Rapid DNA extraction protocol from stool, suitable for molecular genetic diagnosis of colon cancer

BACKGROUND: Colorectal cancer (CRC) is one of the most common forms of cancers in the world and is curable if diagnosed at the early stage. Analysis of DNA extracted from stool specimens is a recent advantage to cancer diagnostics. Many protocols have been recommended for DNA extraction from stool, and almost all of them are difficult and time consuming, dealing with high amount of toxic materials like phenol. Their results vary due to sample collection method and further purification treatment. In this study, an easy and rapid method was optimized for isolating the human DNA with reduced PCR inhibitors present in stool. METHODS: Fecal samples were collected from 10 colonoscopy-negative adult volunteers and 10 patients with CRC. Stool (1 g) was extracted using phenol/chloroform based protocol. The amplification of P53 exon 9 was examined to evaluate the extraction efficiency for human genomic targets and also compared its efficiency with Machiels et al. and Ito et al. protocols. RESULTS: The amplification of exon 9 of P53 from isolated fecal DNA was possible in most cases in 35 rounds of PCR using no additional purification procedure for elimination of the remaining inhibitors. CONCLUSION: A useful, rapid and easy protocol for routine extraction of DNA from stool was introduced and compared with two previous protocols.     

不同方法从大豆不同组织中提取基因组DNA效果的比较

从大豆组织中获得高质量和足够产量的基因组DNA,是进行大豆分子生物学研究的基础.为进行大豆基因组的PCR,RAPD,SSR等分子生物学研究,分别以大豆种子和其叶片为实验材料,采用改良的SDS法和CTAB法对大豆基因组DNA进行了提取.对DNA的提取效果,采用紫外分光光度检测、琼脂糖凝胶电泳分析及DNA的限制性内切酶图谱分析进行了综合比较.结果表明,在以叶片为材料时,SDS法和CTAB法的大豆基因组DNA的提取效果差别不大,SDS法稍好于CTAB法.在以大豆种子为材料时,SDS法的提取效果明显优于CTAB法,SDS法可从大豆种子中提取到能够充分满足各种分子操作的高质量和数量的大豆基因组DNA.     

基因组DNA快速提取及在转基因大豆检测中的应用

基因组DNA的提取是DNA分子水平研究和检测的重要环节.为补充完善现场检测方法,根据硅膜吸附DNA的特性,结合过滤膜和注射器,开发一种现场快速提取植物基因组DNA的方法.选取大豆、棉花、油菜、玉米、水稻5种主要作物的叶片和种子为样品提取DNA,利用PCR和普通重组酶聚合酶扩增(recombinase polymeras...     

利用SSR分子标记快速鉴定杂交水稻种子纯度技术体系的优化

针对目前SSR分子标记技术快速鉴定杂交水稻种子纯度方法商业化应用的限制因素,从DNA提取、PCR反应体系和程序及凝胶电泳等方面进行了技术体系的优化。原方法经优化后,DNA提取时间从每次5h以上减少到2.5min,PCR反应过程从200min缩短至65min,改聚丙烯酰胺凝胶电泳为琼脂糖凝胶电泳,电泳时间由3.0-3.5h缩短为1h,且通过减少dNTP、Taq酶的用量,对琼脂糖凝胶反复利用,大大降低了成本,减少了废弃物的污染,从而建立了准确、简单、快速、成本低廉的快速鉴定杂交稻种子纯度的技术体系。     

适用于SSR分子标记的玉米单粒种子DNA快速提取新方法

研究了玉米单粒种子DNA的提取新方法,利用该方法提取DNA,液氮、研磨、离心、沉淀、SDS、CTAB及氯仿都不用,一个工作人员提取100粒种子(1个检测样品)DNA只需30min左右,一天可以提取1600粒种子(16个检测样品)的DNA,并且提取的DNA分子量大,不降解,完全可以满足利用SSR分子标记进行DNA指纹分析的需要,为DNA指纹技术在玉米种子纯度及真伪快速鉴定中的广泛应用解决了关键性技术难题。     

