A new approach to enhance PCR specificity

A new approach to enhanced specificity and product yield of polymerase chain reaction is proposed. It is based on control of DNA polymerase activity during PCR by changing the magnesium ion concentration, which depends on the temperature of the reaction mixture. A slightly soluble magnesium salt, magnesium oxalate, whose solubility depends on temperature, was used as a source of magnesium ions. During PCR, magnesium oxalate was maintained at saturating concentration by the presence of an insoluble excess of this salt, and the concentration of magnesium ions depended on the salt solubility: binding of magnesium ions at lower temperatures and their release at higher temperatures was shown to affect the DNA polymerase activity and to favor the specific PCR amplification of the target DNA fragment.     

Genetic and physical mapping of new EST-derived SSRs on the A and B genome chromosomes of wheat

The availability of genetic maps and phenotypic data of segregating populations allows to localize and map agronomically important genes, and to identify closely associated molecular markers to be used in marker-assisted selection and positional cloning. The objective of the present work was to develop a durum wheat intervarietal genetic and physical map based on genomic microsatellite or genomic simple sequence repeats (gSSR) markers and expressed sequence tag (EST)-derived microsatellite (EST-SSR) markers. A set of 122 new EST-SSR loci amplified by 100 primer pairs was genetically mapped on the wheat A and B genome chromosomes. The whole map also comprises 149 gSSR markers amplified by 120 primer pairs used as anchor chromosome loci, two morphological markers (Black colour, Bla1, and spike glaucousness, Ws) and two seed storage protein loci (Gli-A2 and Gli-B2). The majority of SSR markers tested (182) was chromosome-specific. Out of 275 loci 241 loci assembled in 25 linkage groups assigned to the chromosomes of the A and B genome and 34 remained unlinked. A higher percentage of markers (54.4%), localized on the B genome chromosomes, in comparison to 45.6% distributed on the A genome. The whole map covered 1,605 cM. The B genome accounted for 852.2 cM of genetic distance; the A genome basic map spanned 753.1 cM with a minimum length of 46.6 cM for chromosome 5A and a maximum of 156.2 cM for chromosome 3A and an average value of 114.5 cM. The primer sets that amplified two or more loci mapped to homoeologous as well as to non-homoeologous sites. Out of 241 genetically mapped loci 213 (88.4%) were physically mapped by using the nulli-tetrasomic, ditelosomic and a stock of 58 deletion lines dividing the A and B genome chromosomes in 94 bins. No discrepancies concerning marker order were observed but the cytogenetic maps revealed in some cases small genetic distance covered large physical regions. Putative function for mapped SSRs were assigned by searching against GenBank nonredundant database using TBLASTX algorithms.     

A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

Background  

Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (Ct) method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the Ct method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample.     

High transferability of bread wheat EST-derived SSRs to other cereals

The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. In this study, 300 primer pairs were designed from 265 wheat ESTs that contain microsatellites in order to develop new markers for wheat. Their level of transferability in eight related species [Triticum durum, T. monococcum, Aegilops speltoides, Ae. tauschii, rye (Secale cereale), barley (Hordeum vulgare), Agropyron elongatum and rice (Oryza sativa)] was assessed. In total, 240 primer pairs (80%) gave an amplification product on wheat, and 177 were assigned to wheat chromosomes using aneuploid lines. Transferability to closely related Triticeae species ranged from 76.7% for Ae. tauschii to 90.4% for T. durum and was lower for more distant relatives such as barley (50.4%) or rice (28.3%). No clear putative function could be assigned to the genes from which the simple sequence repeats (SSRs) were developed, even though most of them were located inside ORFs. blast analysis of the EST sequences against the 12 rice pseudo-molecules showed that the EST-SSRs are mainly located in the telomeric regions and that the wheat ESTs have the highest similarity to genes on rice chromosomes 2, 3 and 5. Interestingly, most of the SSRs giving an amplification product on barley or rice had a repeated motif similar to the one found in wheat, suggesting a common ancestral origin. Our results indicate that wheat EST-SSRs show a high level of transferability across distantly related species, thereby providing additional markers for comparative mapping and for following gene introgressions from wild species and carrying out evolutionary studies.     

Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.     

Construction of a watermelon BAC library and identification of SSRs anchored to melon or Arabidopsis genomes

A bacterial artificial chromosome (BAC) library was constructed for watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus) with an average insert-size of 106 kb, providing 21 haploid genome equivalents. The library was used to identify BAC clones that are anchored to probes evenly distributed on the genomes of melon or Arabidopsis. Twenty eight probes (representing 66% of the tested probes) from melon and 30 probes (65%) from Arabidopsis identified positive BAC clones. Two methods were implemented to identify SSRs from the positively hybridizing BAC clones. First, analysis of BAC end sequences revealed 37 SSRs. For the second method, pooled DNA of BACs identified by the melon probes was used to develop a shotgun library. The library was then screened with synthetic SSR oligonucleotides by hybridization. Sequence analysis of positively hybridizing shotgun clones revealed 142 different SSRs. Thirty eight SSRs were characterized using three watermelon cultivars, five plant introduction (PI) accessions of C. lanatus var lanatus and four PIs of C. lanatus var citroides. Of these, 36 (95%) were found to be polymorphic with up to six alleles per marker. Polymorphism information content values for polymorphic markers varied between 0.22 and 0.79 with an average of 0.53. The methods described herein will be valuable for the construction of a watermelon linkage map with SSRs evenly distributed on its genome that is anchored to the genomes of melon and Arabidopsis.     

