一种快速、简便、高效cDNA合成及克隆策略

以鱼呼肠孤病毒双链ＲＮＡ为模板,建立一种快速、简便、高效的ｃＤＮＡ合成及克隆策略。在一定量模板条件下,采用随机六聚体为引物合成ｃＤＮＡ第一链。以退火方式形成双链ｃＤＮＡ,直接通过琼脂糖凝胶电泳检测其ｃＤＮＡ合成产量。双链ｃＤＮＡ经两端补齐后,平头连接于具有阳性选择标记的载体中,以高效电转化的方式进行快速克隆。     

一种纯化双链DNA测序模板的有效方法

以变性质粒ＤＮＡ为模板的核苷酸序列分析（又称双链ＤＮＡ测序）的结果会受到诸多因素的影响。然而,模板的制备及其纯度显得尤为重要。本试验对以三种方法纯化的质粒为模板的测序结果作了比较,结果显示．能为普通实验室所采纳的二氧化硅吸附法操作上简单、重复性好,其结果可与Ｍ１３顺序分析系统相媲美。     

粘虫核型多角体病毒919bp片段的PCR双链直接序列测定

本文发展了ＰＣＲ克隆和亚克隆技术制备ＤＮＡ测序模板。首先,我们用ｐＵＣ／Ｍ１３系列质粒的通用正反向引物ＰＣＲ扩增出质粒ｐＢｌｕｅｓｃｒｉｐｔＫＳＤＮＡ的多克隆位点及其侧翼序列,用ＥｃｏＲＶ和ＸｈｏＩ消化成为左右两个引物多克隆臂,与粘虫核型多角体病毒（ＬｓＮＰＶ）的ＥｃｏＲＶ和ＸｈｏＩ约４００ｂｐ和５００ｂｐ片段分别连接,经ＰＣＲ扩增,得到两端具有上述正反向引物结合位点的测序模板,用ｄｄＮＴＰ链终止法／ＰＣＲ扩增／银染色,从片段两端测定了全部９１９ｂｐ序列,这种ｄｄＮＴＰ／ＰＣＲ／银染测序法简化了操作,大大缩短了测序模板的制备时间,易于实现自动化操作。     

用温度循环法测定扩增DNA的序列比较汉坦病毒同型间的核酸序列

用温度循环法测定扩增ＤＮＡ的序列比较汉坦病毒同型间的核酸序列作者应用温度循环测序法对汉坦病毒血清型特异性引物扩增所得的ＰＣＲ产物进行序列测定以确定同一血清型几株病毒间序列的相似水平。测序用模板为上游引物和下游引物之间,测得的２８２,２６４和２９１个碱...     

粘虫核型多角体病毒919bp片段的PCR双链直接序列测定

本文发展了ＰＣＲ克隆和亚克隆技术制备ＤＮＡ测序模板。首先,我们用ｐＵＣ／Ｍ１３系列质粒的通用正反向引物ＰＣＲ扩增出质粒ｐＢｌｕｅｓｃｒｉｐｔＫＳ ＤＮＡ的多克隆位点及其侧冀序列,用ＥｃｏＲＶ和ＸｈｏＩ消化成为左右两个引物多克隆臂,与粘虫核多角体病毒（ＬｓＮＰＶ）的ＥｏｏＲＶ和ＸｈｏＩ约４００ｂｐ和５００ｂｐ片段分别连接,经ＰＣＲ扩增,得到两端具有上述正反向引物结合位点的测序模板,用ｄｄＮＴＰ链终点     

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。     

中国人雄激素受体N端转示激活区的测序及突变检测

雄激素受体（ａｎｄｒｏｇｅｎ ｒｅｃｅｐｔｏｒ,ＡＲ）Ｎ端转录激活功能区（ＡＦ１）是ＡＲ发挥转录激活所必需的。用４对引物（Ａ３－Ａ４）ＰＣＲ扩增２０例中国正常男性ＡＲ的ＡＦ１,双链ＤＮＡ循环测序以确定正常中国人ＡＲ的ＡＦ１的核苷酸顺序。在此基础上,用ＰＣＲ－ＳＳＣＰ分析和双链ＤＮＡ循环测序法对２例雄激素抵抗征（ＡＩＳ）患者外周血白细胞和１５例前列腺癌（ＰＣ）患者癌组织中ＡＲ的ＡＦ１区进行突变检测。     

彻底清除转化体中野生型DNA的专一位点诱变方法

我们设计了一种高效的专一位点诱变方法,可从混合体中清除野生型ＤＮＡ。并将所需突变ＤＮＡ转入被转化细胞中,如此合成了与一种ＤＮＡ分子目标序列一致的并带有能产生限制性内切酶位点的插入突变的两条互补的寡核苷酸链。利用野生型ＤＮＡ分子中的两端各有两个限制性位点的一段目标ＤＮＡ片段作为模板,上述合成的两种寡核苷酸引物被延伸、富集并分离。被延伸的产物在聚合酶链式反应中又反过来被用作模板,以获得突变的双链ＤＮＡ片段,此片段通过其两端的限制性位点能很方便地用侧面限制性内切位点克隆到质粒中去。用这些质粒转化的大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）细胞可进行大量的分析。通过集落杂交试验、限制性内切酶分析和ＤＮＡ序列分析,得知这些转化体百分之百含有突变型的ＤＮＡ序列。     

