Résumé
Objective To investigate the influences of drop components on the"coffee ring" test of DNA. Methods The DNA-EB drops containing SDS, NaCl, etc, were evaporated on glass slides. After evaporation, the fluorescent Deposition Pattern (DP) and Integrated Optical Density (IOD) was acquired with a gel imaging system. The dose-effect relationship was analyzed. Result When the non-DNA components concentration is low, the DP was still characteristiced by peripheral rings, subtle component-specific differences, such as tree-ring like structure and radial crack, existed. At high concentrations, DP was various, which may be ring + scattered dots (NaCl), central spot + small weak ring (SDS), concentric/tree-ring (TritonX-100) or ring + spot (H+), et al. Non-DNA components had little effect on IOD(0.5~2 times). Conclusion Non-DNA components in DNA-EB drops influences both the DP and IOD, but rough estimation of DNA concentrations is still effective. Moreover, analyzing the DP can provide more informations about sample purity and residue.
Résumé
Objective To explore the feasibility of "coffee ring" effect in DNA high-throughput evaluation. Methods we mixed uniformly DNA solution and EB solution, then dripped different size of droplets on 5 kinds of substrates. After evaporation, the coffee ring was observed and photographed with LG2020 gel imaging analysis system. Analyzed the correlation among ring formation conditions,ring fluorescence intensity and DNA concentration, and looked for the sensitivity and applicable range of the method. Result DNA - EB solution can form fluorescent ring after surface evaporation on the slide, transparent polystyrene plate carrier and so on. The DNA detection limit can be up to 2ng/μl. DNA concentration and loop integral optical density fitting Logistic equation(R2=0,999) within 2ng/μl~1000ng/μl. Conclusion The coffee ring method is simple, fast and low equipment requirement,which can be used for high-throughput evaluation of DNA solution.
Résumé
Objective To explore the feasibility of hemochrome identification by micro spectrophotometer. Methods We use dip lotion by Centrifugation, reaction with modiifed takayama reagent,measured absorption spectra with a micro spectrophotometer to determine whether samples contain blood. On the basis of optimized parameters, we use diluted human hemoglobin to measure sensitivity of the method; using suspected blood / blood spots, and different storage time and matrix of blood spots to measure speciifcity and samples of adaptability. Result With micro spectroscopic method of hemochromogen,only blood (spot) has speciifc spectral consisting of three absorption peaks at 415,525 and 555nm and no cross suspected common blood / spot.Reaction 2 min, sample volume2.5ul, 1000-fold diluted human blood stably obtains primordial spectrum. By prolonging the immersion time, set the vehicle control, 10 years old blood gauze. Blood stains in colored cloth can be effective detective.. The height of absorption peaks and blood content were significantly corelated, and the change in absorbance at 525 and 555nm consistent trend (y=0.5232x+0.0274, R2=0.9971). Conclusion Micro spectroscopic method of hemochromogen has high sensitivity and speciifcity, quick and easy, can be incorporated into the DNA testing process. There is a good prospect in the actual seizure case. Instead of the traditional crystallization method for teaching, training requirements more in line with the skills and awareness for current students.
Résumé
HIV-1 integrase (IN) is an essential enzyme for retroviral replication. There is no analogue for this enzyme in human cells so that inhibition of IN will not bring strong effect on human body. Thus, HIV-1 IN has become a rational target for therapy of AIDS. This review provides a comprehensive report of alpha, gamma-diketo IN inhibitors discovered in recent years. Compilation of such data will prove to be beneficial in developing QSAR, pharmacophore hypothesis generation and validation, virtual screening and synthesis of compounds with higher activity.
Résumé
Objective To develop a simplified bisulfite genomie sequencing(BGS)method for DNA methylation marker scanning.Methods According to modified BGS protocol,the desalt DNA treated with bisulfite were directly used for bisulfite-PCR(BSP)without alkali treatmenL Complement of the bisulfite modification Wag accomplished by a prolonged pre-denaturation stage.After BSP,a second round PCR was performed with a pair of GC tagged primers to adjust the GC content of the amplieon for direct sequencing.To assess this improved protocol,promotor methylation of TNF-α gene in 3T3-L1 cell and androgen receptor(AR)gene in Hela cell was investigated.The real time BSP for Alu was also used to compare the sensitivity of the modified assay with traditional assay.Results Both the hypermethylated TNF-α promotor and hypomethylated AR promotor were successfully sequenced by improved BGS method,and the results were consistent with that of the traditional assay.The conversion rate reached 100%,while the conversion specificity was higher than 93.75%.The sensitivity of improved BGS method inereaged significantly(t=2.978 2,P＜0.05)and showed good reproducibility.Condusion The improved BGS method is simple and sensitive,facilitating more ambitious genomic methyhtion mapping studies.
Résumé
Objective To study the Application of X-linked differentially methylated polymorphism site in forensic medicine.Methods X-STR HUMARA was chosen as a model locus.PCR procedures were performed after digestion using methylation-sensitive restriction endonucleases.STR polymorphism of HUMARA was analyzed and compared in samples collected from male and female individuals.Result After digestion with methylation-sensitive restriction enzyme HpaⅡ,no PCR products were obtained from male samples,whereas PCR products from the female samples were normally typed.In monoclonal tumor cell samples from females,only one allele was detected.Conclusion The differentially methylated X-STR HUMARA locus is a novel marker for mixture analysis of mixed stains,sex determination and discrimination of tumor tissues.
Résumé
Drowning represents one of the leading causes of unnatural death. The plankton test is still considered as one of the useful methods for medico-legal investigation of death by drowning although controversies concerning the reliability of the test exist. The methods commonly used for detection of planktons generally include microscopic examination after tissue samples are destructed with or without strong acid, and detection of the planktons by molecular biological techniques. The evaluation of the positive results by the former methods are limited by the physical-chemical characters of the planktons. Detection of DNA genetic markers by molecular biological techniques, which can provide abundant information and is applicable to studying various planktons, is a novel method helpful for investigation of drowning. In the present paper, several techniques for detection of planktons were reviewed as references for medico-legal practitioners in investigation of suspected cases of drowning.