一种快速、经济提取外周凝血基因组DNA 方法的建立

目的:建立一种经济、快速且高质量提取人体外周凝血DNA的方法。方法:摸索最佳的匀浆条件,对外周凝血块进行匀浆,采用KI法对匀浆液进行基因组DNA的提取,通过凝胶电泳、单重PCR和多重PCR检测凝血基因组DNA的提取产量和质量,并分别与常规的凝血基因组DNA提取方法,即蛋白酶K消化法,以及提取抗凝血基因组DNA的KI法进行比较分析。结果:最佳的匀浆条件为:39000 rmp,15秒。在此条件下提取的基因组DNA完整性好,纯度和产量与蛋白酶K消化法提取凝血DNA和KI法提取抗凝血DNA的结果相比,没有统计学差异。单重PCR和多重PCR也获得了理想的扩增结果。结论:与常规的外周凝血提取方法相比(蛋白酶K消化法),本方法节省了时间和成本,能快速、经济、有效地提取外周凝血基因组DNA,可用于后续的科研和临床诊断需要,解决了部分科研机构血液基因组DNA的样本来源问题。     

一种快速、经济提取外周凝血基因组DNA方法的建立

目的：建立一种经济、快速且高质量提取人体外周凝血DNA的方法。方法：摸索最佳的匀浆条件,对外周凝血块进行匀浆,采用Ⅺ法对匀浆液进行基因组DNA的提取,通过凝胶电泳、单重PCR和多重PCR检测凝血基因组DNA的提取产量和质量。并分别与常规的凝血基因组DNA提取方法,即蛋白酶K消化法,以及提取抗凝血基因组DNA的Ⅺ法进行比较分析。结果：最佳的匀浆条件为：39000map,15秒。在此条件下提取的基因组DNA完整性好,纯度和产量与蛋白酶K消化法提取凝血DNA和KI法提取抗凝血DNA的结果相比,没有统计学差异。单重PCR和多重PCR也获得了理想的扩增结果。结论：与常规的外周凝血提取方法相比（蛋白酶K消化法）,本方法节省了时间和成本,能快速、经济、有效地提取外周凝血基因组DNA,可用于后续的科研和临床诊断需要,解决了部分科研机构血液基因组DNA的样本来源问题。     

黑翅土白蚁基因组DNA提取方法的优化

目的 为快速地提取到质量较好的黑翅土白蚁基因组DNA进行白蚁种群多样性的研究,对基因组DNA提取方法进行了比较与改进.方法 先初步采取CTAB法与蛋白酶K法对黑翅土白蚁基因组DNA的提取方法进行比较,再利用正交设计法对蛋白酶K法中裂解液、蛋白酶、RNA酶及作用时间4个因素进行优化.结果 蛋白酶K法获得的基因组DNA的质量与产量稍优于CTAB法;较佳的提取步骤组合为:裂解液150 μL,蛋白酶K 6μL,作用时间1h,RNA酶可不添加.结论 采用优化后的方法获得的基因组DNA为模板进行PCR扩增,得到了清晰、稳定的扩增谱带,完全可用于相关后续实验.     

一种从PAXgene全血RNA管内提取基因组DNA的方法

目的:从同一生物样本同步提取RNA和DNA,能提高样本的利用率,而且对于基因组学、转录组学和表观遗传学检测数据之间的比对和匹配分析也十分重要。本研究在不影响RNA样品制备的前提下,建立一种从PAXgene全血RNA管内提取基因组DNA的方法。方法:取一定量PAXgene全血RNA管血液样本,使用QIAamp DNA试剂盒提取血细胞基因组DNA,系统优化提取过程中的离心参数、洗脱量以及初始血液样本量等实验参数,并对提取的基因组DNA质量进行检测。结果:用PAXgene全血RNA管3 mL血液样本能够提取出8.918±1.100μg基因组DNA,紫外分光光度计检测DNA样品的OD 260/280比值为1.89±0.09,琼脂糖凝胶电泳结果显示DNA样品完整无降解。结论:利用本方法提取的DNA样品能够满足下游DNA芯片、DNA甲基化测序等实验要求。该方法有助于从有限的临床血液样本中获取全面的遗传信息,并且提高后续不同实验方法所生成数据之间的可比性和匹配度。     

一种快速提取基因组DNA的方法

采采用氧化硅超顺磁性纳米磁珠和自己设计的试剂体系及提取流程,建立了一种基因组DNA的快速提取方法,该方法以氧化硅磁珠为固相吸附载体,盐酸胍、 -巯基乙醇和SDS为主要裂解吸附试剂。以全血或培养细胞为实验材料进行基因组DNA的提取结果显示用本文建立的方法提取100 L小鼠抗凝血,可得2～3 g基因组DNA, OD260/OD280为1.8 ± 0.05,其纯度可满足后续的酶切和PCR生物操作要求。该方法整个提取过程只需12分钟,不需特殊实验条件同时可省略蛋白酶K的消化过程和离心操作,适用于一般实验室的需求,是一种操作简便、快速高效的提取方法。     

