Standard Polymerase Chain Reaction

Polymerase chain reaction (PCR) is an in vitro method for enzymatic synthesizing and amplification ofdefined DNA sequences outofamixtureofnucleicacids.PCRgenerates sufficient DNA copies for subsequent molecular analysis. The standard PCR is an endpoint reaction, which produce adequate quantity of the PCR prcxiuct suitable to be identified by size electrophoresis analysis, sequencing or by probe hybridization. 

Primers (oligonucieotides) are short single-strand DNA sequences, usually synthesized on automated DNA synthesizer. Upstream (forward or sense) primer and downstream (reverse or antisense) primer are needed for each reaction. Optimal primers concentration ranges between 100 nM and 500 nM (0.1-0.5 pM) of each primer. The molar concentration of the primers can be calculated by the following formula  

Micromolar (pM) concentration of the primer in the reaction = quantity of the primer (in picomoles)/volume of the PCR (in micromoles). 

DNA polymerases are different enzymes able to synthesize a new DNA strand on a template strand. DNA polymerases are important enzymes involved in the DNA repair and replication. The following thermostable polymerases are the most common used enzymes for PGR. 

Reaction Volume The reaction volume ranges over 5-100 pi (5, 25, 50 or 100 pi). For 

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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. 

Fig. 9.4 Establishment of the quantitative methylation-specific polymerase chain reaction (MSP) analytical procedure. The ABI PRISM 7900 HT Sequence Detector system was used to perform real-time polymerase chain reaction (PCR) using MSP primers and bisulfite-modified template DNA. Upper panels Setting up the conditions to obtain the standard curves with 50% (A) or 25% (B) sequential dilution of the template. Lower panels The amplification curves on the left represent P-actin, unmethylated, and methylated MSP products, respectively, for reelin (RELN) (C). Amplification curves were compared at the set threshold before 40 cycles. Amplification curves from various samples are shown in the lower panel right (D)...

Venous blood (8 mL) was collected into 15-mL tubes containing 50 mmol/L disodium EDTA, and genomic DNA was extracted by the standard (phenol/chloroform) method. The genotype frequencies for GSTPl were determined by polymerase chain reaction (PCR)/RFLP-based methods using DNA extracted from peripheral lymphocytes. 

Bortolin, S. Christopoulos, T. K. Verhaegen, M. Quantitative polymerase chain reaction using a recombinant DNA internal standard and time-resolved fluorometry. Anal. Chem. 1996, 68(5), 834-840. 

Table 8.2.2 Polymerase chain reaction (PCR) mixture in a standard 200-[il tube...

Polymerase chain reaction (PCR), on the other hand, has several advantages it can be used to analyze small numbers of tumor cells, DNA from formalin-fixed, paraffin-embedded tumor tissue can be used, and it can be automated and standardized. Quantitative PCR techniques are currently being assessed for their clinical application to HF.R-2 DNA testing (Vona et al., 1999). However, presently the PCR technology is not optimally suited for routine, clinical application (see next section for details of quantitative analysis of HER-2Ineu expression). 

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).

Biodistribution analysis is conducted at the molecular level. The current gold standard is a quantitative polymerase chain reaction (Q-PCR) assay that detects the number of vector copies per microgram of genomic DNA. Biological fluids and tissue samples are carefully harvested (to avoid crosscontamination) from control and gene therapy product-injected animals at... 

Figure 5-19. Applications of the polymerase chain reaction. The diagram illustrates the versatility of the PCR reaction depending on the nature of the primer. Use of a standard primer leads to simple DNA amplification. Primers which include a mismatch can be used to introduce site-directed mutations indeed this is now probably the method of choice for generating...

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