实验11 哺乳类基因组基因文库的构建

一、哺乳类DNA部分酶切片段的回收 1.选定所用的限制性内切酶。将待酶切的哺乳类细胞DNA分别装入6—10个Eppendorf离心管。 2.将限制性内切酶加入装有DNA的离心管。酶量逐级递减,例如1单位/微克DNA,0.5单位/微克DNA,……,37℃保温3—5小时。 3.或将相同的酶量加入装有DNA的离心管,37℃保温。经不同时间终止酶切反应。例如,保温10分钟,20分钟……。     

大鳞副泥鳅线粒体DNA九种限制性内切酶酶切图谱

大鳞副泥鳅ｍｔＤＮＡ经１１种限制性内切酶（ＢａｍＨＩ,ＢｇｌＩ,ＢｇｌⅡ,ＥｃｏＲＩ,ＨｐａＩ,ＫｐｎＩ,ＰｓｔＩ,ＳａｃＩ,ＳａｌＩ,ＸｂａＩ和ＸｈｏＩ）单酶完全酶解获得２３个酶切位点,这些酶酶切位点数依次分别是：２、７、０、３、３、０、３、１、１、２和１。通过琼脂糖凝胶电泳测定大鳞副泥鳅ｍｔＤＮＡ平均分子量为１０．３３±０．２２×１０６ｕ（原子质量）;其分子长约１６７２０±３５０碱基对（ｂｐ）。采用双酶完全酶解法构建了大鳞副泥鳅ｍｔＤＮＡ限制性内切酶酶切图谱。     

鸡肝脏线粒体DNA的限制图谱

本文用六种限制性内切酶对鸡肝脏线粒体DNA(mtDNA)进行了酶解。Eco RⅠ、Bam HⅠ、SalⅠ、HindⅢ、BglⅠ在鸡肝mtDNA上分别有2、2、3、4、4个切点,BglⅡ不能切割鸡肝mtDNA。根据鸡肝mtDNA的单酶、双酶完全酶解以及部分酶解片段的分子量,建立了鸡肝mtDNA的限制图谱。     

铜绿假单胞菌PIC－N萘降解基因的研究

铜绿假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓａｅｒｕｇｉｎｏｓａ）ＰＩＣ－Ｎ对萘、邻苯二甲酸、水杨酸等有较强的氧化能力。发现该菌株以禁为底物可诱导产生芳香烃分解酶系。菌株中存在一个５７．４ｋｂ的质粒,经限制性内切酶ＨｉｎｄⅢ处理可产生７个片段,用限制性内切酶ＥｃｏＲⅠ处理可产生８个片段。将以限制性内切酶ＨｉｎｄⅢ部分酶切的片段克隆至大肠杆菌质粒ｐＭＦＹ４３上,获得２９个克隆株。通过对含有菌株ＰＩＣ－Ｎ质粒ＨｉｎｄⅢ片段的７个重组质粒进行限制酶分析,绘出了该质粒ＨｉｎｄⅢ内切酶７个切点的酶切图谱。     

实时检测限制性内切酶活性的发夹型荧光探针

基于分子信标的原理 ,设计了一种发夹型荧光探针作为限制性内切酶作用的专一底物 ,来研究限制性内切酶的剪切作用。检测BglII和NcoI表明 ,这种探针不仅可以灵敏、实时地指示内切酶的活性 ,采用不同荧光标记的探针还可以同时特异地显示双酶切反应中两个酶各自的活性。并且以Rotor Gene 2 0 0 0实时荧光PCR仪作仪器检测 ,实现了高通量的操作。利用其特有的作变温曲线的功能 ,能很好的区分限制性内切酶与发夹型荧光探针的特异剪切与非特异的结合     

分子生物学技术讲座（二）——分子克隆中所用的酶

限制性内切酶及其它DNA与RNA修饰酶是分子克隆技术的基本工具。1限制性内切醉和DNA甲基化酶限制性内切酶能特异地结合于一段被称为限制性内切酶识别序列的特定的DNA序列或附近的序列,并在此切割DNA双链。它可以分成3类：Ⅰ类和Ⅲ类在同一蛋白分子中兼有修饰（甲基化）作用及依赖于ATP的限制（切割）活性。Ⅰ类酶结合于识别序列,但随机切割DNA;Ⅲ类酶能在识别序列位点切割DNA。在分子克隆中1类和皿类酶都不常用。D类限制一修饰系统限制性内切醇和修饰（甲基化）酶不在同一蛋白分子中,限制性内切酶能专一地切割识别序列,并不依…     