A new approach to primer design for the control of PCR bias in methylation studies

Background

Nerve growth factor (NGF) helps in the healing and survival of ganglion cells, photoreceptors, and optic nerve after injury and has been implicated to have a role in pathophysiology of glaucoma. So far, in animal studies, injury to iris in vitro has revealed an increase in NGF levels in aqueous. There is a great interest in investigating the levels of NGF in human aqueous in glaucomatous eyes, as suggested by animal studies, to gain a better understanding of the pathophysiology of glaucoma.

Findings

In this study, we examined the presence of NGF levels in aqueous humor collected from human eyes and the limitations in determining the NGF levels in human samples. NGF was assessed by ELISA immunoassay in undiluted aqueous samples collected from 32 consecutive patients undergoing surgery for cataract (control) or primary open angle glaucoma (POAG). Recombinant NGF was used as positive control. NGF levels were below undetectable levels in aqueous humor from eyes with POAG and controls by immunoassay. Less than 10% of samples had detectable NGF levels and these were considered outliers.

Conclusion

Our result highlights the undetectable levels of NGF in human aqueous samples.     

A comparative genome approach to marker ordering

MOTIVATION: Genome maps are fundamental to the study of an organism and essential in the process of genome sequencing which in turn provides the ultimate map of the genome. The increased number of genomes being sequenced offers new opportunities for the mapping of closely related organisms. We propose here an algorithmic formalization of a genome comparison approach to marker ordering. RESULTS: In order to integrate a comparative mapping approach in the algorithmic process of map construction and selection, we propose to extend the usual statistical model describing the experimental data, here radiation hybrids (RH) data, in a statistical framework that models additionally the evolutionary relationships between a proposed map and a reference map: an existing map of the corresponding orthologous genes or markers in a closely related organism. This has concretely the effect of exploiting, in the process of map selection, the information of marker adjacencies in the related genome when the information provided by the experimental data is not conclusive for the purpose of ordering. In order to compute efficiently the map, we proceed to a reduction of the maximum likelihood estimation to the Traveling Salesman Problem. Experiments on simulated RH datasets as well as on a real RH dataset from the canine RH project show that maps produced using the likelihood defined by the new model are significantly better than maps built using the traditional RH model. AVAILABILITY: The comparative mapping approach is available in the last version of de Givry,S. et al. [(2004) Bioinformatics, 21, 1703-1704, www.inra.fr/mia/T/CarthaGene], a free (the LKH part is free for academic use only) mapping software in C++, including LKH (Helsgaun,K. (2000) Eur. J. Oper. Res., 126, 106-130, www.dat.ruc.dk/keld/research/LKH) for maximum likelihood computation.     

PCR amplification of microdissected wheat chromosome arms in a simple 'single tube' reaction

A novel method for the adaptor-mediated PCR amplification of microdissected chromosome arms is described. This simple and versatile protocol eliminates the need for enzymatic micromanipulation in nanoliter volumes and permits the efficient amplification of as little as two wheat chromosome arms in a 'single tube' reaction.     

A thermodynamic approach to PCR primer design

We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.     

A new approach to apple proliferation detection: a highly sensitive real-time PCR assay

The present paper describes a new approach for diagnosis of apple proliferation (AP) phytoplasma in plant material using a multiplex real-time PCR assay simultaneously amplifying a fragment of the pathogen 16S rRNA gene and the host, Malus domestica, chloroplast gene coding for tRNA leucine. For the first time, such an approach, with an internal analytical control, is described in a diagnostic procedure for plant pathogenic phytoplasmas enabling distinction between uninfected plant material and false-negative results caused by PCR inhibition. Pathogen detection is based on the highly conserved 16S rRNA gene to ensure amplification of different AP phytoplasma strains. The newly designed primer/probe set allows specific detection of all examined AP strains, without amplifying other fruit tree phytoplasmas or more distantly related phytoplasma strains. Apart from its specificity, real-time PCR with serial dilutions of initial template DNA ranging over almost five orders of magnitude (undiluted to 80,000-fold diluted) demonstrated linear amplification over the whole range, while conventional PCR showed a reliable detection only up to 500-fold or 10,000-fold dilutions, respectively. Compared to existing analytical diagnostic procedures for phytoplasmas, a rapid, highly specific and highly sensitive diagnostic method becomes now available.     

