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

Biotechnology Books

noreply@blogger.com (Blog owner) — Wed, 17 Dec 2008 14:34:00 +0000

Recommended books on biotechnology:

Lab-on-a-Chip Technology: Fabrication and Microfluidics
Publisher: Caister Academic Press
Editor: Keith E. Herold and Avraham Rasooly
Publication date: August 2009
ISBN: 978-1-904455-46-2
"An essential book for biologists and clinicians using LOC technology"

Lab-on-a-Chip Technology: Biomolecular Separation and Analysis
Publisher: Caister Academic Press
Editor: Keith E. Herold and Avraham Rasooly
Publication date: August 2009
ISBN: 978-1-904455-47-9
"A skillful selection of topics of exceptional importance to current science"

Real-Time PCR: Current Technology and Applications
Publisher: Caister Academic Press
Editor: Julie Logan, Kirstin Edwards and Nick Saunders
Publication date: January 2009
ISBN: 978-1-904455-39-4
"a comprehensive guide to the most up-to-date technologies and applications"

Bacterial Polysaccharides: Current Innovations and Future Trends
Publisher: Caister Academic Press
Editor: Matthias Ullrich
Publication date: June 2009
ISBN: 978-1-904455-45-5
"the most important innovations in polysaccharide research and biotechnological applications"

Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis
Publisher: Caister Academic Press
Editor: Karl Wooldridge
Publication date: April 2009
ISBN: 978-1-904455-42-4
"the role of secreted proteins in pathogenesis, drug design and vaccine development"

Microbial Production of Biopolymers and Polymer Precursors: Applications and Perspectives
Publisher: Caister Academic Press
Editor: Bernd H. A. Rehm
Publication date: January 2009
ISBN: 978-1-904455-36-3
"the applications and potential applications of biopolymers in biotechnology"

Plasmids: Current Research and Future Trends
Publisher: Caister Academic Press
Edited by: Georg Lipps
Publication date: July 2008
ISBN: 978-1-904455-35-6
"... a clear and concise text which will be considered an important reference to plasmid researchers at the graduate level and beyond ..." from Expert Review of Vaccines (2009) January Issue

RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity
Publisher: Caister Academic Press
Edited by: Kevin V. Morris
Publication date: March 2008
ISBN: 978-1-904455-25-7
"This book is a well-selected compilation of 14 mostly review-style articles, written by experts in the field ... a well-written, successful endeavour" from ChemBioChem (2008) 9: 2005-2007

Epigenetics
Publisher: Caister Academic Press
Editor: Jorg Tost
Publication date: March 2008
ISBN: 978-1-904455-23-3
"... a significant collection of articles, relating to various aspects of epigenetics" from Microbiology Today (2008)

Other books of interest:

Epigenetics and RNA

noreply@blogger.com (Blog owner) — Sat, 13 Dec 2008 17:23:00 +0000

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Exactly how these epigenetic modifications are directed to the particular gene and the local chromatin has remained enigmatic. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications.

In human cells RNA can specifically direct epigenetic modifications to targeted loci (the promoter regions) and modulate silencing. This regulatory effect is through RNA-associated silencing, can be transcriptional in nature, and is operable through an RNA interference based mechanism (RNAi) that is specifically mediated by the antisense strand of small-interfering RNAs (siRNAs). These recent observations represent a paradigm shift in which a hidden layer of complexity is involved in gene regualtion and is operative via the action RNA essentially epigenetically regulating DNA.

from Kevin V. Morris (2008). RNA Mediated Transcriptional Gene Silencing. In: Morris, K.V. (Ed.) RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press, Norfolk, UK.

Further reading:

Molecular Biology Books for 2009

noreply@blogger.com (Blog owner) — Wed, 10 Dec 2008 16:50:00 +0000

Lab-on-a-Chip Technology: Biomolecular Separation and Analysis
Publisher: Caister Academic Press
Editor: Keith E. Herold and Avraham Rasooly
Publication date: August 2009
ISBN: 978-1-904455-47-9
A skillful selection of topics of exceptional importance to current science ensures that this book will be of major value to a wide range of molecular biologists, clinical scientists, microbiologists, biochemists and anyone interested in LOC technology or developing applications for LOC devices.
further information

Lab-on-a-Chip Technology: Fabrication and Microfluidics
Publisher: Caister Academic Press
Editor: Keith E. Herold and Avraham Rasooly
Publication date: August 2009
ISBN: 978-1-904455-46-2
This comprehensive volume presents the current technologies in the field and includes theoretical and technical information to enable both the understanding of the technology and the reproduction of experiments. The book aims to help the reader to understand current LOC technologies, to perform similar experiments, to design new LOC systems and to develop new methodologies and applications.
further information

