Standard Organic Method Research Articles

Association between breast cancer and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) gene 1595C/T SNP in a Pakistani population.

Aim of the StudyTRAIL-mediated signalling has emerged as an extensively studied biological mechanism reported to differentially induce apoptosis in cancer cells. However, overwhelmingly increasing experimentally verified data is shedding light on resistance against TRAIL-induced apoptosis in cancer cells. Moreover, genetic and epigenetic mutations also exert effects on the functionality of TRAIL and its receptors. In this study we investigated the association between breast cancer and polymorphisms in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in a Pakistani Population.Material and methodsGenotyping for TRAIL gene 1595 C/T polymorphism was done for 363 breast cancer patients and 193 age- and sex-matched healthy controls. DNA was extracted using standard organic methods. PCR-RFLP analysis was done for C/T polymorphism at position 1595 in exon 5 of the TRAIL gene using site-specific primers and restriction enzyme. The results were statistically evaluated by SPSS14.ResultsIn this study, CC homozygotes were 46.3% in patients and 49.7% in controls, p = 0.729 with OR value 0.8705 (95% CI: 0.6137–1.2348). CT was statistically insignificant, p = 0.837 with OR value 0.9242 (95% CI: 0.6494–1.3154). However, the minor allele or risk allele genotype TT had a higher percentage among breast cancer patients (12.1%) than in the control group (6.7%). Since there was a statistically insignificant difference (p = 0.212, OR value 1.9098 with 95% CI 1.0019 to 3.6406) of TT genotype between the two groups, the contrastingly higher percentage of TT genotype in breast cancer patients seems to be a risk factor for the disease. Moreover, the frequency of minor allele T was also found to be higher in the patients (0.329) than in the controls (0.285).ConclusionsThe TRAIL gene 1595 C/T SNP has a contradictory role in cancer development in different populations. In our population group although the percentage of homozygous risk allele TT was higher in patients it was statistically non-significant. The raised T allele and TT genotype in patients may suggest its association with breast cancer in the Pakistani population.

Technical note: Efficiency of total demineralization and ion‐exchange column for DNA extraction from bone

We investigated whether a combination of recently introduced methods, total demineralization and ion-exchange columns, would increase DNA recovery from old bone. Ten bone samples taken after a burial period of approximately 60 years were used in this study. Bone powder was digested using total or incomplete demineralization. DNA was extracted by the standard organic method. The DNA extract was purified with ion-exchange columns or QIAquick spin columns. The efficiency of different DNA extraction methods was compared in terms of DNA concentration, inhibitors generated by real-time PCR, and conventional STR typing results. The mean DNA concentration using the total demineralization method is approximately 3 times higher than that using the incomplete demineralization method. For DNA purification, the method using QIAquick spin columns appeared to yield approximately double the DNA than the method using ion-exchange columns. Furthermore, 2 out of 10 samples showed higher levels of inhibition with C(T) values of IPC > or =30 cycles when using only ion-exchange columns. In STR results, total demineralization yielded more locus profiles by 4.2 loci than incomplete demineralization, and QIAquick spin columns also yielded more locus profiles by 3.5 loci than ion-exchange columns. Total demineralization of bone powder significantly increased DNA yield and improved STR typing results. However, the use of ion-exchange columns was not efficient when compared with the method using QIAquick spin columns. It is suggested that the combination of total demineralization and QIAquick spin columns lead to greatly improved STR typing results.

Distribution of mtDNA Haplogroups in a Population Sample from Poland

DNA was isolated using standard organic method followed by double phenol/chloroform/isoamyl alcohol extraction and its concentration was determined fluorescently with PicoGreen on Fluoroscan Ascent Fl. Amplification was performed in two 8-plex reactions. PCR mixture consisted of 5μL of Qiagen Multiplex PCR Master Mix, 1μL of premixed primers (1), 1–4μL of DNA extract (about 0.5 ng of DNA) and distilled water up to 10μL. Reaction conditions were as follows: pre-incubation step: 95◦C for 15 min; denaturation: 94◦C for 30 s, annealing: 60◦C for 90 s, extension: 72◦C for 90 s; 30 cycles; final extension: 72◦C for 10 min. The samples were amplified using GeneAmp PCR System 9700 or 9600 (PE Applied Biosystems). PCR products were detected by 3% agarose gel electrophoresis. PCR primers and unincorporated dNTPswere removed byMicroconeYM-100Centrifugal Filter Devices (Millipore) according to manufacturer’s protocol. SNPs were detected in two multiplex minisequencing reactions. Each reaction

Synthesis of a Racemic Ester and Its Lipase-Catalyzed Kinetic Resolution

A first-year organic chemistry laboratory experiment is described in which an enzyme is utilized for the kinetic resolution of a racemic ester. In the first step, the racemic ester is synthesized from racemic 1-phenylethanol and acetic anhydride in the presence of pyridine. In the second step, only one of the two enantiomers of the racemic ester is selectively hydrolyzed. This reaction is enzyme-catalyzed and it clearly demonstrates the great ease with which enzymes can be used for the synthesis of enantiomerically-enriched compounds. The two enantiomerically-enriched compounds formed are isolated and analyzed for their optical purity. In these experiments the students are exposed to enzyme chemistry in combination with standard organic methods. Indeed, all the products are purified by techniques that are applied in the research laboratory (column chromatography and distillation), ensuring that these experiments come as close to the research environment as possible. By means of these experiments, topics s...