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:

1. 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.
2. 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:

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

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

Labels: Drosophila, regulation, RNA, RNAi, siRNA

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

Labels: PCR, PCR troubleshooting, real-time PCR

The DNA in a PCR reaction comprises two types:

1. the target sequence to be amplified
2. 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

Labels: PCR, PCR troubleshooting, real-time PCR

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

Labels: epigenetics, regulation, RNA, RNAi, siRNA, yeast

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)

Labels: Drosophila, epigenetics, polycomb, regulation, RNA, RNAi

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:

1. RNA and the Regulation of Gene Expression
2. Epigenetics

Labels: epigenetics, expression, regulation, RNA

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

Labels: DNA, NASBA, real-time PCR, RNA, RT-PCR

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

Labels: biosensor, genomics, hammerhead ribozyme, regulation, ribozyme, ribozymes, RNA, therapeutics

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

Labels: bacteria, pathogen, PCR, plant, real-time PCR

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

Labels: DNA, DNA cleavage, PCR, restriction enzymes

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

Labels: expression, plasmid, plasmids, vector