甜菜碱增强长片段PCR的扩增

聚合酶链式反应 (PCR)作为一项非常成熟的技术可以用于基因组序列的扩增。普通的PCR技术只适合于短片段DNA的扩增 ,一般在 6kb以下。对于 6kb至十几kb甚至几十kb以上的DNA片段的扩增就非常困难。通过添加不同化学物质 ,发现甜菜碱对长片段PCR的扩增有非常有效的增强作用。通过对玉米总DNA以及质粒DNA的扩增 ,发现 1mol L到 25mol L甜菜碱对改进PCR扩增效果明显。通过添加甜菜碱 ,可以从玉米基因组中扩增出 9kb以上的单拷贝片段 ,从质粒中扩增出 16kb以上片段。经过试验 ,发现不同GC含量的引物需要使用不同浓度的甜菜碱。甜菜碱可以减少甚至消除长片段PCR中的非特异性扩增。同时 ,我们发现其它的添加物 ,如DMSO ,甘油 ,甲酰胺对长片段PCR的作用不明显     

长片段PCR技术

长片段ＰＣＲ技术刘建伟,徐湘民（第一军医大学分子生物学研究所,广州５１０５１５）关键词长片段ＰＣＲＰＣＲ技术是１９８３年由Ｍｕｌｌｉｓ首创的一种通过体外酶促扩增反应快速获取特定ＤＮＡ序列的方法,已在分子生物学、分子遗传学、医学和法医学等领域得到广泛应...     

基于PCR的长片段DNA精确合成及应用

长片段DNA的组装及基于PCR的长片段DNA的精确合成(PAS)是基因异源表达时简单快速合成长片段DNA的方法,适于合成含有较高GC含量、重复序列和复杂二级结构的长片段DNA分子(5～6 kb)。简要综述了PAS技术用于长片段DNA序列精确合成的基本原理、操作步骤、常见问题和解决方法,以及该技术的应用。     

PCR专一性研究——PCR的二段扩增法

多聚酶链式反应（polymerasechainreaction,PCR）技术问世几年以来,已经广泛应用于分子生物学的各个领域。通过对此技术方法的改进及与其他技术配合,其应用范围更加扩大,方法更加简化。然而在进行分子生物学研究中,PCR扩增（的）产物的专一性特别重要。本文探更多还原提出了一二段扩增法,很好地解决了引物不理想和由于内切酶位点的引入带来的PCR非专一性扩增.     

大鼠肝脏脂酶基因的PCR扩增,序列分析和克隆

报导了应用聚合酶链式反应技术,扩增大鼠肝脏脂酶基因及其克隆和序列分析的结果,经３１个循环的坟增,得到大鼠肝脏脂酶及其Ｎ端结构域的ｃＤＮＡ,前为１５０８ｂｐ的片段,后为１０７６ｂｐ的片段。克隆事,对此二片估进行一限制性内切酶物理图谱分析,测定了ＤＮＡ序列,并已将它们克隆表达载体ｐＧＴ－ｄ中。     

甜菜碱改善水稻高GC含量DNA序列的PCR扩增北大核心CSCD

聚合酶链式反应(Polymerase chain reaction,PCR)作为一项基本的分子生物学技术,是DNA序列扩增的强有力工具。水稻作为人类最重要的粮食作物之一被广泛研究,然而由于水稻基因组序列的GC含量相对较高,而高GC含量DNA序列的PCR扩增一般比较困难,使得水稻高GC含量序列的功能研究受阻。通过使用不同的添加剂,发现相比于二甲基亚砜和甘油等,甜菜碱对于水稻高GC含量片段的PCR扩增的增强效果最明显。通过对多个高GC含量的水稻DNA片段进行PCR扩增,发现1-2 mol/L甜菜碱对于增强PCR扩增效果显著。此外,甜菜碱对于多种DNA聚合酶作用下的PCR扩增均有增强效果。     

