Polymerase chain reaction electrophoresis

In some experiments, it may be necessary to recover microinjected cells (e.g., for electrophoresis, polymerase chain reaction). One way to do this is to plate cells on small pieces of coverslips placed inside a culture dish, inject all cells on pieces of coverslips and then remove the pieces with forceps. 

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Liu J, Enzelberger M, Quake S (2002) A nanohter rotary device for polymerase chain reaction. Electrophoresis 23 1531-1536... 

G. Muyzer, E. C. de Waal, and A. G. Uitterlinden, Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for I6S rRNA. Appl. Environ. Microbiol. 59 695 (1993). 

Baba, Y., Tomisaki, R., Sumita, C., Morimoto, I., Sugita, S., Tsuhako, M., Miki, T., and Ogihara, T., Rapid typing of variable number of tandem repeat locus in the human apolipoprotein B gene for DNA diagnosis of heart disease by polymerase chain reaction and capillary electrophoresis, Electrophoresis, 16, 1437, 1995. 

Stothard, J.R., Frame, IA. and Miles, MA. (1997) Use of polymerase chain reaction-based single strand conformational polymorphism and denaturing gradient gel electrophoresis methods for detection of sequence variation of ribosomal DNA of Trypanosoma cruzi. International Journal for Parasitology 27, 339-343. 

Fig. 19.1 Differential displays comparing RNAs from saline (S)-, imipramine (I)- or fluoxetine (F)-treated rats. Total RNA was extracted from hypothalami of animals treated with the different drugs for two months. Autoradiograms of amplified -[35S]-dATP-labeled PCR (polymerase chain reaction) products after electrophoresis in 6% polyacrylamide gels are shown for two different primer combinations that identified one upregulated (arrowhead) and one downregulated (arrow) fragment in the groups treated with antidepressants (from [4] with permission).

The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA) play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH) (Amann et al. 1995). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis (Duineveld et al. 2001). 

Analysis of polymerase chain reaction-product by capillary electrophoresis with laser-induced fluorescence detection and its application to the diagnosis of medium-chain acyl-coenzyme A dehydrogenase deficiency. 

To understand how these modem methods work, it is necessary first to review some general laboratory techniques ubiquitous in genetic engineering. Among the most important are gel electrophoresis of nucleic acids, nucleic acid hybridization assays, and the polymerase chain reaction. 

M. Galloway and S.A. Soper, Contact conductivity detection of polymerase chain reaction products analyzed by reverse-phase ion pair microcapillary electrochromatography, Electrophoresis, 23 (2002) 3760-3768. M. Masar, M. Dankova, E. Olvecka, A. Stachurova, D. Kaniansky and B. Stanislawski, Determination of free sulfite in wine by zone electrophoresis with isotachophoresis sample pretreatment on a column-coupling chip, J. Chromatogr. A, 1026 (2004) 31-39. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

Zhou et al. [175] described the determination of severe acute respiratory syndrome (SARS) coronavirus by a microfluidic chip system. The unit included an LIF microfluidic chip analyzer, a glass microchip for both PCR and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. According to the authors, the system allowed efficient DNA amplification of the SARS coronavirus followed by electrophoretic sizing and detection on the same chip. 

Fig. 6. (Opposite page) A high-throughput production method for screening proteins from cDNA libraries. Authentic (A) and glutathione -transferase (GST)-fused (G) proteins in the reaction mixtures after a semi-automated polymerase chain reaction/ transcription and translation from 54 different cDNAs separated by sodium dodecyl sul-fide-polyaciylamide gel electrophoresis and stained with Coomassie Brilliant Blue. T and S, respectively, mark total translation product and the supernatant fraction after centrifugation at 30,000g for 15 min.

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