应用引物延预扩增提高微量DNA拷贝数

采用引物延伸预扩增方法,可普遍提高微量模板ＤＮＡ的考贝数,便于进行基因分析时克服标本时少、来源困难的制约,采用常规扩增、检测２４８ｂｐ的ＤＹＺ１片段体系为观察对象,其最小模板量需１．５ｎｇ／２０μｌ体系。以１５个碱基随机寡核苷酸为引物,对最小模板量进行预扩增,再以其产和１／１０为模板,特异扩增ＤＹＺ１片段。进行了相对定量分析,判断源模板ＤＮＡ拷贝数增加的程度。结果１．５ｎｇ男性ＤＮＡ,经随机扩增后     

一种适用于昆虫痕量DNA模板制备的方法

近年来分子生物学技术在昆虫学各领域中得到了广泛地运用 ,从昆虫样品中有效地获得DNA模板是实验成功的前提。但是由于许多昆虫体形微小 ,许多研究需要取单个个体的样品 ,用传统的酚∶氯仿抽提法难以从痕量样品中获得总DNA ,而某些生物公司的试剂盒相对而言价格昂贵。该文介绍一种快速简便、广泛适用于不同种类昆虫、各种不同保存方法保存的昆虫样品和标本的微量DNA模板制备方法     

一种简单、快速提取DNA的方法

随着分子生物学研究的迅速发展,提取ＤＮＡ已成为一项常规实验。用经典方法提取ＤＮＡ［１］,先用蛋白酶Ｋ、ＳＤＳ消化,然后用酚、氯仿抽提,乙醇沉淀,耗时较多,提取液需要多次转移,易引起交叉污染和ＤＮＡ丢失。本文利用硅藻（ｄｉａｔｏｍ）能够特异性吸附核酸的...     

PCR制备地高辛素标记的探针检测轮状病毒核酸

用聚合酶链反应（ＰＣＲ）技术,制备了轮状病毒第９基因部分片段的地高辛标记的ｃＤＮＡ探针。ＤＮＡ－ＲＮＡ斑点杂交表明该探针具有轮状病毒Ａ组的特异性,可检出１０ｐｇＨＲＶ－ＲＮＡ。实验中选择了粪便标本提取核酸后点膜和粪便上清直接点膜进行斑点杂交,两种方法的结果一致;同时将斑点杂交法与ＰＡＧＥ法的结果进行了比较。实验结果表明用ＰＣＲ技术直接制备地高辛索标记的ｃＤＮＡ探针具有方便、快速、标记率高、特异性强的特点,具有一定的应用前景。     

影响荧光终止法PCR循环测序反应的因素

比较了一些影响荧光终止法ＰＣＲ循环测序反应的因素。实验结果显示在Ｂｅｃｋｍａｎ ＣＥＱ２０００自动测序仪上,可读序列长度随着ｐＵＣ１８模板量增加而逐渐增多,当模板量达到１２５ｎｇ时ＤＮＡ可读序列最长,以后随着ｐＵＣ１８量增加测序长度逐渐下降。当引物量是１μｌ时,其测序结果比用０．５μｌ引物时好。在同样模板量情况下,１０μｌ反应体积比５μｌ反应体积可读序列长。     

应用引物延伸预扩增提高微量DNA拷贝数

采用引物延伸预扩增方法 ,可普遍提高微量模板DNA的拷贝数 ,便于进行基因分析时克服标本量少、来源困难的制约。采用常规扩增、检测 2 4 8bp的DYZ1片段体系为观察对象 ,其最小模板量需 1.5ng/2 0 μl体系。以 15个碱基随机寡核苷酸为引物 ,对最小模板量进行预扩增 ,再以其产物 1/10为模板 ,特异扩增DYZ1片段。进行相对定量分析 ,判断原模板DNA拷贝数增加的程度。结果 1.5ng男性DNA经随机扩增后 ,此DYZ1片段拷贝数增加了 10倍以上 ,大大地提高了特异DNA片段扩增的模板量。表明经随机引物延伸预扩增后 ,微量标本DNA片段拷贝数获得普遍提高 ,增加了微量DNA扩增的敏感度     

地高辛标记核酸探针斑点杂交法检测轮状病毒疫苗中残余MA－104细胞DNA含量的研究

本文报导用地高辛标记核酸探针和分子斑点杂交技术检测轮状病毒基因重配株Ｌ－３株活疫苗中残余ＭＡ－１０４细胞ＤＮＡ含量的方法。提取和纯化ＭＡ－１０４细胞ＤＮＡ,将其ＡｌｕＩ酶切片段用随机引物法引导ＤＮＡ标记为探针。待检样品抽提核酸后点膜进行斑点杂交。此法灵敏度高,可检出０．１４ｐｇ的ＤＮＡ,特异性强,与非同源性ＤＮＡ无杂交。用此法检测轮状病毒基因重配株Ｌ－３株制备的疫苗,ＭＡ－１０４细胞残余ＤＮＡ含量低于１４ｐｇ低于ＷＨＯ限量标准,结果表明此重配株用于研制疫苗是安全的。     

PCR制备地高辛素标记的探针检测轮伏病毒核酸

用聚合酶链反应（ＰＣＲ）技术,制备了轮状病毒第９基因部分片段的地高辛标记的ｃＤＮＡ探针。ＤＮＡ－ＲＮＡ斑点杂交表明该探针具有轮状病毒Ａ组的特异性,可检出１０ｐｇＨＲＶ－ＲＮＡ。实验中选择了粪便标本提取核酸后点膜和粪便上清直接点膜进行斑点杂交,两种方法的结果一致;、同时将斑点杂交法与ＰＡＧＥ法的结果进行了比较。实验结果表明用ＰＣＲ技术直接制备地高辛素标记的ｃＤＮＡ探针具有方便、快速、标记率高、特异性     

乙醇保存的动物标本基因组DNA提取方法的比较

为提高从乙醇长期保存的动物标本中提取大分子DNA的质量,用5种不同方法对动物组织进行预处理实验,然后采用SDS／蛋白酶K裂解,酚一氯仿抽提和乙醇沉淀提取总DNA,通过0．8％琼脂糖凝胶对模板进行电泳和PCR产物作鉴定,经比较,用0．9％NaCL法、PBS法和混合液法进行预处理,消除乙醇对Taq酶的影响以及蛋白质和核酸交联问题,为提取动物基因组DNA的3种更理想方法。     

一种鉴别差异表达基因的新方法：———cDNA示差分析技术

ｃＤＮＡ示差分析技术是继ｍＲＮＡ差异显示技术之后又一种鉴别差异表达基因的方法。该技术利用双链ＤＮＡ模板在ＰＣＲ时呈指数扩增,而单链ＤＮＡ模板为线性扩增原原理,先用常见的限制性内切酶将实验组与对照组消化成平均长度为２５６ｂｐ的ｃＤＮＡ片段,再用ＰＣＲ技术使用组的ｃＤＮＡ片段富集,随后进行３次差减杂交去除共有基因,最后扩增实验组中的特异表达的基因。本概述了该技术的原理、基本方法及其应用前景。     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Principles of organization and evolution of systems of regulation of functions

Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms. The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization followed the way of development of delivery pathways of chemical signal and development of mechanisms of its regulation. The mechanism of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning of physiological systems and organs of the living organism