萌发花生胚轴中蛋白质、RNA和DNA合成及PEG渗调和多胺对其影响

不同活力花生种子的蛋白质、RNA和DNA合成能力不同。用PEG渗调或多胺预处理,可提高劣变花生种子的活力和胚轴大分子合成能力。劣变种子在萌发初期(20小时内),~3H—dT渗入量反比高活力种子增高,表明存在非按期DNA合成,这一增多部份可为咖啡碱所抑制,其剩余部分与高活力种子相似。应用PEG渗调或多胺预处理可使非按期DNA合成部分减弱,而萌发后期的复制部分加强。     

花生不同部位对提取DNA的影响

采用CTAB法和SDS法，分别从花生的根尖、下胚轴、种子、幼叶中提取基因组DNA，所提DNA片段长度都在21 kb以上；同种方法中部位之间的DNA纯度没有差异，产率差异极其显著，从种子、根尖、下胚轴到幼叶产率依次升高，CTAB法中产率从128．5～498．5ng／mg，SDS法中产率从225．O～542．6ng／mg；两种方法中，相同部位之间只有叶片在纯度上差异显著，产率上相同部位之间的差异极其显著。用CTAB法从叶片中提取DNA，采用RAPD方法，对19个花生品种的基因组进行扩增，结果显示所提DNA满足分子分析的要求。RAPD可用于亲本和品种的鉴定。     

利用聚合酶链式反应技术研究单粒古代稻谷(公元800年)离体DNA片段的扩增

 在东南亚地区已发掘出很多古代稻谷，如果能从这些古稻谷中把DNA提出加以分析，可以得到有关栽培稻的系统分化和地理传播方面的直接信息。古代稻种的基因型相当复杂，必须要能从单粒种子开始分析，方能得到有价值的资料。为此，我们进行了从单粒古稻谷中提取DNA的研究工作。本研究采用通常提取植物组织中DNA的方法，从在日本挖掘出的古代稻谷单粒种子中提取出了50-100 ng左右的DNA片段。以这些DNA作为模扳，用聚合酶链式反应(Polymerase Chain Reaction，PCR)技术将DNA加以扩增．由其中几个DNA片段初步合成了相当于水稻光敏色素基因的DNA序列。     

利用聚合酶链式反应技术研究单粒古代稻谷(公元800年)离体DNA片段的扩增

在东南亚地区已发掘出很多古代稻谷,如果能从这些古稻谷中把DNA提出加以分析,可以得到有关栽培稻的系统分化和地理传播方面的直接信息。古代稻种的基因型相当复杂,必须要能从单粒种子开始分析,方能得到有价值的资料。为此,我们进行了从单粒古稻谷中提取DNA的研究工作。本研究采用通常提取植物组织中DNA的方法。从在日本挖掘出的古代稻谷单粒种子中提取出了50—100ng左右的DNA片段。以这些DNA作为模板,用聚合酶链式反应(Polymerase Chain Reaction,PCR)技术将DNA加以扩增。由其中几个DNA片段初步合成了相当于水稻光敏色素基因的DNA序列。     

青稞坚黑穗病LAMP快速检测技术的建立

由大麦坚黑粉菌(Ustilgo hordei)引起的坚黑穗病广泛分布于我国青稞主产区,病原侵染后形成病穗从而造成产量损失。基于前期初步建立的青稞黑穗病快速检测体系,通过对种子洗涤沉淀物的总基因组DNA进行提取,并进行环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)检测人工模拟及实际种子带菌情况,以期建立完善的青稞坚黑穗病快速检测技术。结果表明,裂解液快速提取法可以对样品总DNA进行有效提取,完善后的LAMP快速检测技术可以有效开展青稞种子携带大麦坚黑粉菌的检测。     

