同时抽提DNA和RNA的简便方法

同时抽提ＤＮＡ和ＲＮＡ的简便方法刘定干（中国科学院上海生物化学研究所,２０００３１）关键词ＤＮＡＲＮＡ抽提在研究工作中,常同时需要培养细胞的ＤＮＡ和ＲＮＡ,从同一样品中同时提取ＤＮＡ和ＲＮＡ。目前,已有好几种同时抽提ＤＮＡ和ＲＮＡ的方法［１～４］。作...     

一种改进的快速有效的裸藻DNA抽提方法

大多数裸藻为单细胞游动种类,无细胞壁,细胞质外层特化为表质,部分种类细胞具胶质的囊壳,表质、囊壳的主要成分为蛋白质,使之具有柔韧的类牛皮糖的特性,给裸藻破细胞、DNA抽提及进行下一步工作造成一定的难度,本文在比较、改进的基础上,得到一种快速有效的裸藻DNA抽提方法。       

新生隐球菌基因组DNA不同抽提方法的比较

目的 DNA是进行分子生物学研究的重要基础。在本研究中,我们建立了2种简单快速抽提基因组DNA的方法并可用作PCR扩增的模板。通过比较4种不同的DNA抽提方法以确定哪种更适合进行下一步的基因分析。方法这4种方法是:玻璃珠法,酶法,3%SDS法和氯化苄法。玻璃珠法是用玻璃珠在混漩器上剧烈振荡破碎细胞壁;3%SDS法是将细胞在含10mmol/LDTT的3%SDS溶液中加热,然后用5mmol/LKAc和异丙醇抽提,DNA的产量通过A260测定。结果 3%SDS溶解法、经典酶法、玻璃珠法和氯化苄法的DNA产量分别为0.4154±0.0367、0.8484±0.0756、1.2636±0.2040、0.4070±0.0339(g/L×108CFU/mL)。结论玻璃珠法是最敏感、重复性好、简单、费用合理的抽提方法 。     

有效抽提乙醇中螨标本的核DNA

对如何有效地从乙醇保存的叶螨中抽提核基因组DNA作了探讨。70％乙醇保存的叶螨标本,经过干燥,TEN洗涤,蛋白酶K消化,酚—氯仿抽提,2倍体积无水乙醇沉淀等步骤,得到叶螨核基因组DNA沉淀,再用TE或无菌水溶解后,以其为模板,进行随机引物PCR扩增。结果显示.该方法抽提的DNA无Taq酶抑制物,且其纯度达到进行PCR反应对模板要求的程度。这为采用AP-PCR技术研究螨的系统分类提供了便利。     

基因芯片核酸样品制备方法的优化

基因芯片研究中,基于不同的样品来源,基因含量,检测方法和分析目的,采用的核酸分离。扩增和标记方法各异。本综述了对核酸样品制备条件的优化,主要有核酸的单链化处理,片段化和标记方法,根据具体情况选用合适的处理方法,可显提高基因芯片检测的特异性和重现性。     

噬菌体DNA的快速抽提

介绍一种噬菌体DNA的快速抽提方法.用聚乙二醇沉淀噬菌体颗粒,然后经DEAE纤维素纯化处理和酚抽提.与传统的噬菌体DNA纯化方法相比,改进后的方法方便、快速、经济,可获得高纯度的噬菌体DNA.     

有效提取病毒诱导基因沉默植株中DNA的方法

根据PCR反应的要求,用改良的CTAB法,以番茄植株为材料,实现了微量番茄叶片基因组DNA的快速提取。提取基因组DNA所用的组织量少,所得的DNA经过电泳检测虽有降解,但足以用于PCR检测,以其作模板扩增中国番茄黄化曲叶病毒诱导的硫黄素酶（Su）基因沉默植株中病毒组分中的DNAmβ和1.7 A,片段大小分别为1 300、500 bp。测序结果证明是相应基因的部分片段。该方法的材料不需要使用液氮,可以单人大批量提取,并在基因沉默的番茄植株中能稳定而准确的规模化PCR检测。     

锈菌夏孢子DNA的微量快速提取方法

采用4种方法对毛白杨锈病单个夏孢子堆DNA提取和ITS—PCR扩增表明,钢珠法、玻片法均是锈菌基因组DNA微量快速提取的合适方法,以钢珠法最优,整个过程仅需30min,并且可以获得与CTAB法、氯化苄法相同的PCR扩增结果。钢珠法是在提取液中加入2-3颗钢珠,靠钢珠在涡旋中的相互碰撞将夏孢子破壁,以同时加入NaOH(终浓度3．3％)和Chelex-100(终浓度1．6％)效果最好,ITS-PCR扩增能稳定得到700bp片段。玻片法也能得到相同的结果。分析比较了4种方法的优缺点。     

ＤＮＡ粗提取与鉴定的简易方法

介绍一种在中学可行的ＤＮＡ提取与鉴定的简易方法,该法可以不使用 离心机和一些特殊的昂贵的药品。     

A simple and reliable method for DNA extraction from bivalve mantle

A simple and reliable method was developed for extracting genomic DNA from preserved mantle tissues of Pacific oyster Crassostrea gigas for reproducible PCR amplification. The method was based on destruction of the tissue using Proteinase K, Chelex 100 resin, detergents, and urea, followed by preferential capturing of genomic DNA with silica particles. Approximately 5 mg of mantle tissue provided a sufficient quality and quantity of DNA for several hundreds of PCR reactions amplifying the hypervariable mitochondrial DNA intergenic spacer, which is a useful genetic marker for population structure analysis of Pacific oyster. The method can be applied for DNA preparation from not only fresh and frozen but also ethanol-preserved mantle tissues, so this rapid and economical method can serve for investigating a large number of bivalve specimens collected in the field and next transported in ethanol at ambient temperature.     

