利用Taq DNA聚合酶对PCR产物直接进行顺序分析

Taq DNA聚合酶具有反应速度快、温度作用范围广及良好的续进性等特点,可视为一种理想的DNA顺序分析酶。本文首先对非对称性PCR扩增过程中单、双链DNA产物的积累情况进行了分析,然后采用标记延伸二步法,对Taq DNA聚合酶的性质及影响因素进行分析。为进一步改进Taq DNA聚合酶测序的方法,本反应建立了“Klenow-型”的直接掺入标记同位素测序法,即在反应液中加入与标记核苷酸相应的一定浓度的冷dNTP。此法不但解决了二步法中引物后部分DNA顺序无法读出的缺点,而且简化了反应步骤,亦能得到令人满意的顺序分析结果,每次可读出至少400碱基的序列。     

一种克隆PCR产物的新方法

一种克隆ＰＣＲ产物的新方法周复春,李学伍,吴斌,陈焕春（华中农业大学牧医系,武昌）基因克隆和分离是分子生物学和细胞生物学研究必不可少的组成部分。ＰＣＲ基因扩增可以产生微克级的特异性刀ＮＡ片段,可以简化从基因组ＤＮＡ克隆单拷贝基因片段的步骤。到达这种数...     

PCR产物克隆方法的新进展

ＰＣＲ产物克隆方法的新进展陈尚武,马涧泉（中山医科大学生化教研室．广州）随着ＰＣＲ技术的发展和普及,克隆ＰＣＲ产物已广泛应用于分子生物学的各个领域。通常ＰＣＲ产物不具有粘端,只能进行平端连接的克隆,由于ＴａｑＤＮＡ聚合酶具有非模板依赖性末端转移酶活性...     

PCR产物直接测序技术中影响因素的研究

探讨了PCR产物直接测序技术中的影响因素,结果表明：PCR产物特异性是影响其测序成败的关键因素,PCR反应只有产生惟一扩增产物时,其产物才能被用来直接测序;PCR反应体系残留混合物（dNTP、引物和盐离子等）对其测序质量有明显不利影响,PCR产物纯化后其测序质量能明显提高;同时,PCR产物大小不同,其测序反应的模板用量也不同,在一定长度范围内,最适模板用量随PCR产物长度增加而增加。 Factors that Influence Direct Sequencing of PCR Products XU Zu-yuan1,2,BAO Qi-yu1,NIU Yu-xin1 1.Beijing Genomics Institute / Human Genome Center,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China; 2.Jingzhou Teachers College,Jingzhou,Hubei 434104,China Abstract:Factors influenced direct sequencing of PCR (polymerase chain reaction) products were investigated in this paper.It showed that the specialization of PCR products played a key role in their sequencing reactions and only which could be sequenced directly.It also showed that the PCR reaction residues (including dNTP,primers,and metal ion) affected badly on the sequencing quality,so the purification of PCR products was necessary before sequencing.In addition,the optimum templates amount in sequencing reaction rose with the increasing of their DNA size in a certain range. Key words：polymerase chain reaction(PCR); direct sequencing of PCR product; ABI 377-DNA sequencer; Q20     

一种有效回收短片段PCR产物的方法

目的:为了有效地回收小于200bp的短片段PCR产物。方法:研究了在-20℃条件下,用无水乙醇和3mol/l醋酸钠共沉淀短片段的PCR产物的方法。结果和结论:用这种共沉淀PCR产物的方法,它能够有效地回收小于200bp以下的DNA片段,并且回收到的片段能够有效的进行酶切反应和T-载体连接反应。这种方法也能够回收酶切后的短DNA片段,并对后来的连接反应没有影响。这种共沉淀回收短片段DNA方法相对于其他方法来不仅具有可行性,而且有经济和操作简单的优点。     

耐热DNA聚合酶基因的克隆及在大肠杆菌中的表达

用PCR法从水生栖热菌菌株YT－1中扩增耐热DNA聚合酶基因,得到2．5kb的DNA片段t扩增片段重组到pUCl8中测序证实为Taq DNA聚合酶基因,将该片段重组到pBV221温控表达质粒中,在大肠杆菌中表达出94kDa的重组蛋白,100ml培养物的细胞产酶为1．5×10⁵u,表达的蛋白能催化PCR反应的进行。     

