Mapping and cloning of FAD2 gene to develop allele-specific PCR for oleic acid in spring turnip rape (Brassica rapa ssp. oleifera)

The previously identified QTL for oleic acid content observed in an F2 population from the Brassica rapa ssp. oleifera cross Jo4002 × Jo4072 (a high-oleic-acid individual) was mapped more precisely by adding markers to the linkage group which harbours the locus. In addition, the fad2 gene, which is known to encode the 18:1 desaturase in Arabidopsis, was mapped in Brassica, too. The results are consistent with the QTL corresponding to the Arabidopsis fad2 gene. Comparison of the wild-type and high-oleic-acid allele of the locus revealed only one difference in their nucleic acid sequences leading to an amino acid change. This substitution of leucine by proline most likely affects the fold of the protein and thereby activity of the enzyme. Using this base difference, an allele-specific PCR was designed. The allele-specific markers will be very effective in selection for plants with high-oleic-acid content derived from Jo4072 because they are located exactly at the locus and can differentiate between homo- and heterozygotes.     

The use of whole genome amplification in the study of human disease

The availability of large amounts of genomic DNA is of critical importance for many of the molecular biology assays used in the analysis of human disease. However, since the amount of patient tissue available is often limited and as particular foci of interest may consist of only a few hundred cells, the yield of DNA is often insufficient for extensive analysis. To address this problem, several whole genome amplification (WGA) methodologies have been developed. Initial WGA approaches were based on the polymerase chain reaction (PCR). However, recent reports have described the use of non-PCR-based linear amplification protocols for WGA. Using these methods, it is possible to generate microgram quantities of DNA starting with as little as 1mg of genomic DNA. This review will provide an overview of WGA approaches and summarize some of the uses for amplified DNA in various high-throughput genetic applications.     

Quantification of<Emphasis Type="Italic">Bacillus</Emphasis> species in a wastewater treatment system by the molecular analyses

Bacillus species were observed and quantified by molecular approaches, using the 165 rDNA primers/probes, in a wastewater treatment plant designed for the purpose of stimulating the growth ofBacillus species. The plant has been operating as a test plant since 1997 in the city of Ina, Japan, with excellent treatment performance. Observations byin situ hybridization, usingBacillus-specific probes, indicated thatBacillus strains were inhabited in the plant and their numbers decreased during the treatment process. Similar results were obtained from a quantitative PCR analysis using aBacillus-specific primer set, and the amount of DNA originating from variousBacillus species was maximally 1.91% of the total DNA in the wastewater treatment tank. Clone library analysis using theBacillus-specific primers suggested that, while the population was noticeably increased, the phylogenetic diversity of the increasingBacillus species was very low.     

COX-2 polymorphisms − 765G→C and − 1195A→G and hepatocellular carcinoma risk

Cyclooxygenase-2 (COX-2) is overexpressed in hepatocellular carcinoma (HCC) and considered to play a role in hepatic carcinogenesis. Our aim was to examine the associations between polymorphisms in COX-2 − 765G→C and − 1195A→G and risk of HCC. We conducted a case–control study including 120 patients with HCC and 130 age- and gender-matched controls. Genotypes of the COX-2 polymorphisms − 765G→C and − 1195A→G were determined by polymerase chain reaction-based restriction fragment length polymorphism. No significant difference was observed in the genotype distribution of the − 765G→C polymorphism between patients and controls. The − 1195AA genotype was associated with an increased risk of developing HCC (OR, 2.5; 95%CI, 1.18–5.37). The A allele was present significantly more often in HCC patients (OR 1.5; 95%CI, 1.05–2.14). In conclusion, our results demonstrated that the − 1195AA genotype and A allele have an important role in HCC risk in Egyptian patients.     

Geochemistry and Microbial Populations in Sediments of the Northern Baffin Bay,Arctic

The Northern Baffin Bay between Greenland and Canada is a remote Arctic area restricted in primary production by seasonal ice cover, with presumably low sedimentation rates, carbon content and microbial activities in its sediments. Our aim was to study the so far unknown subseafloor geochemistry and microbial populations driving seafloor ecosystems. Shelf sediments had the highest organic carbon content, numbers of Bacteria and Archaea, and microcosms inoculated from Shelf sediments showed highest sulfate reduction and methane production rates. Sediments in the central deep area and on the southern slope contained less organic carbon and overall lower microbial numbers. Similar 16S rRNA gene copy numbers of Archaea and Bacteria were found for the majority of the sites investigated. Sulfate in pore water correlated with dsrA copy numbers of sulfate-reducing prokaryotes and differed between sites. No methane was found as free gas in the sediments, and mcrA copy numbers of methanogenic Archaea were low. Methanogenic and sulfate-reducing cultures were enriched on a variety of substrates including hydrocarbons. In summary, the Greenlandic shelf sediments contain vital microbial communities adapted to their specific environmental conditions.     

菌落PCR在大规模基因组测序中的应用

一种利用菌落直接PCR扩增DNA并用于测序的实验方法.通过对引物的设计和菌液浓度控制,使PCR反应后的内容物对测序干扰减到最小.与传统的测序过程比较,它省去了抽提模板DNA一步,节省了大量时间和实验成本.另外此方法可对BAC亚克隆库构建时由连接转化过程中导致的假阳性起筛选和鉴定作用.采用该法成功测定了籼稻（Oryza sativa indica）广陆矮4号的L3173号BAC DNA全长序列（约100 kb）,GenBank登录号：AL512542.