Real-Time PCR: Current Technology and Applications
Publisher: Caister Academic Press
Editor: Julie Logan, Kirstin Edwards and Nick Saunders
Publication date: January 2009
ISBN: 978-1-904455-39-4
This essential manual presents a comprehensive guide to the most up-to-date technologies and applications as well as providing an overview of the theory of this increasingly important technique.
further information

Microbial Production of Biopolymers and Polymer Precursors: Applications and Perspectives
Publisher: Caister Academic Press
Editor: Bernd H. A. Rehm
Publication date: January 2009
ISBN: 978-1-904455-36-3
Topics include the biochemistry and genetics of biosynthesis of xanthan, alginate, cellulose, cyanophycin, poly(gamma-glutamic acid), levan, hyaluronic acid, organic acids, oligosaccharides and polysaccharides, and polyhydroxyalkanoates. A recommended book for all biotechnology and microbiology laboratories.
further information

RNA-Mediated Recognition of Chromosomal DNA

noreply@blogger.com (Blog owner) — Mon, 08 Dec 2008 20:14:00 +0000

Designed molecules that recognize specific sequences within chromosomal DNA could provide useful probes for natural cellular processes, tools for laboratory experimentation, and lead compounds for therapeutic development. It was discovered that duplex DNA could be recognized by conjugates consisting of DNA oligonucleotides and cationic proteins or peptides. Similarly efficient recognition by neutral peptide nucleic acids (PNAs) was observed. It was found that duplex RNAs could also mediate efficient recognition of duplex DNA. RNAs can target transcription start sites and either inhibit or activate gene expression. This indicates that promoter-targeted RNAs can be powerful tools for regulating gene expression.

from Corey, DR (2008) RNA-Mediated Recognition of Chromosomal DNA. In: Morris , K.V. (Ed.) RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press, Norfolk, UK.

Wood-Whelan Fellowships

noreply@blogger.com (Blog owner) — Tue, 02 Dec 2008 13:54:00 +0000

The Wood-Whelan Fellowships were established about 1983 by funds provided by an appeal among the world's biochemists for the purpose of granting short-term fellowships to young biochemists to carry out research and training in a laboratory other than their own. This fellowship was named after Harland Wood, former General Secretary and President of IUB, and William Whelan, former General Secretary of IUB and former President of IUBMB. Additions to the funds were made by the American Society for Biochemistry and Molecular Biology.

The fellowships are awarded for travel lasting 1 - 4 months. A fellowship is intended to cover travel and incidental costs, as well as living expenses, to a maximum of $3,000. The costs do not cover research expenses at the laboratory to be visited. They are not awarded to attend courses, symposia, meetings or congresses. Applicants must be residents of countries that are members of IUBMB and should generally be graduate students or young researchers less than 35 years old.

Further information

Pyknon

noreply@blogger.com (Blog owner) — Fri, 28 Nov 2008 09:31:00 +0000

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.

MicroRNA

noreply@blogger.com (Blog owner) — Wed, 26 Nov 2008 12:07:00 +0000

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

Nanotechnology and Nanosensors

noreply@blogger.com (Blog owner) — Fri, 21 Nov 2008 12:12:00 +0000

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

Biosensors

noreply@blogger.com (Blog owner) — Wed, 19 Nov 2008 08:41:00 +0000

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

PCR Troubleshooting: KCl Concentration

noreply@blogger.com (Blog owner) — Tue, 18 Nov 2008 17:19:00 +0000

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:

PCR Troubleshooting: Mg Concentration

noreply@blogger.com (Blog owner) — Wed, 12 Nov 2008 11:05:00 +0000

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:

PCR Troubleshooting: Taq Concentration

noreply@blogger.com (Blog owner) — Sat, 08 Nov 2008 11:35:00 +0000

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:

Plasmid book review

noreply@blogger.com (Blog owner) — Tue, 04 Nov 2008 12:12:00 +0000

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

PCR Troubleshooting: Primer Concentration

noreply@blogger.com (Blog owner) — Sun, 26 Oct 2008 15:51:00 +0000

The recommended primer concentration for PCR is between 0.1μM and 1μM of each primer. The use of higher concentrations of primers can have the following effects:

If the primers are capable of forming dimers, raising their concentration only results in the creation of primer-dimers and does not improve the amplification of the desired PCR product. Primer-derived oligomers will possibly contaminate the reaction.
If the primers do not form primer-dimers, it is likely that raising the primer concentration will lead to non-specific primer binding and the creation of spurious, undesirable PCR products.