两步PCR快速扩增东亚钳蝎BmKIT3 3'cDNA末端序列

对3′RACE方法进行了修改,采用一条特异性引物和一条通用OligodT引物成功地克隆了东亚钳蝎BmKIT33′cDNA末端,与常规3′RACE方法相比,两步PCR方法具有节约成本、节省时间、增加反应特异性等优点,尤其适用于生物活性肽基因的快速克隆和鉴定.     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒(CAV)环形基因组DNA(全长2.3kb)的EcoRI位点和BamHI位点的两侧选择适当序列合成两对引物,用PCR技术,从斑点杂交检测到病毒核酸的CAV感染的MDCC-RP1细胞基因组DNA中,分别扩增出包含EcoRI和BamHI分割开的病毒基因组两部分(1.5kb和0.8kb)约1.5kb和约1.25kb的两个片段.再将其中相应序列拼接克隆进pUC18载体,获得包含CAV全基因组序列DNA片段的克隆质粒pCAV2.4.酶切分析表明,该质粒具有预期的BamHI位点、PstI位点、HindⅢ位点,而预期的EcoRI位点消失.重组质粒插入DNA片段的两端序列分析表明,质粒pCAV2.4是包含CAV全基因组序列的重组质粒,插入DNA片段序列中的EcoRI位点序列发生了一个碱基突变.     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

通过PCR进行长片段DNA的合成

利用PCR合成DNA长片段(Synthesis Large Frament DNA using PCR,SLFD PCR)是一种有效的合成长片段DNA的方法。采用一段已知的500～600bp碱基的DNA片段为PCR模板,根据所要合成的DNA序列可以设计一系列的PCR引物,这些引物都位于模板DNA的5’端,长度为50～60bp,且从5’到3’方向顺序重叠,重叠碱基数目为12～15,全部引物叠加所得到的DNA正是自己所要合成的DNA。这组引物中最3’端的一条含有一个BamH Ⅰ酶切位点,在该位点后面有15碱基与模板DNA5’端一致的序列。另外还设计一条与该模板匹配的下游引物,引物内也含有一个BamH Ⅰ酶切位点。首先采用5’端最右侧的引物与下游引物进行PCR,在PCR进行10个循环后,以此次PCR的产物为下一轮PCR的模板,该轮PCR采用右侧倒数第二个引物为上游引物,下游引物保持不变。采用类似的方法,完成所有的PCR循环,就可以得到所需要合成的DNA长片段。该方法尤其适合100～200碱基左右的长片段DNA的快速合成与克隆。     

Betaine improved restriction digestion

Here we report that supplementation of a common compound betaine (1-carboxy-N,N,N-trimethylmethanaminium inner salt) enhances restriction digestion of DNA molecules being resistant to digestion despite the existence of recognition sites. A previous study reported total isostabilization of DNA was achieved in the presence of 5.2M of betaine, however, we have observed the enhancement of restriction kinetics at 0.3M of betaine, therefore, it likely provided some catalytic proficiency to restriction enzymes rather than the induction of DNA conformational changes. Betaine also enhances catalytic efficiency of PCR, and our result of restriction digestion, taken together, suggests potential application of betaine in other enzymatic reactions in an aqueous solution.     

外源甜菜碱对干旱／盐胁迫下的小麦幼苗生长和光合功能的改善

本文用甜菜碱溶液浸种,研究了外源甜碱对干旱／盐胁迫下的小麦幼苗的生长状况和叶片光合转能的影响,结果表明,外源甜菜碱能使干旱／盐胁迫下的小麦幼苗地上部和根部的干重和含水量增加,使叶片叶绿素a荧光诱导动力学参数Fv/Vo,Fv/Fm和qP增高,qN降低,说明外源甜菜碱有利于植物对光能的捕获和转换,明显促进植物生长,降低干旱／盐胁迫对植物的抑制作用。     