快速可靠的双链PCR产物直接测序法

利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果．     

一种用于PCR模板制备的电泳产物简易回收方法

为了探索一种简便、有效而且能从琼脂糖凝胶中大量回收用于第2次PCR扩增的DNA电泳条带的方法,采用刀片切胶法和牙签插胶法从琼脂糖中回收DNA,并进行了两种方法的比较．结果显示牙签插胶法回收的DNA用作第2次PCR的模板,获得了清晰、稳定的PCR产物电泳条带,用该法成功地制备了一批DNA微阵列探针．由此可见牙签插胶法是一种简便、快速、有效的用于PCR模板的DNA琼脂糖凝胶回收法．     

DNA ligases from thermophilic bacteria enhance PCR amplification of long DNA sequences

Bacterial NAD-dependent Taq and Tth DNA ligases are capable of significantly increasing the yield of long PCR products when the amplification is carried out using bacterial family A DNA polymerases, e.g. Taq or Tth DNA polymerases, or with enzymatic blends containing these polymerases. We also show that Taq and Tth DNA ligases improve the results of PCR in the absence of NAD and therefore in the absence of DNA ligase activity. These observations suggest that bacterial DNA ligases can interact with these DNA polymerases, presumably as accessory proteins, thereby enhancing the efficiency of DNA polymerization. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 5, pp. 685–690.     

A New Approach to Enhanced PCR Specificity

A new approach to enhanced specificity and product yield of polymerase chain reaction is proposed. It is based on control of DNA polymerase activity during PCR by changing the magnesium ion concentration, which depends on the temperature of the reaction mixture. A slightly soluble magnesium salt, magnesium oxalate, whose solubility depends on temperature, was used as a source of magnesium ions. During PCR, magnesium oxalate was maintained at saturating concentration by the presence of an insoluble excess of this salt, and the concentration of magnesium ions depended on the salt solubility: binding of magnesium ions at lower temperatures and their release at higher temperatures was shown to affect the DNA polymerase activity and to favor the specific PCR amplification of the target DNA fragment.     

普通Taq DNA聚合酶扩增幽门螺杆菌尿素酶基因长片段

ＰＣＲ反应扩增较长的ＤＮＡ片段比较困难。本实验对幽门螺杆菌染色体ＤＮＡ上的２７Ｋｂ尿素酶基因用普通ＴａｑＤＮＡ聚合酶进行扩增,扩增结果以琼脂糖凝胶电泳证实,得到所需的２７Ｋｂ的片段。该实验为今后幽门螺杆菌工作的进一步研究提供了经济,有效,简捷的条件。     

Thermally reversible inactivation of Taq polymerase in an organic solvent for application in hot start PCR

In the past, Taq polymerase was reversibly inactivated by modification with a dicarboxylic acid anhydride in aqueous media, to enable ‘hot start PCR’. However, there are various constraints in using such a method including temperature and concentration. Here we describe an alternative method whereby Taq polymerase may be reversibly inactivated following incubation with an excess of citraconic anhydride at elevated temperatures, in an anhydrous non-protic organic solvent – tert-butyl methyl ether – by first drying the enzyme with a salt or carbohydrate excipient to form an amorphous powder. Reactivation of the enzyme is due to the instability of the chemical modification at low pH following a short incubation in a suitable buffer.     

Taq DNA聚合酶功能区域的定位

通过参Ｕ法定点突变产生了ＴａｑＤＮＡ聚合酶Ｎ端分别缺失３个,２３５个,２８７个和４４３个氨基酸的４个缺失体,利用Ｂａｌ－３１连续缺失法产生了ＴａｑＤＮＡ聚合酶的Ｃ端分别缺失了２个、１６个、２９个、３２个、３４个氨基酸的５个缺失体．经ＤＮＡ聚合酶活性测定表明Ｎ端缺失３个,２３５个,２８７个氨基酸后活力和完整的Ｔａｑ相近,而缺失４４３个氨基酸后则失去了ＤＮＡ聚合酶活力;Ｃ端的５个缺失体都失去了ＤＮＡ聚合酶活性．据此ＴａｑＤＮＡ聚合酶的功能区域被定位在２８７～８３２氨基酸之间．