Raising the primer concentration does not therefore cause an increase in the effective concentration of the primers. Low primer concentration generally ensures cleaner product and lower background.

However, to amplify short PCR target sequences, careful calculation of the optimum primer concentration is required. For example, if the target fragment length is 100bp, a greater number of PCR product molecules is required to provide a specified amount of amplified DNA (in nanograms) than for a larger target fragment. In order to generate the required number of PCR product molecules, a greater number of primers may be needed. Therefore, concentration of primers higher than 1μM may be necessary, and desirable, for short target sequences.

Further reading:

Gene Regulation in Drosophila - short RNAs

noreply@blogger.com (Blog owner) — Wed, 22 Oct 2008 16:57:00 +0000

Short RNAs play multiple roles in affecting gene expression at many levels as illustrated by work in Drosophila. RNA interference uses double stranded RNA which is cleaved by Dicer to produce small interfering RNAs (siRNAs) as guides to cleave homologous mRNAs. This process occurs in the cytoplasm and is used in the endogenous process of viral resistance. In addition, many of the same gene products are also involved in transcriptional gene silencing processes. This was first documented for cosuppression of white-Alcohol dehydrogenase transgenes, which is associated with the Polycomb repressive complex of chromatin proteins. Genetic studies of RNA silencing genes also implicate a role in heterochromatin silencing. Some gene products involved in RNAi are also involved in the formation of repeat associated small RNAs (rasiRNAs), whose formation appears to be Dicer independent and critical for repressing transposon expression particularly in the germline. Roles for small RNAs are also implicated in chromatin insulator activity, the integrity of the nucleolus and long-range associations of homeotic genes.

from Kavi et al in RNA and the Regulation of Gene Expression

PCR Troubleshooting: Inadequate dNTPs

noreply@blogger.com (Blog owner) — Fri, 17 Oct 2008 15:36:00 +0000

An incorrect concentration of deoxynucleotidetriphosphates (dNTPs) can cause problems for the PCR procedure. The usual dNTP concentration is between 40μM and 200μM of EACH of the four dNTPs. Excessive dNTP concentrations can inhibit the PCR preventing the formation of product. However, concentrations up to 400 μM each dNTP have been reported to work adequately. Low primer, target, Taq, and dNTP concentrations are preferable as these generally ensure cleaner product and lower background. For longer PCR-fragments a higher deoxynucleotidetriphosphate concentration may be required. A large change in the dNTP concentration may require a corresponding change in the concentration of MgCl₂.

Suboptimal concentration of nucleotides can cause incomplete primer elongation or premature termination of DNA synthesis during the elongation step of the PCR cycle.

from PCR Troubleshooting: The Essential Guide

Further reading:
PCR Books
Real-Time PCR: Current Technology and Applications

PCR Troubleshooting: The Template DNA

noreply@blogger.com (Blog owner) — Wed, 15 Oct 2008 11:21:00 +0000

The DNA in a PCR reaction comprises two types:

the target sequence to be amplified
the non-target DNA (also called the "burden" DNA

The amount of total DNA in a PCR has a marked effect on the outcome of a PCR procedure. Using too much total DNA results in packed DNA in the confined space of the reaction vessel and can lead to false priming and even poor DNA synthesis due to the obstructed diffusion of large Taq polymerase molecules. However the ratio of target DNA to burden DNA is also important. The concentration of the target DNA should be balanced with the number of cycles in the reaction. Using an elevated concentration of the target combined with the normal, or higher than normal, number of cycles can cause the accelerated accumulation of nonspecific products. The accumulation of nonspecific products is often observed in a reamplification PCR, when the high initial concentration of the PCR fragment is accompanied by a high number of cycles. Reducing the number of cycles may help. However, low concentrations of primer, target, Taq, and nucleotides are recommended as these generally ensure cleaner product and lower background.

Problems also occur when the ratio of the target DNA to the burden DNA is very low, for example the amplification of a 500 bp fragment from the human genome (1 to 6 x 10⁶). A better ratio is between 1:1 and 1:1 x 10⁴. A ratio of 1:1 is achieved in a reamplification reaction and a ratio of about 10⁴ is achieved when amplifying from the Escherichia coli genome.