Oxidation of Betaine Aldehyde by Betaine Dehydrogenase from Spinach Leaves

Betaine content in leaves of fifteen plant species was determined. The results showed higher betaine levels in those salt-, drought-, and chilling-resistant species. Betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8 ) was isolated and partially purified from spinach leaves. Some properties of this enzyme were studied. BADH was precipitated by 60% saturation of (NH4)2SO4. Its activity was not detected in 70% saturation of (NH4)2SO4. BADH has two isoenzymes. The activity of BADH was quite stable below –80℃. It was inhibited by 0.125–1.0 mol/L NaG1 or KC1 but not by Mn2+ and Mo6+, and slightly increased by Mg2+.     

Betaine lipids

Diacylglyceryltrimethylhomoserine (DGTS) and diacylglyceryl hydroxymethyltrimethyl-β-alanine (DGTA) belong to a new type of glycerolipids called betaine lipids, which are composed of diacylglycerol andN-permethylated hydroxyamino acids, that are linked by an ether linkage. Betaine lipids are widely distributed in lower plants and algae as well as in some non-photosynthetic microorganisms. They are no longer regarded as “unusual” lipids but are important constituents of membranes of lower organisms. The present state of knowledge of the phylogenetic distribution, the fatty acid composition, the thermal properties, and the biosynthesis of the bataine lipids is briefly summarized in this review in a perspective of a future search for the biological roles and activities of betaine lipids. Recipient of the Botanical Society Award of Young Scientists, 1991.     

Cloning and Expression of a Betaine/GABA Transporter from Human Brain

Abstract: A cDNA clone encoding a human γ-aminobutyric acid (GABA) transporter has been isolated from a brain cDNA library, and its functional properties have been examined in mammalian cells. The nucleotide sequence predicts a transporter with 614 amino acids and 12 putative transmembrane domains. The highest degree of amino acid identity is with a betaine/GABA transporter originally cloned from the dog termed BGT-1 (91%) and a related transporter from mouse brain (87%). These identities are similar to those for species homologues of other neurotransmitter transporters and suggest that the new clone represents the human homologue of BGT-1. The transporter displays high affinity for GABA (IC₅₀ of 30 µM) and is also sensitive to phloretin, l -2,4-diaminobutyric acid, and hypotaurine (IC₅₀ values of ～150–400 µM). The osmolyte betaine is ～25-fold weaker than GABA, displaying an IC₅₀ of ～1 mM. The relative potencies of these inhibitors at human BGT-1 differ from those of mouse and dog BGT-1. Northern blot analysis reveals that BGT-1 mRNA is widely distributed throughout the human brain. The cloning of the human homologue of BGT-1 will further our understanding of the roles of GABA and betaine in neural function.     

Betaine Improves Freezing Tolerance in Wheat

The accumulation of the osmolyte betaine was found to be correlatedwith the development of freezing tolerance (FT) of two wheatcultivars where it increases by about three fold during thecold acclimation period. Exogenous betaine application resultedin a large increase in total osmolality mostly due to betaineaccumulation. Plants that accumulated betaine are more tolerantto freezing stress since a four day exposure to 250 mM betaineresulted in a LT₅₀ of –8°C (in spring wheat Glenlea)and –9°C (in winter wheat Fredrick) compared to –3°C(Glenlea) and –4°C (Fredrick) for control non-exposedplants. Betaine treatment (250 mM) during cold acclimation increasedFT in an additive manner since the LT₅₀ reached –14°C(Glenlea) and –22°C (Fredrick) compared to –8°C(Glenlea) and –16°C (Fredrick) for plants that arecold acclimated in the absence of betaine. These results showthat betaine treatment can improve FT by more than 5°C inboth non-acclimated and cold-acclimated plants. The betainetreatment resulted in the induction of a subset of low temperatureresponsive genes, such as the wcor410, and wcor413, that arealso induced by salinity or drought stresses. In addition tothese genetic responses, betaine treatment was also able toimprove the tolerance to photoin-hibition of PSII and the steady-stateyield of electron transport over PSII in a manner that mimickedcold-acclimated plants. These data also suggest that betaineimproves FT by eliciting some of the genetic and physiologicalresponses associated with cold acclimation. (Received April 23, 1998; Accepted September 4, 1998)     

