分子诊断实验室去除核酸污染的方法学研究

核酸检测因具有良好的灵敏度和特异性而被广泛应用于体外诊断、动植物商品检疫、法医鉴定等领域.然而操作过程中易受到核酸污染导致的假阳性结果,严重影响了检测准确性.因此寻找一种有效的防止和清除核酸污染的方案对于实验室正常运转及保障检测结果的可靠性具有重要意义.文中比较了几种不同清除核酸污染的方法,确认了 84消毒液和PCRg...     

Regeneration of commercial nucleic acid extraction columns without the risk of carryover contamination

Nucleic acid extraction is a basic requirement in a molecular biology laboratory. In terms of purity and yield, commercial nucleic acid extraction columns are superior; however they are expensive. We report here an efficient strategy to regenerate diverse commercial columns for several rounds without altering the binding capacity of the columns or changing the properties of the nucleic acids purified. Plasmids purified with regenerated columns were functionally identical in super-coiled nature, restriction analysis, expression of the encoded reporter genes, or amplification of the viral RNA in real-time PCR. To ensure that the regenerated columns were free of the residual DNA, we used two different plasmids with different drug-resistance markers. By colony plating and PCR amplification of the encoded genes, we show that the regeneration process is absolute. Using radiolabeled DNA, we demonstrate that DNA exposed to the regeneration reagent is fragmented to molecular weight below 36 bp. Our data collectively prove regeneration of the commercial columns without the concern of carryover contamination. A procedure to permit safe and efficient regeneration of the commercial columns is not only of great advantage to extend the lifetime of these columns but also makes them commercially more affordable, especially in a resource-poor setting.     

紫杉醇产生菌的细胞总核酸提取

树状多节孢（Nodulisporium sylviforme)为中国新记录属种,首次发现它可产生特效抗癌药物紫杉醇,国内外尚未见其DNA提取等方面的研究报道。为了获得高质量的DNA,利用单因素试验对其细胞总核酸的提取方法进行了探索。找到了一套适合紫杉醇产生菌DNA的提取较为简便易行可靠的方法。用此法直接从紫杉醇产生菌中提取DNA,并将所提取DNA进行随机引物扩增（RAPD）,得到了较清晰的扩增图谱。     

Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes

A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed. Individual micro-colonies of P. aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in P. aeruginosa rRNA. Within each micro-colony, reaction of the peroxidase with a chemiluminescent substrate generated light that was subsequently captured by film or with a digital camera system. Each spot of light represented one micro-colony of P. aeruginosa. Sensitivity and specificity for the identification of P. aeruginosa were 100% as determined by testing 28 P. aeruginosa strains and 17 other bacterial species that included closely related Pseudomonas species. Furthermore, the number of micro-colonies of P. aeruginosa represented by light spots correlated with counts of visible colonies following sustained growth. We conclude that PNA CISH speeds up traditional membrane filtration techniques and adds the specificity of PNA probe technology to generate fast and definitive results.     

Ring-closing metathesis reactions in nucleic acid chemistry-cyclic dinucleotides for targeting secondary nucleic acid structures

Cyclic dinucleotides are synthesized using a ring-closing metathesis protocol and incorporated into oligonucleotides. A stabilization of a three-way junction is observed by an oligodeoxynucleotide containing a central 2'-C to 3'-phosphate connection.     

Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration

A system for rapid point-of-use nucleic acid (NA) analysis based on PCR techniques is described. The extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface-area-to-volume ratios. Short (500 bp) and medium size (48,000 bp) DNA have been captured, washed, and eluted using the silicon dioxide surfaces of these chips. Chaotropic (GuHCl) salt solutions were used as binding agents. Wash and elution agents consisted of ethanol-based solutions and water, respectively. DNA quantities approaching 40 ng/cm2 of binding area were captured from input solutions in the 100-1000 ng/mL concentration range. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50 percent, and concentration factors of about 10x using bacteriophage lambda DNA as the target. Rapid, multichannel PCR thermal cycling modules with integrated solid-state detection components have also been demonstrated. These results confirm the viability of utilizing these components as elements of a compact, disposable cartridge system for the detection of NA in applications such as clinical diagnostics, biowarfare agent detection, food quality control, and environmental monitoring.     

Polyethyleneimine derivative for nucleic acid model

Water-soluble polyethyleneimine (PE) derivatives containing nucleic acid bases and hydrophilic amino acids such as homoserine (Hse) and serine were prepared by the activated ester method as nucleic acid models. From spectroscopic measurements, the polymers were found to interact with DNA accompanied by an induction of conformational change. Hypochromicity in UV spectra indicated that a stable polymer complex was formed between poly (A) with PEI-Hse-Ura by complementary hydrogen bonding with equimolar nucleic base units (adenine∶uracil=1∶1). The induced conformation of DNA by the interaction with the polymer containing uracil and homoserine (PEI-Hse-Ura) was concluded to be a super triple helical structure. The formation of the polymer complex, DNA:PEI-Hse-Ura, was found to be affected by the presence of metal ions such as Ca²⁺ and Cu²⁺.     

PNA biosensors for nucleic acid detection

Biosensor devices, based on the conversion of nucleic acid recognition reactions into useful electrical signals, offer considerable promise for DNA diagnostics. The unique hybridization properties of solution-phase PNA can be extrapolated onto transducer surfaces in connection with the design of remarkably specific DNA biosensors. This article reviews the development of PNA biosensors, and discusses common PNA-biosensing protocols along with their prospects in DNA biosensor technology.     

Paradigms for computational nucleic acid design

The design of DNA and RNA sequences is critical for many endeavors, from DNA nanotechnology, to PCR-based applications, to DNA hybridization arrays. Results in the literature rely on a wide variety of design criteria adapted to the particular requirements of each application. Using an extensively studied thermodynamic model, we perform a detailed study of several criteria for designing sequences intended to adopt a target secondary structure. We conclude that superior design methods should explicitly implement both a positive design paradigm (optimize affinity for the target structure) and a negative design paradigm (optimize specificity for the target structure). The commonly used approaches of sequence symmetry minimization and minimum free-energy satisfaction primarily implement negative design and can be strengthened by introducing a positive design component. Surprisingly, our findings hold for a wide range of secondary structures and are robust to modest perturbation of the thermodynamic parameters used for evaluating sequence quality, suggesting the feasibility and ongoing utility of a unified approach to nucleic acid design as parameter sets are refined further. Finally, we observe that designing for thermodynamic stability does not determine folding kinetics, emphasizing the opportunity for extending design criteria to target kinetic features of the energy landscape.     

Search for a prion-specific nucleic acid

Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID50) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP(Sc) molecules and ID50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID50 unit is approximately 25 nucleotides in length. In the same fractions, there were approximately 3,000 protease-resistant PrP(Sc) molecules per ID50 unit after accounting for clearance of PrP(Sc) following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of approximately 1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.     

Watersoluble synthetic nucleic acid analogs--polyethyleneimine derivatives containing nucleic acid bases--conformation and interactionwith nucleic acids

Water soluble polyethyleneimine derivatives containing nucleic acid bases were found to interact with polynucleotides, DNA, RNA. The conformational change by formation of complex was observed by CD spectra and was discussed with the hypochromicity in UV spectra. The rates of interactions between nucleic acid bases in polymers were slow as shown by UV spectra, but the conformational changes of the polynucleotides were fast as shown by CD spectra. In the case of the uracil derivative (PEI-Hse-Ura), high value of CD spectra [theta] 2.80 = -8.0 x 10(-4) for the complex with DNA might be caused by psi type conformation of DNA.