When the total amount of the DNA in a PCR reaction is extremely small, there is an increased likelihood of its loss owing to any conceivable cause (clotting, adsorption, chemical or enzymatic degradation). Furthermore, a small amount of target DNA leads to an increased risk from contaminating DNA from impurities on anything that can come into contact with the DNA solution. In this respect, both the DNA diluent, the dust floating in the air, exhalations and even particles of skin or hair from your body should not be disregarded, as these can carry both the DNA and the DNA-degrading substances. Nucleases are probably as the major cause of DNA degradation in a PCR procedure. They are abundant on the surface of the human skin and can be present everywhere else too. Mild autoclaving of the DNA diluent and everything that comes in regular, occasional, or accidental contact with buffers and solutions will destroy both the nucleases and comtaminating DNA. If you suspect problems of this nature, wear gloves, a surgeon's cap, and a face mask. Also, wash the working space with an oxidizing substance such as (6% H₂0₂).

from PCR Troubleshooting: The Essential Guide

Further reading:
PCR Books
Real-Time PCR: Current Technology and Applications

Heterochromatin and RNAi

noreply@blogger.com (Blog owner) — Tue, 14 Oct 2008 07:26:00 +0000

Heterochromatin is a prevalent chromatin state among eukaryotes that has critical functions in chromosome segregation, control of genomic stability and epigenetic regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, two RNAi complexes, the RNAi-induced transcriptional gene silencing (RITS) complex and the RNA-directed RNA polymerase complex (RDRC), are part of a RNAi machinery involved in the initiation, propagation and maintenance of heterochromatin assembly. These two complexes localize in a siRNA-dependent manner on chromosomes, at the site of heterochromatin assembly. RNA polymerase II (RNApII) has a central role in RNAi-dependent heterochromatin assembly. RNApII synthesizes a nascent transcript that serves as an RNA platform to recruit RITS, RDRC and possibly other complexes required for heterochromatin assembly. Both RNAi and an exosome-dependent RNA degradation process contribute to heterochromatic gene silencing. Recently reported findings challenge the widely accepted view that heterochromatic gene silencing is caused strictly by chromatin compaction. As RNAi-dependent chromatin modifications have been observed throughout the eukaryotic kingdom the mechanisms described here may be utilized in a large range of eukaryotes.

from Vavasseur et al in RNA and the Regulation of Gene Expression

Further reading: Epigenetics

Polycomb Mediated Control of Gene Expression

noreply@blogger.com (Blog owner) — Fri, 10 Oct 2008 15:45:00 +0000

Regulation of gene expression is a complex, multi-layered process that is crucial to correctly drive and maintain cell identity during development and adult life. RNA interference (RNAi) and the Polycomb system are two well-conserved gene silencing pathways. RNAi participates in post transcriptional as well as transcriptional gene silencing of natural genes as well as transposons and viruses. Polycomb group (PcG) proteins are well-known for their role in silencing HOX genes through modulation of chromatin structure. Both mechanisms are involved in specific epigenetic processes like cosuppression in Drosophila melanogaster and the formation of C. elegans mes and SOP-2 complexes. There are molecular links between RNAi components and Polycomb-mediated silencing in human cells and in Drosophila. RNA polymerase II and Argonaute 1 interact to bring about chromatin modifications on endogenous Polycomb target gene promoters in human cells, while Drosophila RNAi components modulate the nuclear organization of PcG target DNA elements, thereby affecting the strength of PcG-mediated silencing. Several microRNAs and non coding RNAs have been found in human and fly HOX gene loci. They may regulate HOX gene expression both post-trascriptionally and co-transcriptionally.

from Portoso and Cavalli in RNA and the Regulation of Gene Expression (Chapter 3)

Epigenetic Regulation of Gene Expression

noreply@blogger.com (Blog owner) — Thu, 09 Oct 2008 07:58:00 +0000

Epigenetics is the study of meiotically and mitotically heritable changes in gene expression which are not coded for in the DNA. Three distinct mechanisms appear to be intricately related and implicated in initiating and/or sustaining epigenetic modifications; DNA methylation, RNA-associated silencing, and histone modifications. While chromatin remodeling and DNA methylation have been studied for several years now far less is know about how these epigenetic marks are directed to each particular gene. Recently, however the role of RNA in epigenetic gene regulation has begun to become apparent.

from Kevin V. Morris in "Chapter 2 Epigenetic Regulation of Gene Expression" from RNA and the Regulation of Gene Expression

Further reading:

RNA and the Regulation of Gene Expression

Epigenetics

NASBA nucleic acid amplification

noreply@blogger.com (Blog owner) — Mon, 06 Oct 2008 11:12:00 +0000

NASBA is an isothermal nucleic acid amplification method which is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. The initial NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been applied to a wide range of RNA and some DNA targets. Real-time NASBA has become a sensitive and specific method for detection, quantification and differentiation of RNA and DNA targets. Molecular beacons have been used in real-time NASBA in commercially-available kits in published assays. The increase in availability of fluorimeters suitable for real-time NASBA ensures that this methodology will become a realistic alternative to real-time reverse transcriptase PCR.