西藏小型猪SLA-DQA基因外显子2的PCR-RFLP多态性分析

目的分析西藏小型猪SLA-DQA基因第2外显子不同酶切位点的基因型多态性以及等位基因的多态性,检验这些酶切位点上的基因频率是否达到Hardy-Weiberg平衡态。方法采用EcoRⅠ和AluⅠ两种限制性内切酶对西藏小型猪SLA-DQA目的基因的第1和第2内含子部分序列以及完整的外显子2进行PCR-RFLP分析。结果经EcoRⅠ酶切后,以纯合子BB基因型居多,BB、AB、AA基因型频率分布为45.000%、31.667%和23.333%,其中B为优势等位基因（60.833%）;经AluⅠ酶切后,MN基因型频率（50.000%）分别高于MM型（30.000%）和NN型（20.000%）,     

分子信标检测限制性内切酶活性的新型荧光分析方法

利用分子信标被切割后荧光信号的变化.发展了一种简便的检测限制性内切酶活性的新型荧光分析方法.以限制性内切酶Xsp I为例.将其识别位点嵌入具有茎环结构的分子信标探针(Molecular Beacon,MB)的环状部分,利用这种分子信标在溶液中形成的瞬时二聚体结构,实现了限制性内切酶对分子信标的切割.在优化的条件下,反应初速度与酶的浓度成正比,线性范围为0.05～50 U/mL,检测限为0.05 U/mL.通过改变分子信标环部的识别位点的序列.还可以检测其他限制性内切酶如Alu I的活性.     

一种测定癌基因c-Ha-ras点突变的简易方法——PCR-RFLP法

利用多聚酶DNA链延伸反应与限制性内切酶酶解片段长度多态性分析相结合,可简单、迅速、准确地对胃癌组织及细胞株DNA中癌基因c-Ha-ras第12位密码子是否存在点突变进行测定。这个方法是使用非同位素方法对单拷贝基因点突变进行检测的首次报道。     

农药

862738降解对硫磷(一六O五)的缺陷假单胞菌质粒的大小及其酶切物理图谱〔英〕/MeDaniel,C.S.…了Abstr.Annu.Meet。Am.Soe。Mierobiol一1985,85,Meet。一12〔译自DBA,2985,4(9),85一04457」缺陷假单胞菌质粒pCS_1可用限制性内切酶酶解降解农药。该质粒编码催化降解一六O五的磷酸醋酶。     

塔拉豆荚壳中单宁的提取-微波辅助萃取法

利用微波辅助萃取技术对塔拉单宁进行提取,通过不同的起始温度、加蒸馏水量、提取次数、功率大小等实验并用1%的FeCl3溶液检测提取效果,确定较佳的提取温度、次数、加蒸馏水量和微波辅助萃取仪的功率等因素。结果表明,微波提取塔拉单宁的最佳提取条件为:每次提取加水150~200 mL,微波功率400 w,60℃提取20 min,反复提取4次;再升温至70℃提取20 min 2次,即可提取完全。原料中的单宁含量微波浸提比水浴锅浸提稍高。因此认为用此提取方法可替代水浴浸提法。     

次氯酸钠离析法观测甘蓝叶片脉序的处理条件优化筛选

次氯酸钠（NaClO）离析法主要用于植物叶片表皮的观测,在研究过程中发现该法也可用于叶片脉序的观测。以甘蓝（Brassica oleracea var.capitata）为实验材料,采用二次正交设计方法对水煮时间、NaOH浓度、NaOH处理时间、NaClO浓度、NaClO处理温度和处理时间等各种处理条件进行优化筛选,以期得到适合于甘蓝叶片脉序观测的最佳处理条件组合。实验结果表明,新鲜甘蓝叶片水煮3分钟,10%的NaOH溶液60°C水浴处理2.5小时,3%的NaClO溶液40°C水浴离析2小时,叶片脉序的观测效果最佳。     

次氯酸钠离析法观测甘蓝叶片脉序的处理条件优化筛选

次氯酸钠(NaClO)离析法主要用于植物叶片表皮的观测, 在研究过程中发现该法也可用于叶片脉序的观测。以甘蓝(Brassica oleracea var. capitata)为实验材料, 采用二次正交设计方法对水煮时间、NaOH浓度、NaOH处理时间、NaClO浓度、NaClO处理温度和处理时间等各种处理条件进行优化筛选, 以期得到适合于甘蓝叶片脉序观测的最佳处理条件组合。实验结果表明, 新鲜甘蓝叶片水煮3分钟, 10%的NaOH溶液60°C水浴处理2.5小时, 3%的NaClO溶液40°C水浴离析2小时,叶片脉序的观测效果最佳。     

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.     

