Synthesis of polyanionic C5-modified 2'-deoxyuridine and 2'-deoxycytidine-5'-triphosphates and their properties as substrates for DNA polymerases

Modified 2'-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor

Phosphate fertilization and phosphorus forms in an Oxisol under no-till

Under no-till phosphorus (P) accumulates in a few centimeters of the topsoil layer. Plant residues left on the soil surface release P and organic acids, which may improve P availability and fertilizer efficiency, including both soluble (such as triple super phosphate) and less soluble sources (such as reactive natural phosphates). In this study, soybean response to P fertilizer and P forms in the top 40 cm of an Oxisol were evaluated after surface application of different phosphates in a 5-year-old no-till system. Treatments consisted of 0 or 80 kg ha-1 of total P2O5 applied on the soil surface, both as natural reactive phosphate (NRP) or triple super phosphate (TSP). In addition, 80 kg ha-1 of P2O5 were applied to subplots, in furrows below and beside the soybean (Glycine max L.) seeds, in different combinations of NRP and TSP. Soil samples were taken before and after the soybean growth, down to 0.40 m and soil phosphorus was chemically fractionated. The responses to NRP were similar to TSP, with an increase in P reserves at greater depths, even in non-available forms, such as P-occluded. After the soybean harvest, P-occluded levels were lower at the surface layer, but an increase was observed in the soluble, organic and total P down to 40 cm. An improved P distribution in soil depth, especially regarding the soluble and organic forms, resulted in higher soybean yields, even when the phosphates were applied to the soil surface.Em semeadura direta o fósforo (P) acumula-se na camada mais superficial do solo, mas os resíduos deixados na superfície liberam P e ácidos orgânicos, que podem melhorar a disponibilidade e a eficiência de fertilizantes como o superfosfato triplo e fosfatos naturais reativos. Neste estudo, a resposta da soja à adubação com P e as formas de P até 40 cm de profundidade do solo foram avaliadas após a aplicação de fosfatos em um sistema conduzido em semeadura direta há cinco anos. Os tratamentos consistiram de 0 ou 80 kg ha-1 P2O5 total, aplicados na superfície do solo como fosfato natural reativo (FNR) ou superfosfato triplo (SFT). Nas subparcelas foram aplicados, no sulco de semeadura, 80 kg ha-1 de P2O5, em diferentes combinações de FNR e SFT. Amostras de solo foram coletadas até 0.4 m, antes e depois do cultivo da soja (Glycine max L.), para fracionamento do P. As respostas ao FNR foram semelhantes às do SFT, com aumento das reservas de P em profundidade, mesmo em formas não-disponíveis como P-ocluso. Após a colheita da soja, os teores de P-ocluso diminuíram na camada mais superficial, mas foi observado um aumento nas formas solúvel, orgânica e P - total em toda a espessura de solo estudada. A melhor distribuição do P no solo, principalmente em formas solúvel e orgânica, resultou em maior produtividade da soja, mesmo quando o fertilizante foi aplicado na superfície do solo