NASBA technology is an alternative method to standard proceduresfor the amplification and detection of a range of nucleic acid targets. The majority of applications have been developed for detection and analysis of RNA targets including viral genomes, viroids, ribosomal RNA (rRNA) and messenger RNA (mRNA). Advantages of NASBA over methods such as reverse transcriptase PCR include fast amplification kinetics and selective amplification of RNA in a background of DNA. The amplification is isothermal and thus there is no requirement for thermocycling during the procedure. Single-stranded RNA amplicons are produced by NASBA which can be used directly in subsequent rounds of amplification or probed for detection without the need for denaturation or strand separation.

Real-time NASBA assays are rapid, specific and sensitive with RNA amplification and a target-specific fluorescent signal achieved simultaneously in one tube with measurements obtained through a fluorimeter. Qualitative, quantitative, monoplex and multiplex formats of real-time NASBA have now been described. The methodology seems to be a suitable alternative to other amplification procedures without the need for expensive thermocyclers. Since the original reports of real-time NASBA in 1998 the number of applications, available kits and expansion to include DNA targets is apparent and likely to continue over the next few years.

from Fox et al in "Chapter 12: Real-Time NASBA" from Real-Time PCR: Current Technology and Applications

Further reading: Real-Time PCR: Current Technology and Applications

The Hammerhead Ribozyme Revisited

noreply@blogger.com (Blog owner) — Sat, 04 Oct 2008 15:37:00 +0000

Hammerhead ribozymes are the smallest known naturally occurring ribozymes which are capable of catalyzing the endonucleolytic trans-esterification of RNA. A recent re-examination of the catalytic properties of naturally-derived hammerhead ribozymes has resulted in a better understanding of the catalytic efficiency of this enzyme in vitro and in vivo. The minimal trans-cleaving hammerhead ribozyme has been a ubiquitous tool in both genomics and therapeutics research over the last twenty years and these new insights into hammerhead ribozyme biochemistry may offer hope for the generation of improved trans-cleaving ribozymes which function effectively in vivo. Next-generation hammerhead ribozymes may play an important role as therapeutic agents, as enzymes which tailor defined RNA sequences, as biosensors, and for applications in functional genomics and gene discovery.

from Hean and Weinberg in Chapter 1 from RNA and the Regulation of Gene Expression

Further reading: RNA and the Regulation of Gene Expression

Molecular Tools for Pathogen Detection in Plants

noreply@blogger.com (Blog owner) — Fri, 03 Oct 2008 07:51:00 +0000

Plant pathogenic bacteria, phytoplasmas, viruses and viroids cause harmful, widespread and economically important diseases in a very broad range of plant species worldwide. Damage is often sufficient to cause significant yield losses in cultivated plants. The two main effects on agriculture are decreased production and, in a less direct way, the need for the implementation of expensive management and control procedures and strategies.

The lack of suitable chemical control methods means that prevention is necessary to avoid the dissemination of the pathogens. Prevention measures require pathogen detection methods of high sensitivity, specificity and reliability, because many phytopathogenic bacteria and viruses can remain latent, in low numbers, or in special physiological states in propagative plant material and in other reservoirs. Accurate detection of phytopathogenic organisms is crucial for virtually all aspects of plant pathology, from basic research on the biology of pathogens to the control of the diseases they cause.

