR , PCR Methods Appl., 1, 25-33 (1991). 

An extension of the PCR method is RT-PCR. Here mRNA is extracted from the cells or tissue, converted to cDNA using the enzyme reverse transcriptase and PCR is then carried out This method is used to detect the expression of specific mRNA sequences in cells or tissues. 

Another possibility to enhance the specific DNA-fragment is to perform a nested PCR. With this PCR method, sequences of very low abundancy can be amplified, but the risk to enhance the background at the same time is very high. For a nested PCR, a small aliquot of the first PCR reaction is used together with gene-specific primers which bind within the initial PCR product. 

Quantitative PCR has been widely used to determine the amount (number of molecules) of DNA molecules in a test sample. The best quantitative PCR method involves the addition of known amounts of a similar DNA or RNA fragment, such as one containing a short deletion or specific mutation, to the test sample before amplification. Such internal standards must be precisely calibrated to ensure that they are amplified and detected in a form and manner that are similar to the test sample. The ratio of the internal standard and the targeted template will depend on the amount of internal standard added and allows for the determination of the amount of the targeted molecule in the test sample. Therefore, the ideal standard for quantitative amplification based assays should have a structure that is comparable to the template of interest and which allows for the simultaneous amplification of both template and standard using a single primer pair. 

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). 

The cDNAs, which are assumed to be synthesized at the end of the RT reaction, are subjected to amplification reaction by following two methods. The first, Indirect in situ PCR method, involves the incorporation of unlabeled nucleotides (40). In the indirect method, the target cDNA is amplified by unla-... 

For the styrene-butadiene example, the use of the PCR method to develop a calibration for di-butadiene is summarized in Table 12.6. It should be mentioned that the data were mean-centered before application of the PCR method. Figure 12.12 shows the percentage of explained variance in both x (the spectral data) andy (the c/i-butadiene concentration data) after each principal component. After four principal components, it does not appear that the use of any additional PCs results in a large increase in the explained variance of X or y. If a PCR regression model using four PCs is built and applied to the calibration data, a fit RMSEE of 1.26 is obtained. 

---