紫外线为何能杀死病菌？

紫外线为何能杀死病菌？答：紫外线照射引起病菌细胞的核酸ＤＮＡ链的磷酸二脂键和氢键断裂,胸腺嘧啶（Ｔ）与胞嘧啶（Ｃ）的水合作用以及胸腺嘧啶的二聚体（ＴＴ）形成,这样便引起ＤＮＡ双链结构扭曲变形,阻碍碱基间的正常配对,破坏ＤＮＡ模板的正常功能,影响双链的...     

利用PCR技术行反义寡核苷酸体外抗丁型肝炎病毒基因组RNA中核酶的研究

设计一对ＰＣＲ引物,其中上游引物的５’端除目的基因外,还加Ｔ７ＲＮＡ聚合酶启动子序列,以质粒（ｐＳＶＬＤ３）为模板,通过ＰＣＲ扩增出带有Ｔ７ＲＮＡ聚合酶启动子序列的１３９ｂｐ的ｃＤＮＡ片段,它含有丁型肝炎病毒（ＨＤＶ）基因组ＲＮＡ中核酶（Ｒｉｂｏｚｙｍｅ）区的ｃＤＮＡ该核酶具有自身裂解功能,经测序发现该ｃＤＮＡ有２个碱基变异,以此ＰＣＲ产物为模板,通过Ｔ７ＲＮＡ聚合酶,转录出核酶的前体,并观察到其     

Maximizing specificity and yield of PCR by the quantum dot itself rather than property of the quantum dot surface

We found that semiconductor quantum dots (QDs) dramatically improved both product yield and specificity of PCR. The concentration of QDs is important for improving PCR amplification. In the presence of appropriate concentration of mercaptoacetic acid (MAA)-coated QDs, specificity and yield of PCR were enhanced. Also, strong nonspecific bands and weaker smeared bands were eliminated. At lower annealing temperatures (25–45 °C), addition of MAA-coated QDs into the PCR reagent produced specific PCR products without nonspecific sequence amplification. MAA alone did not improve PCR amplification. Streptavidin (SA) surface modified QDs with different size also effectively improved the specificity of PCR, demonstrating that the observed effect was not due to property of the QD surface but instead due to the QD itself. Bovine Serum Albumin (BSA) could relieve Taq polymerase from MAA-coated QDs in PCR by interaction with QDs and therefore imply that QDs improve specificity of PCR by interaction with Taq polymerase. These results demonstrate that QDs, added to reaction mixes at appropriate concentrations, can increase PCR yield and improve PCR specificity, even at low annealing temperatures. We assume that many different surface modified polymeric nanoparticles might have similar effects.     

Gene walking using sequential hybrid primer polymerase chain reaction

We developed a simple and robust method for removing nonspecific amplification produced during gene walking with a gene-specific primer and a degenerate primer. The primary walking polymerase chain reaction (PCR) was followed by two or three PCR rounds, each incorporating a low concentration of a reverse hybrid primer, where the 3′ end was bound to a target sequence generated in the preceding PCR round and the 5′ end was a new sequence that generated a target sequence for the next PCR round. The low concentration of the hybrid primer and the extent of amplicon stem-loop formation inhibited nonspecific amplification and enabled successful walking along three genes.     

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.     

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.     

Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis

Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products.     

改进的反向PCR技术克隆转移基因的旁侧序列

对传统的反向PCR技术作了一些改进：用巢式PCR扩增含量极少的靶序列;PCR反应体系中加入5%的甲酰胺以减少非特异性扩增.结果表明,改进的反向PCR体系是克隆人基因组已知片段旁侧序列的高度灵敏、高度特异的方法.     

An abnormally long HIV-1 env DNA PCR product due to altered sequences of primer binding sites

To investigate the generation of an abnormally long HIV-1 env PCR DNA product the latter was cloned and sequenced followed by sequence analysis of HIV-1 primer binding sites. We found that the formation of an abnormally long PCR product was due to HIV-1 env sequence alteration (a) in the reverse primer binding site resulting in faulty primer binding and (b) downstream from the forward primer sequence resulting in a new binding site with reverse complementary sequence with respect to the forward primer at the opposite end of the PCR product. Both changes led to amplification of a longer PCR product with forward primer alone. Our results indicate that the HIV-1 genetic diversity in the env gene can lead to amplification of a specific PCR product of unexpected size which can be disregarded in the absence of its cross-validation.     

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.