Effect of repeated microwave disinfections on bonding of different commercial teeth to resin denture base

doi: 10.1111/j.1741‐2358.2011.00516.x Effect of repeated microwave disinfections on bonding of different commercial teeth to resin denture base Objective: To verify the influence of repeated microwave disinfections on the shear bond strength of two commercial types of teeth to acrylic resin, when the ridge lap surfaces were unmodified, bur abraded, bur grooved or etched by monomer. Material and methods: Eighty specimens (n = 10) were adhered to the tooth ridge lap surface, polymerised in a water bath at 74°C for 9 h. Microwaved specimens were individually immersed in 150 ml of water and submitted to five simulated disinfections in a microwave oven calibrated at 650 W for 3 min. Control specimens were not microwave treated. Shear bond strength tests were performed in an Instron machine with a cross‐speed of 1 mm/min. The fracture load values were transformed into shear bond strength as a function of the bonding area (0.28 cm²). Data were submitted to anova and Tukey’s test (α = 0.05). Fractured areas were classified as adhesive, cohesive (resin or tooth) or mixed failures. Results: Repeated microwave disinfections increased the shear strength of the tooth/resin bond. Mechanical retention in microwaved and non‐microwaved procedures improved the shear bond strength. Conclusions: The different commercial types of teeth influenced shear bond strength values, with Biotone teeth showing the lower values.     

Microwave-mediated enzymatic modifications of DNA

Here we report microwave-induced specific cleavage, ligation, dephosphorylation, and phosphorylation of nucleic acids catalyzed by restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase. The microwave-mediated method has dramatically reduced the reaction time to 20 to 50 s. In control experiments, the same reactions failed to give the desired reaction products when carried out in the same time periods but without microwave irradiation. Because the microwave method is rapid, it could be a useful alternative to the time-consuming conventional procedure for enzymatic modification of DNA.     

Destruction and injury of Escherichia coli during microwave heating under vacuum

AIMS: To study the effect of 2450 MHz microwave radiation under vacuum (vacuum microwave or VM) on survival and injury of Escherichia coli and to search for possible nonthermal effects associated with VM. METHODS AND RESULTS: Destruction kinetics of E. coli in peptone water were determined in a continuous-flow vacuum system, heated by convection heating in a water bath or with microwaves (VMs). Vacuum was used to control the boiling point of water and to maintain temperature in the bacterial suspensions at specified levels (49-64 degrees C). CONCLUSIONS: z-Value in the water bath treatment was 9.1 degrees C while for VM at 510 and 711 W it was 6.2 and 5.9 degrees C, suggesting that E. coli is more sensitive to temperature changes under microwave heating. Arrhenius calculations of the activation energies of the destruction reactions suggest that the mechanism of destruction in VM may be different from that of conventional heat. The number of injured micro-organisms showed no significant differences among treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of temperature on E. coli destruction was different when microwaves were the medium of heat transfer, suggesting the existence of factors other than heat contributing to the lethal effect of VM.     

香菇多糖微波辅助提取及体外免疫学活性研究

利用微波辅助水浸提法优化影响香菇多糖提取的单因子试验(料液比、浸提温度、浸提时间、微波处理时间等)和多因子的正交试验[L9(34)]。结果表明,香菇多糖的最佳提取条件为:料液比1∶30,浸提温度90℃,浸提时间2h,微波处理3m in。在此条件下,香菇多糖提取率为4.75%。香菇多糖粗品的体外免疫学研究的结果表明,在25~1600μg/mL的浓度范围内,多糖都具有促进小鼠脾淋巴细胞体外增殖的作用;在25~400μg/mL的浓度范围内,多糖对脾淋巴细胞增殖活性呈现一定的剂量依赖性。     

Sulfonation of polyvinylidene difluoride resin and its application in extraction of restriction enzymes from DNA digestion solutions

Sulfonation of polyvinylidene difluoride (PVDF) resin was achieved by incubation of the resin with sulfuric acid at a moderately high temperature. The sulfonated PVDF (SPVDF) resin was studied for its ability to extract restriction enzymes from DNA digestion solutions. The SPVDF resin was effective in adsorbing restriction enzymes such as EcoRI and BamHI and the extraction procedure was easy and simple to perform. The adsorption depended upon the amount of the resin added. We found that 1 mg of the SPVDF resin could completely remove all restriction enzyme activity routinely used in DNA digestion within 2 min after its addition. Treatment of a digestion solution with the SPVDF resin did not change the reaction solution and the same digestion buffer could be used for another digestion of the same DNA with other enzymes. We also found that, in comparison with normal PVDF, the SPVDF resin adsorbed less DNA, resulting in less loss of DNA in the extraction step. The potential application of the SPVDF resin in other procedures of molecular cloning and enzyme purification is discussed.     

Microwave‐assisted 18O labeling of Fmoc‐protected amino acids

Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of ¹⁸O‐labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave‐assisted procedure. This approach resulted in reduction of reaction time to 15 min and increased reaction efficiency. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.