Molecular Biology Blog: The blog for research scientists and advanced students

A pyknon is a new type of putative regulatory motif that named from the greek adjective for dense. By definition, pyknons are variable length sequences with a statistically significant number of intact copies in the intergenic and intronic regions of the genome and additional copies in the untranslated or amino acid coding regions of known transcripts. Even though the original presentation discussed pyknons in the context of the human genome, pyknons likely represent a more general architectural component of eukaryotic genomes. The exact role of pyknons is currently unclear but the findings so far support a regulatory responsibility. The possibility has been raised that pyknons hint at a previously unseen layer of cell process regulation.

from Rigoutsos, I (2008) Pyknons as putative novel and organism-specific regulatory motifs In: Morris , K.V. (Ed.) RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press, Norfolk, UK.

Labels: gene regulation, pyknon, regulation, RNA

MicroRNAs are short, ~22 nucleotide regulatory RNAs, first discovered in Caenhorhabditis elegans. Hundreds of microRNAs have been identified in plants and animals. Based on the current number of predicted microRNAs, one to three percent of genomic DNA is believed to encode these small, regulatory RNAs.

MicroRNAs inhibit protein synthesis by binding to their target mRNAs and regulating gene expression in a post-transcriptional manner. The exact mechanism by which target gene expression is down-regulated is unclear; however, experimental evidence has led to several different theories to explain microRNA-mediated mRNA repression. These possible mechanisms include target degradation, localization to P-bodies, inhibition of translation initiation or elongation, mRNA deadenylation, and mRNA destabilization.

from Chin and Slack in RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity

Further reading: RNA and the Regulation of Gene Expression

Labels: gene regulation, microRNA, mRNA, regulation, RNA

Nanotechnology will play an important role in future biosensor development. Nanotechnology is now making possible development of in vivo sensors, i.e. nano-sized devices envisioned to be ingested or injected where they could act as reporters of in vivo concentrations of key analytes. These engineered nanoparticle devices imbedded in the cytosol of individual tissue specific cells will be capable of transmitting recognition events, that is, the binding to biorecognition elements of target analytes of clinical relevance to an external data capture system. Nanosensors will enable compartmental analyses of metabolite levels and metabolic activity. Nanosensor prototypes have been expressed in Yeast and in mammalian cell cultures for determination of carbohydrate homeostasis in living cells with subcellular resolution. Nanosensors can be selectively expressed under the control of tissue specific promoters. The clinical relevance arising from constant, real-time metabolic vigilance via sensor based ligand specific biorecognition elements is immense. Virus-based nanoparticles have been developed for tumor specific recognition, targeting, imaging and destruction.

Of particular note, DNA conjugate materials have been prepared which can recognize DNA fragments with one-base specificity for reliable genotyping of single nucleotide polymorphisms, while bacterial magnetic particles have been integrated into functional nanomaterials by assembling enzymes, antibodies and receptors onto nano-sized bacterial magnetic particles for use in applications such as determination of human insulin.

The emerging ability to control patterns of matter on the nanometer length scale can be expected to lead to entirely new spatial positioning schemes of biorecognition elements using a variety of new materials. Although current technologies such as microstructure fabrication, surface modification, integration of detection and optimization of chemistry can not effectively complete with current, well established detection instrumentation, the need for high throughput diagnostic/detection methods will continue. If pursued, array technology should open the door for commercializing sensor platforms utilizing a variety of biorecognition elements for general diagnostic/detection purposes.

from Chambers et al. in Curr. Issues Mol. Biol. (2008) 10: 1-12
abstract     full article pdf

Labels: biosensors, nanosensors, nanotechnology

A biosensor is a compact analytical device or unit incorporating a biological or biologically derived sensitive recognition element integrated or associated with a physio-chemical transducer. Since the first biosensor was developed many new biosensors have been studied and the range of applications extended.

Molecular recognition is central to biosensing. Initially, biosensor recognition elements were isolated from living systems. However, many biosensor recognition elements now available are not naturally occurring but have been synthesized in the laboratory. The sensing of targets, i.e. analytes of interest, is being influenced by the availability of new engineered binding proteins. Employing the techniques of modern biotechnology, it is now possible to construct DNA polynucleotides at will, thus opening new possibilities for the generation of biosensor recognition elements arising from paths not taken by nature.

