PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate.

GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

Hybridisation of N4-methylcytosine-containing amplicons on DNA microarrays.

5'-Cy5-labelled PCR amplicons containing the analogue base, N(4)-methylcytosine, instead of cytosines were compared in microarray hybridisation experiments with the corresponding amplicons containing the canonical set of bases, with respect to the intensity of the fluorescence signal obtained, and cross hybridisation to non-corresponding probes. In general, higher hybridisation temperatures resulted in reduced signal intensities, particularly in the case of the N(4)-methylcytosine containing amplicons. At the lower hybridisation temperatures tested (40 °C, 30 °C), these modified amplicons gave about equal or stronger fluorescence signal than the corresponding regular amplicons. With the two GC-richest amplicons tested, in one instance the N(4)-methylated target gave a dramatically higher signal intensity than the unmodified amplicon, interpreted as reflecting the reduced formation of hairpin structures in the target sequence, due to the lower thermodynamic stability of the G:N(4)-methylC base pair, making the target more accessible, while in the other case no hybridisation was observed with either version of the amplicon, probably due to interference from a G-tetrad structure. Both for the regular and the N(4)-methylated amplicons, no significant cross hybridisation was seen in these experiments.

Chemically pretreating slaughterhouse solid waste to increase the efficiency of anaerobic digestion.

The combined effect of temperature and pretreatment of the substrate on the anaerobic treatment of the organic fraction of slaughterhouse solid waste was studied. The goal of the study was to evaluate the effect of pretreating the waste on the efficiency of anaerobic digestion. The effect was analyzed at two temperature ranges (the psychrophilic and the mesophilic ranges), in order to evaluate the effect of temperature on the performance of the anaerobic digestion process for this residue. The experiments were performed in 6 L batch reactors for 30 days. Two temperature ranges were studied: the psychrophilic range (at room temperature, 18°C average) and the mesophilic range (at 37°C). The waste was pretreated with NaOH before the anaerobic treatment. The result of pretreating with NaOH was a 194% increase in the soluble chemical oxygen demand (COD) with a dose of 0.6 g NaOH per g of volatile suspended solids (VSS). In addition, the soluble chemical oxygen demand/total chemical oxygen demand ratio (sCOD/tCOD) increased from 0.31 to 0.7. For the anaerobic treatment, better results were observed in the mesophilic range, achieving 70.7%, 47% and 47.2% removal efficiencies for tCOD, total solids (TS), and volatile solids (VS), respectively.

Capacity of N4-methyl-2'-deoxycytidine 5'-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases.

The dCTP analog N4-methyl-2'-deoxycytidine 5'-triphosphate (N4medCTP) was evaluated for its performance in the polymerase chain reaction (PCR). Using the HotStart Taq DNA polymerase with a standard thermal protocol, test segments 85 or 200 bp long were amplified equally well using dCTP or N4medCTP:dCTP mixtures ranging in molar ratio from 3:1 to 10:1, while complete replacement of dCTP by N4medCTP gave clearly lower amplicon yields and higher Cq values. Comparable yields with N4medCTP or dCTP were achieved only by using a slowdown protocol. Post-PCR melting analyses showed decreasing Tm values for amplicons obtained with increasing N4medCTP:dCTP input ratios; for the 200-bp amplicon, complete replacement of dCTP by N4medCTP in the reaction reduced the Tm by 11 °C; for the 85-bp amplicon the Tm reduction was 7 °C. In experiments aiming at the 200-bp amplicon, Pfu exo(-) DNA polymerase did not sustain PCR when dCTP was fully replaced by N4medCTP, even with the slowdown protocol, except at elevated N4medCTP concentrations, and, compared to PCR conducted exclusively with dCTP, the use of N4medCTP:dCTP mixtures gave reduced yields and distinctly higher Cq values, regardless of the thermal program employed. PCR experiments with 9°N DNA polymerase using N4medCTP in the conventional thermal protocol failed to produce the 200-bp amplicon.