Uniform amplification of multiple DNAs by emulsion PCR

When several DNAs are amplified by PCR in one PCR tube, biased amplification is known to occur because amplification efficiency differs from one DNA to another. Therefore, we conducted PCR in the water in oil-emulsion (W/O emulsion) to examine whether the procedure allows the uniform amplification of several DNAs. In the amplification of a model library consisting of two clones, the emulsification of the PCR mixture successfully reduced the difference in its amplification efficiency to approximately one-seventh the value obtained without emulsification. Furthermore, we conducted repeated PCR to amplify a model library consisting of ten short hairpin RNA (shRNA) expression vectors as a model experiment for gene discovery using an shRNA expression library. Consequently, the emulsification of the PCR mixture successfully reduced PCR bias. Our results indicate that emulsion PCR is capable of uniformly amplifying libraries of shRNA, ribozyme, cDNA, and others, and is useful also for gene discovery using these libraries.     

A new approach to estimating the pool of stable organic matter in soil using data from long-term field experiments

It is a necessity to have a successful method to separate, quantify and define the active and passive soil organic matter pools for appropriate verification of models. In this study, the organic carbon content of long-term bare fallow soils was used as an indicator of the size of the stable soil organic matter pool. Although soil texture and soil structure are widely accepted as having an influence on the stable pool, most soil organic models neglect the relationship between soil structure and carbon stabilization. Therefore, the aim of this presentation is to estimate the size of the stable carbon pool and to relate it to soil texture and structure properties. It was calculated that over 50 yr, under bare fallow conditions, the relative decrease in the amount of carbon (C) for the most stable pools ranged between 2 and 12%. In comparison, for the less stabilized pools the relative decrease was calculated from 50 to 100%. This indicates that the organic carbon content of long-term bare fallow soils should be very similar to the size of the most stable C pool. We also observed that the amounts of carbon associated with primary particles <20 μm for numerous soils with contrasting carbon content, soil texture, and management practices showed a lower and an upper limit. Both these limits and the carbon content of long-term bare fallow soils (which were assumed to be similar to the size of the stable pool) were related to the content of primary particles <20 μm in the soil. To calculate these relationships, an equation was used including one term to describe the influence of soil texture and another to describe that of soil structure. The calculated regression for the bare fallow soils corresponded very well to the lower limit of carbon content associated with primary particles <20 μm. The upper limit was estimated only by increasing the regression parameter which is related to the amount of C per unit primary particles <20 μm. Considering the many published results of the influence of soil texture and structure on carbon stabilization processes in soil, the stable pool may be defined as the capacity of soils to sorb C. The upper limit of carbon content associated with primary particles <20 μm may be interpreted as the capacity of soil to protect C. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date.     

A novel system for large-scale sequencing of cDNA by PCR amplification.

We have developed a method for constructing a library containing the 3' end fragment of cDNA for large-scale sequencing of cDNA clones. The average size of the insert was 270 bp. Cell lysates that carry plasmids having the cDNA insert were subjected to PCR amplification of the cDNA moiety and the products were subjected to sequencing analysis using an autosequencer. With this protocol, sample preparation became a non-limiting step, that allowed us to sequence as many samples as the autosequencer could handle.     

A compound decision approach to covariance matrix estimation

Covariance matrix estimation is a fundamental statistical task in many applications, but the sample covariance matrix is suboptimal when the sample size is comparable to or less than the number of features. Such high-dimensional settings are common in modern genomics, where covariance matrix estimation is frequently employed as a method for inferring gene networks. To achieve estimation accuracy in these settings, existing methods typically either assume that the population covariance matrix has some particular structure, for example, sparsity, or apply shrinkage to better estimate the population eigenvalues. In this paper, we study a new approach to estimating high-dimensional covariance matrices. We first frame covariance matrix estimation as a compound decision problem. This motivates defining a class of decision rules and using a nonparametric empirical Bayes g-modeling approach to estimate the optimal rule in the class. Simulation results and gene network inference in an RNA-seq experiment in mouse show that our approach is comparable to or can outperform a number of state-of-the-art proposals.     

Increasing the physical markers of wheat chromosomes using SSRs as FISH probes

In plants the marker sequences used to identify chromosomes are mainly repetitive DNA probes. Simple sequence repeats (SSRs) are major components of many plant genomes and could be good markers for chromosome identification. In a previous work, we reported the physical distribution of 4 oligonucleotides, (AG)12, (CAT)5, (AAC)5, and (AAG)5, on Triticum aestivum L. chromosomes. The distinctive distribution pattern found suggested that SSR in situ hybridization is useful as a diagnostic tool in wheat cytogenetics. To check whether that finding is generally applicable, we analyzed the chromosomal distribution of the rest of the 14 possible classes of di- and tri-nucleotide repeats by FISH. A detailed knowledge of the sequence content of hexaploid wheat chromatin was acquired based on the hybridization signals, which also provide a rich set of chromosome markers for chromosome identification. Except for (AT)10 and (GC)10, for which the chromosomal distribution could not be accurately determined, and (AC)8 and (GCC)5, which were found dispersed throughout the chromosomes, the remaining repeats were observed as clusters on specific chromosome sites. (AGG)5, (CAC)5, (ACG)5, (AAT)5, and (CAG)5 exhibited a preferential distribution in the pericentromeric regions of the B genome chromosomes. The richest patterns of intercalary signals on several A and B genome chromosomes were produced by (ACT)5. A karyotype based on the SSR probes providing the best FISH patterns was constructed for T. aestivum 'Chinese Spring'.