Plasmid-to-plasmid Southern blot analysis validates the presence of nucleotide binding site (nbs) sequences in cloned plasmids.

Southern blot analysis is an important molecular biology technique for identifying a specific sequence in DNA samples. Although it is no longer used extensively in recent years, the steps and underlying principles of Southern blot are applicable to modern biology. High sensitivity and limited background are keys to successful Southern blots, whereas obtaining good quality and quantity of genomic DNA as starting materials and detecting a single/low copy target sequence in the genome can be challenging. To ensure student success in performing the technique for the first time, a modified "plasmid-to-plasmid" Southern blot was implemented to confirm the presence of grape nucleotide-binding site (nbs) sequences in cloned plasmids like those described previously. The plasmid DNA and a control plasmid, pSCA7 (T1-T3-W6) containing a known grape nbs sequence, were digested with restriction enzymes, followed by agarose gel electrophoresis. The DNA band corresponding to the nbs sequence of the pSCA7 (T1-T3-W6) was extracted from the gel for PCR digoxigenin (DIG) probe synthesis. At the same time, the cloned plasmid DNA and its digested DNA fragments were blotted from the gel onto nylon membranes to be hybridized with the DIG probe followed by the detection for nbs sequences. Students successfully performed Southern blots to confirm the presence of nbs sequences in their cloned plasmids and wrote up the results following the format of scientific research papers. They learned the principles and applications of Southern blot and gained hands-on experience with associated techniques.

RT-qPCR demonstrates light-dependent AtRBCS1A and AtRBCS3B mRNA expressions in Arabidopsis thaliana leaves.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in diagnosis and research to determine specific mRNA expressions in cells. As RT-qPCR applications increase, it's necessary to provide undergraduates hands-on experience of this modern technique. Here, we report a 3-week laboratory exercise using RT-qPCR to demonstrate the light-dependent expressions of AtRBCS1A and AtRBCS3B genes encoding two Arabidopsis thaliana small subunits of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In the first week, students purified and quantified total RNA from leaves of A. thaliana pretreated in the dark for 96 hr and untreated controls. In the second week, RNA samples were separated by formaldehyde gel electrophoresis and used for RT-qPCR. Students calculated expressions of the two genes in dark treated leaves as percentages of those of the controls by using the 2(-ΔΔC) T method and the collected CT s. In the third week, class CT s, melting curves, students' calculations, and factors affecting the reliability of RT-qPCR results were summarized and discussed. Students' results show that (i) relatively pure and intact RNA samples are obtained; (ii) ACTIN2 is a better reference gene than the 18S rRNA; (iii) the dark treatment reduces both gene expressions to < 1%; (iv) the reduction in the expression of AtRBCS3B is significantly more than that of the AtRBCS1A. Results from pre- and post-lab tests indicate that besides the theory, this exercise helps students learn the applications and associated techniques of RT-qPCR. Future modifications and new experiments that can be developed based on students' learning outcomes and assessment are also discussed. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):405-411, 2016.

Blue genes: An integrative laboratory to differentiate genetic transformation from gene mutation for underclassmen.

The ability of students to understand the relationship between genotype and phenotype, and the mechanisms by which genotypes and phenotypes can change is essential for students studying genetics. To this end, we have developed a four-week laboratory called Blue Genes, which is designed to help novice students discriminate between two mechanisms by which the genetic material can be altered: genetic transformation and gene mutation. In the first week of the laboratory, students incubate a plasmid DNA with calcium chloride-treated Escherichia coli JM109 cells and observe a phenotype change from ampicillin sensitive to ampicillin resistant and from white color to blue color on plates containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) and isopropyl ß-D-thiogalactopyranoside (IPTG). Over the course of the next three weeks, students use a battery of approaches including plasmid DNA isolation experiments, restriction maps, and PCR to differentiate between mutation and transformation. The students ultimately come to the conclusion that the changes in phenotypes are due to genetic transformation and not mutation based on the evidence generated over the four-week period. Pre-laboratory tests and post-laboratory tests indicate that this set of exercises is successful in helping students differentiate between transformation and mutation. The laboratory is designed for underclassmen and is a good prerequisite for an apprentice-based research opportunity, although it is not designed as a class based research experience. Potential modifications and future directions of the laboratory based upon student experiences and assessment are presented.

A laboratory exercise illustrating the sensitivity and specificity of Western blot analysis.

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the separated polypeptides from the gel is created on the membrane, which is then probed with antibodies or other ligands to identify specific polypeptide(s). Here, we report an undergraduate laboratory exercise involving Western blotting. During the 3-week laboratory exercise, students investigated the likelihood of the presence of a serum albumin in fruit fly (Drosophila melanogaster) homogenate, bovine calf serum (BCS), and fetal bovine serum (FBS). Students isolate proteins from fruit fly larvae and perform sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic blotting, and immunoassay comparing those proteins with BCS and FBS proteins. Their results indicate that serum albumin is present in BCS and FBS but is absent in fruit flies. In the process, the specificity and sensitivity of Western blot analysis is demonstrated. The laboratory exercise can be easily incorporated into any college-level biochemistry or molecular techniques laboratory. The procedure used can be easily adapted to study other proteins.

PCR cloning of partial nbs sequences from grape (Vitis aestivalis Michx).

Plants defend themselves against pathogens via the expressions of disease resistance (R) genes. Many plant R gene products contain the characteristic nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. There are highly conserved motifs within the NBS domain which could be targeted for polymerase chain reaction (PCR) cloning of R genes. Here, we report a 4-week undergraduate laboratory exercise that mimics the research environment to PCR-clone partial nbs sequences using degenerate primers corresponding to the phosphate-binding loop (P-loop) and GLPL motifs within the NBS domain of potential R gene products from the North American grape, Vitis aestivalis Michx. Students were able to complete the laboratory procedures successfully and obtained four different clones, among which three are new. Through the laboratory exercise, students learned a variety of important molecular techniques including genomic DNA isolation, DNA quantification, PCR, agarose gel electrophoresis, DNA extraction from agarose gel, ligation, bacterial transformation, and plasmid DNA isolation and purification. They also used currently available web-based bioinformatic programs for sequence analysis. The laboratory exercise provides students the hands-on experience on PCR cloning and shows them how it is done in a research environment. The clones obtained may be further tested for their potential use as markers to differentiate resistant cultivars from the susceptible ones, a useful tool in breeding programs.

An undergraduate laboratory exercise to study the effect of darkness on plant gene expression using DNA microarray.

DNA microarrays are microscopic arrays on a solid surface, typically a glass slide, on which DNA oligonucleotides are deposited or synthesized in a high-density matrix with a predetermined spatial order. Several types of DNA microarrays have been developed and used for various biological studies. Here, we developed an undergraduate laboratory exercise using an Arabidopsis DNA microarray to study the gene expression of Brassica rapa, Wisconsin Fast Plant. Genes involved in senescence, cell wall loosening/degradation, and sugar transport were the most upregulated, while those involved in photosynthesis, the elimination of reactive oxygen intermediates associated with photooxidative stress and auxin synthesis, were the most downregulated. Students were able to complete the experiment successfully. Throughout the exercise, they learned various important molecular techniques including RNA isolation, quantification, reverse transcription, cRNA synthesis, labeling and purification, and microarray hybridization, washing, scanning, and feature extraction. The exercise can be integrated into a college-level molecular biology laboratory. The procedure used can be adapted to examine other effects on other organisms.

Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.