利用微卫星DNA标记进行杂交水稻种子纯度鉴定的研究

利用微卫星分子标记，对湖南目前推广的6个杂交稻组合及其亲本之间的多态性进行了研究。在已筛选的178对引物中，有52对能在一个或多个组合中显示稳定的多态性，杂种表现为父母本互补带型。不同组合间多态性的比例具有差异。用表现多态性的两对引物分别对掺假的威优46和金优207进行纯度鉴定，都能有效地将掺入组合中的假种子区分开。该方法具有准确可靠、多态性高、稳定性好、易于操作的特点。     

玉米成熟子粒RNA提取方法的改良与优化

玉米成熟子粒含有较多的多糖、蛋白质和脂类,传统的RNA提取方法难以提取到高质量的RNA。以授粉后28 d灌浆后期的玉米子粒为试材,以授粉后7 d的玉米子粒为对照,对目前国内外常用的Trizol法、Biozol法、RNAiso Plus法、三种硅胶柱商业试剂盒总RNA提取方法以及本研究建立的改良提取方法进行比较。结果表明,以授粉后7 d的玉米子粒为材料时,各种方法都能获得较好提取效果;以灌浆后期接近成熟玉米子粒为试材时,采用本研究改良或新建立的提取法,可以大幅降低提取成本,快速提取高质量和高得率的RNA。     

Effects of fruit collection date on Phillyrea latifolia L. seed germination

This study was conducted to determine the effects of date of fruit collection on the germination of Phillyrea latifolia L. Fruits were collected between September and December of 2007, in Egirdir, Turkey. It was found that the one thousand seed weight for the species seeds was 400 g, with significant differences among dates of fruit collection. The highest germination percentage of 58% was obtained from the seeds collected on the ground from previous years on the 1st of September 2007. The seeds from crown on the 1st of September 2007 had a germination percentage of 42%. Moreover, seeds from crown on the 1st of November and December 2007 and 15th of October and November 2007 did not germinate. In conclusion, P. latifolia seeds collected on the ground from previous years could be sown in early autumn to obtain a high germination rate.     

Anti-Influenza Virus Activity of Adlay Tea Components

Plant Foods for Human Nutrition - Our previous study showed anti-influenza virus activity in adlay tea prepared from adlay seeds, naked barley seeds, soybean, and cassia seeds. In this study, we...     

The effect of seed extraction methods on seed quality of two cultivar's tomato (Solanum lycopersicum L.)

In order to compare of different methods and identify the optimum condition for tomato seed extraction, factorial experiments with 3 replications was conducted. In the first experiment, pulp of two tomato cultivars (Faraon, Dominator) were fermented at two temperatures (25, 35 degrees C) and six periods (24, 48, 72, 96, 120, 144 h). The germination of seeds in laboratory as well as seedling emergence and preliminary growth in greenhouse were studied and measured. The results showed that effect of cultivar on traits (except of seedling emergence) was significant. Also the effect of temperature of fermentation, duration of fermentation and also interaction effects of them on seed germination were significant. Totally seed quality decreased with increasing temperature and duration of fermentation and the fermentation duration from 24 to 48 h at temperature 25 degrees C, is recommended. In the second experiment, tomato seeds were extracted by HCL (pH was arranged to 1, 2, 3 for 10, 20, 30 min), H2SO4 (pH was arranged to 1, 2, for 15, 30 min), Sodium carbonate (5, 10% for 24 and 48 h) and fermentation. Percentage germination, germination rate, length of radicle and length of plumule were used for seed quality assessment. The results showed that interaction effect between pH and duration of HCL treatments was significant for seed germination (percentage and rate) and there was an interaction effect between concentration and duration for germination rate in alkali treatments. Different extraction methods had not detrimental effect on percentage germination, but acid treatments produce very bright clean seeds in compare to other treatments.     