一种简单、有效的适于PCR操作的放线菌DNA提取方法

目的:利用改良酶法发展了一种从微量(几百微升)发酵液中快速安全的提取放线菌基因组DNA的方法。方法:利用溶菌酶破壁,蛋白酶K和SDS除蛋白,成功提取较高质量的放线菌基因组DNA,所得的DNA可作为PCR反应的模板进行16SrRNA等基因有效扩增。结果:能从海绵和土壤分离的放线菌中成功提取基因组DNA。结论:该方法操作简单、费用低廉、不使用酚、氯仿等有毒害作用有机试剂,非常适于长期从事放线菌操作的研究人员。为大量放线菌菌株的快速鉴别、高通量筛选和系统分类研究创造了条件。     

湖泊沉积物基因组DNA的提取方法比较研究

水体沉积物为一新型微生物种质资源库,不依赖于培养的菌种多样性研究的首要条件是获取优质的基因组DNA。本研究以改进的氯仿异戊醇法、蛋白酶K+SDS法、玻璃珠+蛋白酶K+SDS法提取湖泊基因组DNA,并与试剂盒提取结果进行比较,为沉积物分子生态学的研究提供参考和借鉴。结果表明,改进后的氯仿异戊醇法提取的DNA纯度较低,腐殖质明显,纯化后PCR产量较低;蛋白酶K+SDS法获得的DNA完整性较好,浓度高,PCR扩增应用效果最好;玻璃珠+蛋白酶K+SDS法提取的基因组DNA效果次之;试剂盒法提取的基因组DNA浓度较低,片段长度略小。蛋白酶K+SDS法为除试剂盒外适合于湖泊沉积物基因组DNA提取的较好方法。     

适于涡虫基因组DNA快速制备的改良的CTAB法

提取得到高质量的DNA样品是进行分子生物学研究的必要前提。为了找到一种适用于提取涡虫基因组DNA的常规方法,我们以东亚三角头涡虫为材料,分别用改良的CTAB法、SDS法、SDS-蛋白酶K法对涡虫的基因组DNA进行了制备,并对3种方法制备的涡虫基因组DNA进行了检测与比较。根据比较结果,我们认为改良的CTAB法最适合于涡虫基因组DNA的快速制备,为涡虫的分子生物学研究打下了基础。     

血液基因组DNA的快速提取方法

目的:研究血液中基因组DNA的简便快速提取方法。方法:取新鲜抗凝血,以红细胞裂解液除去红细胞,再破碎白细胞,除去杂蛋白,获得基因组DNA。结果:所得基因组DNA完整、无断裂,含量和纯度均较高。以所提基因组DNA作为模板能很好的扩增出p21因子启动子序列,因此该法所提取的DNA是完整可靠的。结论:该法能简便、快速、安全、廉价的提取血液中的基因组DNA,并适用于临床检测和分子生物学研究。     

三种提取冬虫夏草菌丝体基因组DNA方法的比较

采用改良氯化苄法、CTAB法、蛋白酶K裂解法这三种方法,对冬虫夏草菌丝体基因组DNA进行提取,将提取的基因组DNA经过紫外分光光度计检测纯度和计算浓度、琼脂糖凝胶电泳分析及PCR扩增,结果表明,3种方法均能提取到符合分子生物学的DNA,其中蛋白酶K裂解法纯度最高,含蛋白质少,改良氯化苄法、CTAB法次之。而浓度则是改良氯化苄法最高,CTAB法、蛋白酶K裂解法次之。3种方法所提取的DNA均能扩增出理想条带。     

Efficient DNA Extraction from Nail Clippings Using the Protease Solution from <Emphasis Type="Italic">Cucumis melo</Emphasis>

Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however, has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR–RFLP and Real Time PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology, medical diagnostics, and forensic science.     

Effects of DNA and urea on the specificity for H1 histone of the neutral protease B partially-purified from rat liver chromatin

A neutral protease, named protease B in the previous report (Tsurugi, K. & Ogata, K. (1982) J. Biochem. 92, 1369-1381), was partially purified from rat liver chromatin by gel filtration through Sepharose 6B followed by DE-Sephadex column chromatography. The proteolytic activity on total histones of the partially-purified protease B was increased about two fold by addition of DNA and again increased by further addition of 2 M urea. Analysis of the hydrolysed products showed that out of five species of histones, only H1 was degraded in the presence of an amount of DNA equivalent to the amount of histones, whereas core histones were also degraded in the absence or presence of one-tenth amount of DNA. Urea accelerated the selective degradation of H1 histone because H1 histone was preferentially degraded in the presence of even a low amount of DNA. In contrast, core histones became resistant to the protease B in the presence of DNA and/or urea. Heat-denatured DNA stimulated the degradation of H1 histone even in the absence of urea to almost the same extent that native DNA did in the presence of urea. Thus, protease B efficiently degrades H1 histone when its association with DNA is destabilized by either addition of urea or pretreatment of DNA with heat.     