In the future, the ability to "recognize" and "detect" electrically and magnetically will be radically transformed. The emergence of magnetoelectronics is a promising new platform technology for biorecognition element/sensor development.

from Chambers et al. in Curr. Issues Mol. Biol. (2008) 10: 1-12
abstract     full article pdf

Labels: biosensors, nanotechnology

Potassium chloride (KCl) is normally used in a PCR amplification at a final concentration of 50mM. To improve the PCR amplification of DNA fragments, especially fragments in the size range 100bp to 1000bp, a KCl concentration of between 70mM and 100mM is sometimes recommended. For the amplification of longer products a lower salt concentration appears to be better. But the PCR amplification of short products works better at higher salt concentrations. This is probably because an increase in salt concentration permits shorter DNA molecules to denature preferentially to longer DNA molecules. Shorter molecules are therefore amplified better at higher salt concentration. It should be remembered however that a salt concentration above 50mM can inhibit the Taq polymerase.

If you are finding unwanted, long, non-specific products an increase in KCl concentration may reduce the appearance of these products. Similarly, to get rid of short, non-specific products you can decrease the KCl concentration to about 35 or 40mM. In either case do not change the MgCl₂ concentration. To improve the yield of a product you can try adjusting the KCl concentration: increase it for a desired product less than 1000bp; lower it for a desired product greater than 1000bp.

Further reading:

1. PCR Books
2. Real-Time PCR: Current Technology and Applications
3. Real-Time PCR in Microbiology: From Diagnosis to Characterization
4. PCR Troubleshooting: The Essential Guide

Labels: PCR, PCR troubleshooting, real-time PCR

Magnesium is a required cofactor for thermostable DNA polymerases. Mg²⁺ in the PCR mixture stabilizes dsDNA and raises the Tm. Mg²⁺ concentration therefore is an important for controlling the specificity of the reaction. A low Mg²⁺ concentration requires more stringent base pairing in the annealing step. Too few Mg²⁺ ions result in a low yield of PCR product; too many Mg²⁺ ions increase the yield of non-specific products and promote misincorporation.

Insufficient Mg²⁺ concentration in a PCR mixture can causes failure of the reaction. Excess magnesium (or the presence of manganese) will cause the fidelity of DNA polymerases to be reduced and may cause the generation of unwanted products. On a gel this can appear as a ladder or smear. The MgCl₂ concentration should normally be between 1mM and 4mM. Since dNTPs sequester Mg²⁺ ions, a major change in the dNTP concentration in a rection would require a change in the concentration of MgCl₂. Similarly, changing the KCl-based buffer concentration or any other component of the PCR mix may require adjustment of the Mg²⁺ concentration in the reaction mixture.

Further reading:

1. PCR Books
2. Real-Time PCR: Current Technology and Applications
3. Real-Time PCR in Microbiology: From Diagnosis to Characterization
4. PCR Troubleshooting: The Essential Guide

Labels: PCR, PCR troubleshooting, real-time PCR

In a PCR experiment approximately 1 unit of the Taq enzyme should be used for a 25μl reaction. Suboptimal concentration of the Taq enzyme can cause incomplete primer elongation or premature termination of the PCR product synthesis during the elongation step of a PCR cycle.

Too much Taq will result in an excessive background of unwanted DNA fragments (a smear on a gel) while a huge excess may cause the reaction to fail with no product being detected. A Taq concentration of 1 unit per 25μl reaction ensures a cleaner product and lower background.

Further reading:

1. PCR Books
2. Real-Time PCR: Current Technology and Applications
3. Real-Time PCR in Microbiology: From Diagnosis to Characterization
4. PCR Troubleshooting: The Essential Guide

Labels: PCR, PCR troubleshooting, real-time PCR, Taq

Writing in the journal Expert Review of Vaccines, David B. Weiner, Chair, Gene Therapy and Vaccine Program, CAMB University of Pennsylvania, and Abhishek Satishchandran review a book on Plasmids published by Caister Academic Press:

"Dr. George Lipps of the University of Bayreuth in Germany has assembled a clear and concise text which will be considered an important reference to plasmid researchers at the graduate level and beyond. The book has been fluently divided into 4 major topics of discussion, each further subdivided into increasing complex subcategories. The theme of cloning and expression vectors is maintained through the first three chapters. Appropriately, the basics plasmid biology are graciously provided in a series of lists and tables, easy enough for the most novice of graduate students ... This volume represents an important reference to bridge knowledge gaps and provide useful descriptions rooted in the fundamentals of plasmid biology. Dr. Lipps has done an excellent job in creating a useful, informative, and focused volume that should grace the shelf of many a molecular biologist well into the future."

Further reading: Plasmids: Current Research and Future Trends

Labels: plasmid, plasmids