Milkweed seed wing removal to improve oil extraction

Milkweed is now being grown commercially mainly for the production of floss used as hypoallergenic fillers in pillows and comforters. More recently, the use of milkweed seed oil in soaps and personal care products is being explored. The oil used in this effort was obtained by screw pressing whole milkweed seeds. The milkweed seed has a considerable amount of paper-thin wing around the edge of the hull. The light wing contributes greatly to the low bulk density of the seeds and the efficiency of oil extraction. This study explored the feasibility of removing the wings from the seeds to reduce the amount of material going into oil extraction. Hand-fractionation of the seeds showed that the wings, hulls, and kernel accounted for 12.2%, 51.2%, and 36.5% of the seed weight, respectively. The wing contained 1% of the total oil. Most of the oil is in the kernel (73%), but a significant amount is also found in the hulls (22.4%). Mechanical removal of seed wing was evaluated using an impact huller. Seeds (1 kg) with 4%, 7.2%, and 10% moisture were passed through the huller running at 1250 and 1750 rpm impeller speeds. The seeds discharged from the huller were screened to separate the intact seeds and partially dewinged seeds, dewinged seeds, and fines. Seed wings were effectively removed at seed moisture contents and impeller speed combinations of 7% and 1250 rpm or 10% and 1700 rpm. This was verified using 100 kg seeds. Removing the wings reduced the weight of the seeds by 13%, reduced the volume by 46%, and increased the bulk density by 63% while losing less than 5% of the total oil. The oil content of the dewinged seeds was 16.6% higher than the whole seeds. These reductions in seed weight and volume can significantly increase the output of the oil extraction equipment.     

橡胶林土壤微生物2种DNA提取方法的比较

比较了橡胶林土壤微生物CTAB-SDS提取法和SDS提取法的效果。结果表明,2种提取方法均获得约 23.1kb的DNA片段,2种提取方法在颜色、提取量和纯度上存在较大差异。CTAB-SDS提取法提取DNA的纯度较好,不需要纯化可以直接用于PCR扩增、酶切等后续操作;SDS提取法提取的纯度低,不能直接用于PCR扩增。2种方法都适合橡胶林土壤DNA的提取,如果要求获得纯度高的DNA,CTAB-SDS提取法是最佳选择。橡胶林土壤微生物DNA的提取,推荐使用CTAB-SDS提取法。     

玉米功能性Insertｉon/Deｌetｉon(InDeｌ)分子标记的挖掘及其在杂交种纯度鉴定中的应用

InDel作为重要的遗传标记已被广泛用于作物连锁图谱的构建及多样性研究。通过生物信息学方法从玉米全基因组水平进行InDel标记的挖掘并对其在玉米杂交种纯度鉴定中的应用进行分析,根据B73全基因组序列(第二版)及Mo17的二代电子拼接序列发现了40 000多个InDel位点,其中约有11 400个含有InDel位点的序列可用于特异性引物的开发。新发展的143个代表基因(<1 kb)或基因内部的功能性InDel标记在B73及Mo17间得到了验证并用于玉米杂交种纯度的鉴定。经过分析,共有13个共显性InDel标记在6个杂交种亲本间表现出明显的长度多态(>50 bp)。结合玉米子粒DNA快速提取方法,利用这些共显性InDel标记对6个杂交种1 400多个样本纯度进行鉴定,结果显示该方法可以快速、准确、经济地鉴定玉米杂交种纯度。     

Flowering and true seed production in potato (Solanum tuberosum L.). 1. Effects of inflorescence position,nitrogen treatment,and harvest date of berries

Summary Flowering and true seed production from different order inflorescences in potato (Solanum tuberosum L.) were evaluated in two experiments. The number of flowers per inflorescence, berry set, berry weight, number of seeds per berry and seed weight generally decreased from primary inflorescences to tertiary inflorescences and inflorescences on lateral stems. The possible relation with carbohydrate distribution is discussed. Quality of the seeds produced from the different orders of inflorescences was a function of seed size; larger seeds showed better germination, emergence and seedling growth. Late harvest of berries increased berry weight and number of seeds per berry from primary inflorescences. Application of additional nitrogen during the flowering period did not affect flowering or differences between inflorescence positions in seed production, but it significantly decreased the number of seeds per berry. The practical implications of these results for true seed production are discussed.