检测CHO基因工程乙肝疫苗残余DNA方法的建立和应用

对样品的处理方法进行了探索,最后选用一个疫苗剂量的蛋白酶Ｋ同时消化样品和标准ＤＮＡ,并将消化处理后的样品和标准ＤＮＡ用地高辛标记的探针进行检测,灵敏度达１０ｐｇ。共检测２１批检品,除一批ＤＮＡ残余量超过１００ｐｇ／ｄｏｓｅ以外,其它２０批样品的ＤＮＡ残余量均低于１００ｐｇ／ｄｏｓｅ。     

DNA提取的应用与相关技术分析

为了分析影响提取DNA的有关因素,采用标准酚—氯仿抽提法和蛋白酶消化法提取DNA。结果表明,平均每mL全血可提取200 ~ 300μgDNA;新鲜组织及OCT包埋冰冻组织提取DNA,平均每0.2g组织可获得200~300μgDNA;直接将石蜡标本提取物制作模版,PCR扩增良好。从4种组织中均获得较为纯净的DNA;OCT对组织DNA无不良影响。 Abstract:To explore influence factors of DNA extraction,the protease and phenol-chloroform method was used to extract DNA in whole blood,fresh tissues,frozen tissues embedding with OCT and tissues embedding in paraffin.It results that 200~300μg DNA was extracted from 1ml whole blood or 0.2g fresh tissues or frozen tissue embedding with OCT.DNA extracted from paraffin specimen can be directly used in PCR amplification.Purity DNA can be extracted from four kinds of different tissues.OCT hasn't harmful effect on tissue DNA extraction.     

Dentilisin Activity Affects the Organization of the Outer Sheath of Treponema denticola

Prolyl-phenylalanine-specific serine protease (dentilisin) is a major extracellular protease produced by Treponema denticola. The gene, prtP, coding for the protease was recently cloned and sequenced (K. Ishihara, T. Miura, H. K. Kuramitsu, and K. Okuda, Infect. Immun. 64:5178–5186, 1996). In order to determine the role of this protease in the physiology and virulence of T. denticola, a dentilisin-deficient mutant, K1, was constructed following electroporation with a prtP-inactivated DNA fragment. No chymotrypsin-like protease activity was detected in the dentilisin-deficient mutant. In addition, the high-molecular-mass oligomeric protein characteristic of the outer sheath of the organism decreased in the mutant. Furthermore, the hydrophobicity of the mutant was decreased, and coaggregation of the mutant with Fusobacterium nucleatum was enhanced compared to that of the wild-type organism. The results obtained with a mouse abscess model system indicated that the virulence of the mutant was attenuated relative to that of the wild-type organism. These results suggest that dentilisin activity plays a major role in the structural organization of the outer sheath of T. denticola. The loss of dentilsin activity and the structural change in the outer sheath affect the pathogenicity of T. denticola.     

50-kb Chromatin Fragmentation in the Absence of Apoptosis

Treatment with ionic detergents of nuclei isolated from various continuously growing cell lines generally yields chromatin samples of high viscosity. Extensive treatment with nuclease-free proteinase K or pronase solubilized the viscous lysates with >90% of the DNA migrating at 50 kb. Freshly prepared human peripheral blood T cells also yield a substantial fraction of their DNA in an 50- to 100-kb band. The cleavage sites may coincide with a class of DNase I-hypersensitive regions, since digestion of chromatin by DNase I at 10 U/ml, without protease, also yields fragments of preferentially 50-kb size. Occasionally, the oligonucleosomal ladder was also detected together with high molecular weight degradation products. Remarkably, all of these fragmentation patterns were seen in healthy, resting or proliferating cells, i.e., in the absence of apoptosis. Tritiated thymidine incorporation could be readily detected in the 50-kb DNA fragments. The effect of an apoptotic intracellular milieu on the integrity of isolated chromatin is apparently imitated by the extensive protease treatment used in our DNA isolation protocol.     

Selection-by-function: efficient enrichment of cathepsin E inhibitors from a DNA library

A method for efficient enrichment of protease inhibitors out of a DNA library was developed by introducing SF-link technology. A two-step selection strategy was designed consisting of the initial enrichment of aptamers based on binding function while the second enrichment step was based on the inhibitory activity to a protease, cathepsin E (CE). The latter was constructed by covalently linking of a biotinylated peptide substrate to each of the ssDNA molecule contained in the preliminarily selected DNA library, generating 'SF-link'. Gradual enrichment of inhibitory DNAs was attained in the course of selection. One molecule, SFR-6-3, showed an IC(50) of around 30 nM, a K(d) of around 15 nM and high selectivity for CE. Sequence and structure analysis revealed a C-rich sequence without any guanine and possibly an i-motif structure, which must be novel to be found in in vitro-selected aptamers. SF-link technology, which is novel as the screening technology, provided a remarkable enrichment of specific protease inhibitors and has a potential to be further developed.