Difluoroacetic acid: an alternative acid in the detritylation reaction for the solid-phase synthesis of oligonucleotides.

Dichloroacetic acid or trichloroacetic acid are commonly used in the detritylation reaction of the automated solid-phase synthesis of oligonucleotides. Dichloroacetic acid, however, is often contaminated with trichloroacetaldehyde (chloral), leading to the formation of inseparable impurities in the final oligonucleotide product. In this work, three different sequences, namely T18, d(TAA)6, and an 18-mer mixed sequence, were used as models to compare the deprotection efficiency of three acids: trichloroacetic acid, dichloroacetic acid, and difluoroacetic acid. Comparable purities of full-length products were obtained for the synthesis of the three model sequences when dichloroacetic acid or difluoroacetic acid were used during the detritylation reaction, however, conditions need to be optimized for the synthesis of purine-rich sequences. Therefore, difluoroacetic acid is a potential alternative to dichloroacetic acid in the solid-phase synthesis of oligonucleotides to avoid the impurity formation due to presence of chloral.

"Difficult" deoxyribonucleotide sequences in the solid-phase synthesis by the phosphoramidite chemistry.

This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite chemistry-based solid phase synthesis. In total, 76 sequences with different dinucleotide and trinucleotide repeats were synthesized, and the fully-deprotected products were analyzed by denaturing anion exchange HPLC. Overall, sequences containing more 2'-deoxyadenosine residues were obtained in relatively lower yields, likely due to the relative ease of 2'-deoxyadenosine to undergo depurination during the detritylation reaction. Furthermore, dinucleotide steps, such as d(CG)/d(GC) and d(AG)/d(GA), likely contribute the overall lower yields of full-length products as well.

Conservation genetics of an endangered lady's slipper orchid: Cypripedium japonicum in China.

Knowledge about the population genetic variation of the endangered orchid, Cypripedium japonicum, is conducive to the development of conservation strategies. Here, we examined the levels and partitioning of inter-simple sequence repeat (ISSR) diversity (109 loci) in five populations of this orchid to gain insight into its genetic variation and population structure in Eastern and Central China. It harbored considerably lower levels of genetic diversity both at the population (percentage of polymorphic loci (PPL) = 11.19%, Nei's gene diversity (H) = 0.0416 and Shannon's information index (I) = 0.0613) and species level (PPL = 38.53%, H = 0.1273 and I = 0.1928) and a significantly higher degree of differentiation among populations (the proportion of the total variance among populations (Φpt) = 0.698) than those typical of ISSR-based studies in other orchid species. Furthermore, the Nei's genetic distances between populations were independent of the corresponding geographical distances. Two main clusters are shown in an arithmetic average (UPGMA) dendrogram, which is in agreement with the results of principal coordinate analysis (PCoA) analysis and the STRUCTURE program. In addition, individuals within a population were more similar to each other than to those in other populations. Based on the genetic data and our field survey, the development of conservation management for this threatened orchid should include habitat protection, artificial gene flow and ex situ measures.

Shoot organogenesis and plant regeneration from leaf explants of Lysionotus serratus D. Don.

The gesneriaceous perennial plant, Lysionotus serratus, has been used in traditional Chinese medicine. It also has a great development potential as an ornamental plant with its attractive foliage and beautiful flowers. An efficient propagation and regeneration system via direct shoot organogenesis from leaf explant was established in this study. High active cytokinin (6-benzyladenine (BA) or thidiazuron (TDZ)) was effective for direct organogenesis of initial induction. Murashige and Skoog (MS) growth media containing 0.5 mg L(-1) BA alone or with combination of 0.1 mg L(-1) α-Naphthaleneacetic acid (NAA) were the most effective for shoot proliferation. High BA concentration (1.0 mg L(-1)) in the media caused high percentage of vitrified shoots though they introduced high shoot proliferation rate. Histological observation indicated that adventitious shoot regeneration on the medium containing 0.5 mg L(-1) BA alone occurred directly from leaf epidermal cells without callus formation. Regenerated shoots rooted well on medium containing half-strength MS medium with 0.5 mg L(-1) indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA), and the plantlets successfully acclimatized and grew vigorously in the greenhouse with a 94.2% and 92.1% survival rate.