PCR amplification

RT-PCR A method used to quantitate mRNA levels that relies upon a first step of cDNA copying of mRNAs prior to PCR amplification and quantitation. 

Figure 2 Comparison of cloning and expression methods. In the conventional strategy (left), dehydrogenase genes obtained by PCR amplification of the original source DNAs are cloned into overexpression plasmids and verified by sequencing. Those with the desired structure are individually transformed into suitable host strains and the proteins are obtained, either as crude extracts or as purified samples. In the proposed streamlined approach (right), full-length dehydrogenase genes obtained by chemical synthesis are used directly in coupled transcription/translation reactions to obtain the proteins of interest.

Figure 5. 1% agarose gel showing PCR amplification products for PG cDNA obtained from mRNA isolate. Lanes 1, 2, 4 and 6 apple pectin culture medium, lanes 3, 5 and 7 glucose culture medium, lanes 1, 2 and 3 to isolate r,3 and lanes 4, 5, 6 and 7 to isolate rz. Lane 8 corresponds to the amplification of the 742 pb probe cloned in the vector PCR TM (In-vitrogen) digested with Eco R1 and lane 9 A digested with Pstl. 

The detection of mRNA of PG gene(s) by cDNA-PCR amplification assays in non-inducing conditions would agree with the hypothesis of probable basal levels of mRNA and discard a de novo induction in presence of the inductor. However, a substantial increasing of mRNA and proteins and, subsequently, of activity were observed in presence of pectine. 

A site at the Agricultural Experimental Station (Ithaca, NY) was treated in microcosms with C-labeled glucose, phenol, caffeine, and naphthalene. Levels of C02 were measured to assess utilization of the substrates, and the populations analyzed by separating the C-labeled DNA by density centrifugation, followed by PCR amplification and sequencing of 16S rRNA (Padmanabhan et al. 2003). Populations contained relatives to a range of bacteria that varied with the substrate. Only relatives of Acinetobacter were found in all samples, and for caffeine only Pantoea. 

Li S, Thompson SA, Woods JS. 1996. Localization of gamma-glutamylcysteine synthetase mRNA expression in mouse brain following methyhnercury treatment using reverse transcription in situ PCR amplification. Toxicol Appl Pharmacol 140 180-187. 

Tab. 5.2 Primer sets used for PCR amplification of human mtDNA and differences with the anderson sequence found in three templates at NIST for SRM 2392...

B. Henrion, G. Chevalier, and F. Martin, Typing truffle species by PCR amplification of the ribo.somal DNA spacers. Mycol. Re,s. 9S 37 (1994). 

Figure 19 Sample gel of the results of a PCR. Lane 1 is a 100-bp molecular marker lanes 2-6 are samples. The presence of the top bands (the species-specific endogenous gene) demonstrates that the PCR amplification was successfiil. Lack of the middle band (the introduced effect gene) and the bottom band (the selectable marker gene) in lane 3 indicates that sample is negative for the effect gene. Presence of all three bands in the remaining lanes indicates the samples are positive for the effect gene...

These reference genes demonstrate that the DNA isolated was of sufficient quality and quantity for PCR amplification. It is assumed that in the course of food processing, the species-specific reference gene and the transgene are degraded in a similar manner. It is also assumed that effects of the matrix on PCR amplification will be similar. The reduced amplification efficiency of both genes presumably has no effect on the ratio of their amounts, which reflects the ratio of modified and unmodified DNA. 

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. 

Liu, W., Ahlert, J., Gao, Q. et al. (2003) Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model. Proceedings of the National Academy of Sciences of the United States of America, 100, 11959. 

GeneReleaser set of proprietary polymeric materials, which facilitates the DNA release from cells or other genetic materials, suitable for PCR amplification leading to a PCR ready DNA/RNA protocol made in about 5 min. 

Sarkar G, Sommer SS. Haplotyping by double PCR amplification of specific alleles. Biotechniques 1991 10 436, 438, 440. 

Ruano G, Kidd KK. Direct haplotyping of chromosomal segments from multiple heterozygotes via allele-specific PCR amplification. Nucleic Acids Res 1989 17[20] 8392. 

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