A simple method for extracting DNA from Cryptosporidium oocysts using the anionic surfactant LSS

Detection of low amounts of Cryptosporidium oocysts in raw water sources is considered an important component in the management, prevention and control of Cryptosporidium in drinking water supplies as Cryptosporidium causes massive waterborne outbreaks worldwide. As Cryptosporidium has a robust oocyst that is extremely resistant to chlorine and other drinking water disinfectants, both the freeze-thaw method and DNA extraction kits have been commonly used for extracting and purifying DNA from the oocyst. However, the DNA extraction procedures are time consuming and costly. Therefore, a simple and low-cost method to extract and purify DNA from the robust oocyst has been required. In this study, we discussed a simple method for detecting Cryptosporidium DNA with the anionic surfactant, n-lauroylsarcosine sodium salt (LSS) using the loop-mediated isothermal amplification (LAMP) to eliminate the need for the freeze-thaw method and the DNA extraction kits. As a result, Bst DNA polymerase was inhibited by 0.1% LSS but not 0.01% LSS and 5% Triton X-100 or Tween 20. Although DNA was extracted from the oocysts by incubating with 0.1% LSS at 90°C for 15 min, Bst DNA polymerase was inhibited by 0.1% LSS. The inhibition by 0.1% LSS was suppressed by adding 5% of the nonionic surfactants, Triton X-100 or Tween 20. The concentration of LSS in a LAMP tube was 0.01% while that in an incubation tube was 0.1%, because LSS in an incubation tube was diluted by a factor of 10 at the DNA amplification process. Therefore, we found that ten oocysts of Cryptosporidium parvum could be detected by incubation with 0.1% LSS, without removing LSS or adding the nonionic surfactants in the LAMP method.     

A simple method for the direct extraction of plasmid DNA from yeast

Summary A rapid and simple method for the small scale isolation of shuttle plasmid DNA from Saccharomyces cerevisiae is described. It uses glass beads to break cells and reagents which are also used in bacterial mini-preps to yield plasmid DNA without chromosomal contamination in sufficient quantities to enable direct visualisation on agarose gels.     

A simple method for extraction of fungal genomic DNA

We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze-dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8-32 microg of high molecular weight DNA per 30 mg of freeze-dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow-growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min(-1) in the presence of CsCl. The modified method yielded 1.5-2 microg of high molecular weight DNA per colony.     

A simple method of DNA extraction for Eimeria species

A new, simple method is described for extracting DNA from coccidia (Eimeriidae) oocysts. In our hands this method works well for all Eimeria oocysts and, presumably, will work equally well for oocysts of other coccidia genera. This method combines the two steps of breaking oocyst and sporocyst walls, and dissolving the sporozoite membrane in one step. This greatly simplifies the currently used DNA extraction procedures for Eimeria species and overcomes the disadvantages of existing DNA extraction methods based on glass-bead grinding and sporozoite excystation procedures. Because all the procedures are done in a 1.5-ml microfuge tube, which minimizes the loss of DNA in the extraction procedures, this method is especially suitable for samples with small number of oocysts. In addition, this method directly lyses the oocyst and sporocyst walls as well as the sporozoite membrane in a continuous incubation; therefore, it does not require the sporozoites to be alive. The results of PCR experiments indicate that this method generates better quality of DNA than what the existing glass-bead grinding method does for molecular analysis, and is suitable for both large or small number (<10(2) oocysts) of living or dead oocyst samples.     

A cost-effective,simple and high-throughput method for DNA extraction from insects

Abstract We present a high-throughput cost-effective method to extract DNA suitable for polymerase chain reaction (PCR) from insect tissue. The method uses standard 200 μL-deep 96-well plates in which samples are ground, digested and subsequently purified. The test extraction using four different insect species and controlling for potential contamination showed that the method yields good-quantity and quality DNA. PCR with mitochondrial and nuclear primers was reliable. The proposed extraction protocol combines the speed of commercial 96-well plate methods with the economies associated with readily available and cheap laboratory chemicals, consumables and equipment. Therefore, this method is particularly suitable for low-budget research projects and for laboratories with only basic equipment present.     

A simple method for DNA extraction from marine bacteria that produce extracellular materials

We present a simple method for extracting DNA from the marine bacteria Hahella chejuensis, a Streptomyces sp., and a Cytophaga sp. Previously, DNA purification from these strains was hindered by the presence of extracellular materials. In our extraction method, the marine bacteria are lysed by freezing and grinding in liquid nitrogen, and treated with SDS. The extracted DNA is purified using a phenol/chloroform mixture, and precipitated in isopropanol. The extracted DNA is of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion, genomic DNA blot hybridization, and genomic DNA library construction. We used this method to extract genomic DNA from several other marine bacteria. Our method is a reproducible, simple, and rapid technique for routine DNA extractions from marine bacteria. Furthermore, the low cost of this method makes it attractive for large-scale studies.