中间偃麦草单条染色体分离及体外扩增

本研究利用微细玻璃针法从减数分裂中期I的花粉母细胞中分离回收了携带小麦抗黄矮病基因的中间偃麦草染色体2Ai－2。将目标染色体放入装有蛋白酶K消化液的0.5ml小离心管中,用Sau3AI酶切,在DNA片段的末端连接接头后,进行 PCR扩增。扩增产物经琼脂糖凝胶电泳分析,DNA片段的大小在150～3000bp之间,而大部分集中在200～1500bp之间。 Abstract:A simple method was used to adapt refined needles for the collection of Th.intermedium 2Ai－2 chromosome carrying BYDV (barley yellow dwarf virus) resistance gene from meiosis－metaphase spreads .The aimed chromosome was put into a 0.5ml Eppendorf tube,deproteinized with proteinase K,digested with Sau3AI,and link－adaptors were ligated to the ends of the DNA fragments.After amplification by PCR,size distributions of the PCR products were analyzed by agarose gel electrophoresis and smears of DNA were revealed that the size ranged from 150bp to 3000bp with predominant fragments at about 200～1500bp.     

Betaine Significantly Improves Multiplex Tetra-Primer ARMS-PCR Methods

The tetra-primer ARMS-PCR method offers significant advantages over the commonly used methods to genotype single nucleotide polymorphisms. It offers fast and cost-effective detection and requires minimum level of expertise and basic instrumentation. The benefits of TP-ARMS-PCR increase exponentially upon multiplexing. However, several complications preclude the common use of multiplex TP-ARMS-PCR methods, primarily the lack of robustness and the difficulty of optimization. We have previously developed triplex and quadruplex TP-ARMS-PCR methods involving the simultaneous detection of up to three SNPs in a single reaction and utilized Betaine, a PCR additive used to enable amplification of GC-rich templates with strong secondary structures, in an attempt to facilitate method development and optimization. In the present communication, we introduced experimental data demonstrating the important effects of Betaine on our previous methods and its potential to overcome the ruggedness and robustness issues commonly found in TP-ARMS-PCR methods, and highlighted the general benefits of Betaine with respect to TP-ARMS-PCR. Our data support the routine inclusion of Betaine in all TP-ARMS-PCR methods, especially when multiplexing is concerned.     

Protective Effect of Betaine on Respiratory Enzymes

Effects of betaine and NaC1 in various concentrations on the activities of enzymes in tricarboxylic acid cycle (isocitric dehydrogenase, malic dehydrogenase, succinic dehydrogenase and fumarase), terminal oxidation (cytochrome oxidase) and photorespiratory pathway (glycolate oxidase and hydroxypyruvate reductase) have been studied. Betaine, in contrast to electolyte NaC1 was non-inhibitory to these enzymes up to 500 mmol/L. Partial protection against NaC1 inhibition to the activities of these enzymes were afforded by betaine. These results were consistent with the postulated role of betaine in cytoplasmic osmoregulation. These results showed that betaine was a superior compatible solute.     

Betaine activates a hyperpolarizing chloride conductance in squid olfactory receptor neurons

Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl⁻ and could be reversibly blocked by the Cl⁻ channel blockers 4-acetamido-4′-isothio-cyanatistilbene-2,2′disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl⁻]_i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl⁻ conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K⁺ or Na⁺ ruled out the possibility that the Cl⁻ conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca²⁺ imaging experiments showed that betaine did not affect [Ca²⁺]_i, suggesting that the Cl⁻ current is not Ca²⁺ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons. Accepted: 20 March 1998