Rapid and accurate methods for detection and diagnosis of plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools. Those based on PCR or Real-Time PCR are routinely used worldwide. However, nucleic acid extraction is still necessary in many cases and inhibition problems are decreasing the sensitivity of molecular detection. Integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques are advisable. Overall, molecular techniques based on different types of PCR amplification and especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture.

from Lopez et al in Curr. Issues Mol. Biol. (2009) 11: 13-46

Further reading:
Plant Pathogenic Bacteria: Genomics and Molecular Biology
Real-Time PCR: Current Technology and Applications
Real-Time PCR in Microbiology: From Diagnosis to Characterisation

Cleavage of Mispaired DNA by Restriction Enzymes

noreply@blogger.com (Blog owner) — Fri, 03 Oct 2008 07:24:00 +0000

The utility of restriction endonucleases as a tool in molecular biology is in large part due to the high degree of specificity with which they cleave well-characterized DNA recognition sequences. The specificity of restriction endonucleases is not absolute, yet many commonly used assays of biological phenomena and contemporary molecular biology techniques rely on the premise that restriction enzymes will cleave only perfect cognate recognition sites. In vitro, mispaired heteroduplex DNAs are commonly formed, especially following PCR amplification. A recent study into the Cleavage of Mispaired Heteroduplex DNA Substrates by Numerous Restriction Enzymes investigated a panel of restriction endonucleases to determine their ability to cleave mispaired heteroduplex DNA substrates. Two straightforward, non-radioactive assays were used to evaluate mispaired heteroduplex DNA cleavage: a PCR amplification method and an oligonucleotide-based assay. These assays demonstrated that most restriction endonucleases are capable of site-specific double-strand cleavage with heteroduplex mispaired DNA substrates, however, certain mispaired substrates do effectively abrogate cleavage to undetectable levels. These data are consistent with mispaired substrate cleavage previously reported for EcoRI and extend the curren knowledge of mispaired heteroduplex substrate cleavage to 13 additional enzymes.

from Langhans and Palladino in Current Issues in Molecular Biology

Further reading: Cleavage of Mispaired Heteroduplex DNA Substrates

Plasmid-based Expression Systems for Mammalian Cells

noreply@blogger.com (Blog owner) — Thu, 02 Oct 2008 07:29:00 +0000

Many attempts to construct different expression vector systems for mammalian cells have been made in recent years. These vector systems can be categorized in terms of vector administration, mechanisms of vector replication and mechanisms to achieve nuclear persistence of the vectors.

Episomal vectors, either based on viral plasmid replicons or on chromosomal elements, are invaluable tools for basic and applied science. The biochemistry and cell cycle dependent regulation of mammalian DNA replication has been extensively studied using SV40-based vectors and more recently, EBV-based vectors have been used to isolate putative mammalian origins of replication. The non-viral vectors whose functioning depends on the insertion of a chromosomal S/MAR sequence represent a minimal system to study the epigenetic regulation of mammalian DNA replication and the relevance of global functional nuclear architecture. The construction of mammalian artificial chromosomes has had a considerable impact on our understanding of the functional elements of the eukaryotic chromosome, telomeres, replication origins and centromeres. Similarly, the analysis of the biochemistry and regulation of transcription was greatly facilitated by the use of episomal vectors. These constructs will find even further applications for the analysis of basic biological phenomena, such as protein-protein interactions, signal transduction pathways, cell movements and many more.

Long term expression of transgenes in the absence of selection has been reported for EBV-based vectors, S/MAR-based vectors and mammalian artificial chromosomes, making these vectors attractive systems for the production of pharmaceutical relevant proteins in mammalian cells. Mammalian artificial chromosomes and S/MAR-based vectors have been successfully used for the genetic modification of mammalian organisms, such as mice and pigs, and have proven to be significantly more efficient than currently used integrating vectors.

Episomal vectors and especially vectors based on chromosomal elements may represent alternatives to the currently used viral vectors for genetic therapy. The major risks associated with virus-based vectors involve problems associated with insertional mutagenesis, imunogenicity and cytotoxicity. These problems were highlighted by the first therapy related fatality, in which death was caused by an inflammatory reaction that was attributed to the use of adenoviral vectors. Insertional mutagenesis events related to the use of integrating vectors have been reported for retroviral as well as AAV vectors. Since episomal vectors based exclusively on chromosomal elements do not need any virus-encoded protein for their function and furthermore do not integrate into the host cell genome, they should avoid the major safety risks that have been associated with virus-based vectors. Their use has been demonstrated in vitro and some have been propagated in animal systems. Their present main limitation is their low transfection efficiency compared to systems based on replication deficient viruses. However, recent success in the construction of non-viral episomal vectors and steady improvement of DNA transfection techniques makes it likely that these limitations can be overcome in the near future, so that optimized episomal vectors will be available for clinical trials.

fromBaiker et al in Chapter 3. Plasmids: Current Research and Future Trends

Further reading: Plasmids: Current Research and Future Trends