Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays

A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.     

High throughput parallel synthesis of oligonucleotides with 1536 channel synthesizer

A 1536 channel oligonucleotide synthesizer, the MultiSyn, was developed with the capability to simultaneously synthesize 1536 oligonucleotides of 20mer length in 10 h. The instrument was designed to synthesize different sequences of various lengths in micro-wells and has synthesized oligonucleotides as long as 119 nt with reasonably good yields using CPG beads of 1000 Å pore size. The instrument consists of four 384 channel synthesis modules. Phosphoramidite chemistry was employed and step yields as high as 99.3% were achieved. The enhancement of oligonucleotide synthesis throughput is accomplished by increasing the spatial density of reaction wells. We have identified several parameters that are critical in achieving a good synthesis yield and negligible failure rate in small reaction wells. The coefficient of variation (CV) of product yields in 1536 reaction wells was 20%. The quality of the product was examined by capillary electrophoresis and mass spectrometry. The instrument has robustly synthesized oligonucleotides of various lengths for use as primers and probes for PCR amplifications, oligonucleotide microarrays and genotyping applications. This high throughput oligonucleotide synthesizer is a useful instrument for genomic applications, which require tens of thousands of probes or primers in a short time.     

Evidence for an interaction effect during <Emphasis Type="Italic">in vitro</Emphasis> rooting of oil palm (<Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq.) somatic embryo-derived plantlets

In vitro rooting of oil palm shoots derived from somatic embryos was achieved through a single-phase protocol in which three shoots are cultured in the same culture tube on an α-naphtaleacetic acid-enriched culture medium. Rooting performance was dependent on both the genetic origin and initial size of the shoot explants. All shoots from a given tube showed a tendency to give roots of the same type, independent of the original size of the explant. Whatever the clonal line, longer-size shoots (L-type: >9 cm) showed higher rooting rates than medium-size (M-type: 7–9 cm) and short-size ones (S-type: 5–7 cm). When groups of three shoots from the same clonal line were rooted together in the same culture tube, the combination of plant size within the group impacted overall quality of rooting. Within triplets of shoots containing more than one short individual, the probability of obtaining adequate rooting was low. Similarly, when more than one long shoot was included in the triplet rooting, quality was also poor. By avoiding such combinations, the rate of well-rooted plantlets increased by 25%, with a maximum of 66% when triplets of S/M/L combination were used. Smaller shoots, which usually showed poor rooting performance, were therefore found to benefit from the presence of their neighbors. This interaction between the sizes of individuals in a given tube was found to be associated with a within-tube correlation effect, a phenomenon previously described as “event coupling,” which was estimated using a distorted binomial-type distribution of probabilities. The resulting calculation of a coupling factor (average r = 0.60) explains the behavior of shoots within the same culture tube and their average rooting performance. Modeling of the interactions that occurred during in vitro rooting is described here and is recommended for improvement of this critical step in micropropagation.     

Carcinome urothélial de l’urètre: une nouvelle observation et revue de la littérature

Urothelial carcinoma of the male urethra is a rare tumour. We report a new case of urothelial carcinoma of the urethra in a 64-year old man who was examined due to a dysuria. Retrograde and voiding urethrocystography carried out had shown images of bulbopenile urethral stricture treated by urethroplasty. Histological examination of the urethrectomy part revealed a urothelial carcinoma of the urethra. The patient refused any additional treatment and survived 14 months after diagnosis.     

A modeling approach of the in vitro conversion of oil palm (Elaeis guineensis) somatic embryos

A mathematical model for the growth and conversion of somatic embryos was developed with the aim of monitoring the large scale production of oil palm microplants. The predicted biomass of somatic embryos obtained and subcultured (B _n ), together with the number of harvested shoots (Sh _n ) – two key parameters for production forecasts – have been modeled for seven different shoot harvesting procedures. For the four different clonal lines studied, observed differences between experimental B _n values at the end of each culture cycle and their theoretical counterpart generated by mathematical models were found to range between −30% to +14% at the end of the first 6-weeks culture cycle, then from −50% to +70% after the 6th subculturing operation (36 weeks). Concerning the predicted number of shoots harvested after conversion of somatic embryos (Sh _n ), average variations between experimental and theoretical values ranged between −45% and +41%. Predicted values for biomass (B _n ) between two culture cycles were found to vary slightly (+6% to +10%) indicating that the production of embryo biomass, as predicted by the model, was rather stable, for a given clonal line, from one 6-week cycle to another. The established model could thus be regarded as valid and the variations observed for B _n and Sh _n were found to be acceptable when compared to the those described by other models. Taken as a whole, predicted values for the two studied production parameters were in agreement with the corresponding experimental data (correlation=0.98).     

Construction of a linkage map of the Rennell Island Tall coconut type (Cocos nucifera L.) and QTL analysis for yield characters.

AFLP and SSR DNA markers were used to construct a linkage map in the coconut (Cocos nucifera L.; 2n = 32) type Rennell Island Tall (RIT). A total of 227 markers were arranged into 16 linkage groups. The total genome length corresponded to 1971 cM for the RIT map, with 5-23 markers per linkage group. QTL analysis for yield characters in two consecutive sampling periods identified nine loci. Three and two QTLs were detected for number of bunches and one and three QTLs for number of nuts. The correlation of trait values between characters and evaluation periods is partially reflected in identical QTLs. The QTLs represent characters that are important in coconut breeding. The cosegregation of markers with these QTLs provides an opportunity for marker-assisted selection in coconut breeding programmes.     

Profiling Expression Patterns and Isolating Differentially Expressed Genes by cDNA Microarray System with Colorimetry Detection

A high-density cDNA microarray with colorimetry detection system to simultaneously monitor the expression of many genes on nylon membrane is described and characterized. To quantify the expression of genes and to isolate differentially expressed genes, the southern hybridization process on filter membranes was employed. The levels of gene expression were represented by color intensities generated by colorimetric reactions in place of hazardous radioisotopes or costly laser-induced fluorescence detection. The gene expression patterns on nylon membranes were digitized by devices such as an economical flatbed scanner or a digital camera. The quantitative information of gene expression was retrieved by image analysis software. Quantitative comparison of the northern dot-blotting method with the microarray system is described. Applications employing single-color detection as well as dual-color detection to isolate differentially expressed genes among thousands of genes are demonstrated.     

Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l^-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l^-1), BA (0.1 mg l^-1) and GA₃ (0.1 mg l^-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l^-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine - GA₃ gibberellic acid - MCPA methyl- chlorophenoxyacetic acid - NAA naphthalen-acetic acid - PCPA P-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5 T 2,4,5-trichlorophenoxyacetic acid