Traditional Polyacrylamide Gel Electrophoresis Research Articles

High-throughput iSpinach fluorescent aptamer-based real-time monitoring of in vitro transcription

In vitro transcription (IVT) is an essential technique for RNA synthesis. Methods for the accurate and rapid screening of IVT conditions will facilitate RNA polymerase engineering, promoter optimization, and screening for new transcription inhibitor drugs. However, traditional polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography methods are labor intensive, time consuming and not compatible with real-time analysis. Here, we developed an inexpensive, high-throughput, and real-time detection method for the monitoring of in vitro RNA synthesis called iSpinach aptamer-based monitoring of Transcription Activity in Real-time (STAR). STAR has a detection speed at least 100 times faster than conventional PAGE method and provides comparable results in the analysis of in vitro RNA synthesis reactions. It also can be used as an easy and quantitative method to detect the catalytic activity of T7 RNA polymerase. To further demonstrate the utility of STAR, it was applied to optimize the initially transcribed region of the green fluorescent protein gene and the 3T4T variants demonstrated significantly enhanced transcription output, with at least 1.7-fold and 2.8-fold greater output than the wild-type DNA template and common transcription template, respectively. STAR may provide a valuable tool for many biotechnical applications related to the transcription process, which may pave the way for the development of better RNA-related enzymes and new drugs.Graphical iSpinach aptamer-based monitoring of transcription activity in real-time(STAR).

Combined analysis of serum γ-glutamyl transferase isoenzyme II, α-L-fucosidase and α-fetoprotein detected using a commercial kit in the diagnosis of hepatocellular carcinoma

γ-glutamyl transferase isoenzyme II (GGT-II) is a sensitive biomarker of hepatocellular carcinoma (HCC). However, numerous disadvantages of the traditional manual method affected its application. The commercial kit provided a convenient and fast method for the determination of GGT-II levels. The purposes of the present study were to compare the reproducibility and sensitivity between the manual and commercial kit methods and to evaluate the diagnostic efficiency for HCC with the combined analysis of GGT-II, α-L-fucosidase (AFU) and α-fetoprotein (AFP). In patients with various liver diseases (HCC, liver cirrhosis and chronic hepatitis) and normal subjects, GGT-II was detected by manual and commercial polyacrylamide gel electrophoresis (PAGE). The levels of AFU and AFP were assayed by colorimetry and a chemiluminescence immunoassay, respectively. The commercial PAGE had equal diagnostic efficiency with traditional manual PAGE and no significant differences were observed in intra- and average-gel reproducibility and GGT-II sensitivities between the manual and commercial PAGE (P>0.05). The incidence of GGT-II detected by commercial PAGE in HCC patients was 84.1% and <8% in benign liver disease. The levels of AFU and AFP in the benign liver diseases and normal subjects were lower than those in HCC. According to the cut-off value obtained by receiver operating characteristic curves, a total of 56.6 and 59.3% of HCC patients (64 out of 113 and 67 out of 113) had AFU >636.5 μmol/l h and AFP >44.0 μg/l, respectively. There were no significant correlations between GGT-II and AFU or AFP. Combined detection of GGT-II with AFU or AFP increased the diagnostic sensitivity to 92.9 and 93.8%, respectively. These results suggest that commercial PAGE provides a simple and reproducible method for GGT-II detection. Combined determination of GGT-II with AFU or AFP exhibited superior sensitivity and specificity for the diagnosis of HCC.

Silver staining DNA in polyacrylamide gels

This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (approximately 1 h) and is implemented using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely used method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.

Differentiation of pathogenic Bartonella species by infrequent restriction site PCR.

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. Bartonella isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease HhaI in conjunction with an infrequently cutting endonuclease, EagI, SmaI, or XbaI. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with an EagI, SmaI, or XbaI site in combination with an HhaI site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3' ends of the EagI-, SmaI-, or XbaI-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations EagI-HhaI and SmaI-HhaI. However, the combination of XbaI-HhaI produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic Bartonella species.