The authors note that the detailed studies of the molecular mechanisms of DNA repair pathways were made possible by using site-specifically modified oligonucleotides and that the availability of phosphoramidites to synthesize oligonucleotides with DNA lesions has contributed to the field. They illustrate the article using primarily structural studies in the following examples:
Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 50
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Gebhardt et al. showed application of emodin (82) and its ethylenediamine analog 83 as non-ATP competitive inhibitors of GSK-3 (Table ). Addition of the ethylenediamine group on the emodin nucleus increased potency of inhibition (IC50 0.56±0.02 µM, 83), reduced cytotoxicity and generated an insulin sensitizing effect mediated by increasing hepatocellular glycogen and fatty acid biosynthesis. Selectivity's of compounds 82 and 83 were evaluated against twelve protein kinases including eleven of human protein kinases. Compound 83 showed high selectivity towards GSK-3β but 82 failed to do so.
There are often challenges in synthetic chemistry or radiochemistry to obtain the desired probe once the biochemical and physiological characteristics are optimized. Depending on the imaging modality, the synthetic chemistry requirements may vary considerably. For example, optical probes are challenged to maximize photon yield while minimizing photobleaching. PET probes are challenged by their shorter half-life and dependence on automation to reduce radiation exposure. For a PET imaging agent to succeed, a reliable and efficient method of synthesis that is translatable across radiochemistry facilities must be established. Furthermore, the synthesis method must be amenable to current Good Manufacturing Practice (cGMP) methods with a comprehensive quality assurance program if the probe is to be applied in human subjects.
I think most people don't show it because they don't bother to check it, and it's generally not of high scientific interest. I certainly never waste my cDNA by running it on a gel, it's just too precious. I use it in PCR directly after RT with the assumtion that RT worked, and run several internal controls to assess the quality of the cDNA. (beta actin, etc.) I never have any problems.
The intensity of the smear depends on how much you load. Try to calculate how much RNA and cDNA you would expect to have in your sample, and then use this to estimate how bright you expect your band to be. (Hint: RNA and ssDNA don't bind EtBr strongly anyway, so I would never expect a bright smear.)
Random hexamers also generate shorter cDNAs, especially if their concentration is high, so the shape of the smears might be different.
Can I ask why you are so preoccupied with verifying the products of your RT? If you are doing normal RT-PCR, don't bother running a gel; just proceed directly to PCR and do some internal PCR controls. If you are preparing a cDNA library, it would be preferable to prepare radiolabeled first-strand cDNA, run an alkaline gel alongside a labelled ladder, and then do autoradiography to determine the integrity of the cDNA alone (you won't see RNA in the radiograph).
Good question. However, I think the number of polymerase molecules in a reaction are limiting in this respect, and I think that there are a number of reasons strand displacement won't contribute to a significant increase in the number or cDNA molecules. (No time to explain, I have to go do some experiments!)
Hope this helps a bit,
Mukesh K. Pandey, PhD, received his BSc (H) and MSc in chemistry from Banaras Hindu University. He completed his MPhil and PhD in natural product and synthetic organic chemistry at the University of Delhi. After receiving his doctorate, he joined the University of Massachusetts at Lowell for his post-doctoral training on the design and synthesis of natural product-based polymeric material for targeted drug delivery. In 2009, he moved to Harvard Medical School for radiochemistry and molecular imaging training as a research fellow with Dr Timothy DeGrado. In 2012, he joined the Molecular Imaging Research program at Mayo Clinic in Rochester, Minnesota, as an Assistant Professor of Radiology. His research interests include the design, synthesis, and evaluation of novel molecular PET imaging probes for neurodegenerative diseases.