连续PCR扩增过程中异常现象的发生机理研究

聚合酶链式反应（Polymerase Chain Reaction，PCR）技术从其发明以来，因为其操作的简单方便和高效率而在生物学研究的各个领域得到了广泛的应用，包括序列扩增、序列的人工突变、疾病诊断、法医学鉴定、基因的表达分析等等。从PCR技术发明以来，如何提高反应的特异性和反应的效率一直是人们所共同关心的题目，为此也发展了相当数量的各种方法，如热启动PCR、降落PCR、巢式PCR以及在反应体系中添加一些有益的附属物等。而适合不同目的的PCR技术也得到了充分的发展，如多重PCR、反转录PCR、定量PCR、原位PCR、PCR突变、毛细管PCR技术等等。并且，包括随机引物扩增多态、扩增片段长度多态性、简单重复序列多态性、单核苷酸多态性等这些在PCR技术基础上发展而来的各种分子标记技术极大地方便了遗传分析和遗传图谱的构建等工作。在PCR技术发明了20年后的今天，提高PCR的反应性能、发展适合新领域的PCR技术和新的分子标记技术仍然是研究者关心的题目和努力的方向。 　　PCR实验中已经观察到多种异常现象，除了常见的扩增失败（没有产物）、扩增产物特异性不强（有非特异产物出现）、引物多聚体产物扩增、扩增效率低等现象以外，还包括PCR介导的重组、跳跃、复制滑动等等。阐明这些异常现象的发生机理和过程，避免或缓解这些异常现象在扩增过程中对目的产物扩增的影响，以及促进和利用一些特殊的异常PCR扩增都是PCR技术研究所关心的话题。各种研究工作中经常需要扩增一些长片段的序列，但是在进行长片段PCR时经常会发现扩增目标序列的长度是有限的、扩增效率比较低、扩增产物检测中有很强的背景弥散等现象;同时长片段PCR需要一些特殊的反应体系组成和反应条件。如何更加有效地实现更长序列的PCR扩增也是人们所关心的话题之一。 　　常见的PCR产物重复扩增（以上一轮扩增产物为模板进行新的PCR扩增）扩增轮数少，通常仅进行一次重复扩增;同时，在重复扩增中常使用的策略是使用巢式引物。而连续PCR扩增实验（用相同的引物以产物为模板进行多轮次的连续重复PCR扩增）从未见于文献报道。我们第一次系统地进行了连续PCR扩增实验;同时，在实验过程中我们观察到了一种新的PCR扩增异常现象——用不同来源的模板（病毒、细菌质粒或真核生物来源的DNA序列）进行连续PCR扩增不同长度的靶序列，经过有限次数的重复扩增后，最终都会导致扩增失败;这种扩增失败都表现为在常规琼脂糖电泳检测时特异产物条带的消失和不能泳动出点样孔之复杂异常产物的出现;这种扩增产生的异常产物能够被稳定地重复扩增。用λ和细菌质粒序列为模板连续扩增不同长度靶序列的结果表明：连续PCR扩增失败的时期具有扩增靶序列长度的依赖性，越长的靶序列在连续PCR中扩增失败的时期越早。 　　对不同连续PCR扩增的扩增过程观察表明扩增产物经历了一个从高效特异性扩增到低效率特异性扩增，再到扩增产生复杂异常产物的过程。对复杂异常产物的甲酰胺辅助变性处理和变性胶电泳（尿素变性聚丙烯酰胺胶电泳和NaOH碱变性琼脂糖电泳）检测表明扩增产生的复杂产物主要由连续分布的小于靶序列长度的具有相当程度多样性的非全长链组成。连续PCR产生的复杂产物在内部具有局部的双链区域和大量的单链区域及外部单链分支，能够被单链特异的S1核酸酶消化，但是不能被双链特异的限制性内切酶消化。用DNase I或限制性内切酶处理连续扩增早期产生特异扩增产物形成不同长度序列组成的混合物，或者直接用不同扩增反应产生的不同长度的核酸序列组成混合物，混合物在经历变性-复性后都表现出类似连续PCR失败所产生的异常产物电泳行为。这些证据都表明PCR扩增过程中形成的非全长链成分是导致这种异常现象的关键因素，多个不同长度的非全长链复性形成“杂种分子”（具有较大且不一致的分子量和复杂的分支结构），最终表现为常规琼脂糖电泳异常的复杂产物。同时，异常产物组成非全长链成分和全长链成分是其能够实现稳定重复扩增的基础。 　　实验结果表明：对于特定长度的靶序列而言，导致复杂异常出现的根本原因是连续PCR扩增体系中所经历的总PCR热循环数目（每一轮PCR扩增所使用的循环数目多，成功连续扩增的轮数就少）;而扩增体系中的引物浓度、DNA聚合酶用量的多少、扩增程序中时间参数等对此影响较小;巢式PCR和单引物-互补引物PCR的结果表明这些处理对于缓解或延迟异常产物的出现有一定的作用。人工处理（DNase I或限制性内切酶处理）完整模板双链形成的非全长链长产物，然后把非全长链长产物以不同比例同完整模板混合模拟连续扩增后期产物，这种人工混合模板表明连续PCR扩增中同源的非全长链成分对PCR扩增有严重的干扰作用，是导致复杂异常产物出现的直接原因。 　　已有的研究表明：PCR介导重组、长片段PCR难于操作有共同的产生基础——扩增过程中非全长链成分的产生和非全长链成分对后续扩增过程的干扰作用。这一点和导致连续PCR失败的原因是一致的。非全长链成分的出现是PCR扩增过程中不可避免的，其最初产生的可能来源有三个：模板的损伤（扩增前的模板损伤或扩增热循环过程中的损伤）、聚合酶的忠实性、以及聚合酶的进行性。根据聚合酶的特性而调整扩增程序中延伸时间的实验表明，聚合酶的进行性不是导致连续PCR扩增失败的最主要原因。这种非全长链成分产物从无到有且不依赖于体系中非全长链成分的过程我们称之为非全长链成分的初级合成;而已经存在的非全长链成分干扰后续合成形成非全长链成分的过程我们称之为非全长链成分的次级合成。非全长链成分的初级合成和次级合成共同导致了连续扩增的失败和异常产物的形成。 　　从已有的研究结果看，任何降低PCR扩增过程中非全长链成分产生的措施，特别是聚合酶忠实性的提高，都能缓解异常扩增产物的出现和利于长片段PCR操作。 　　

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B. maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1: 1 molar ratio. The equilibrium dissociation constants (K-D) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys(53)-Cys(62)), which comprises the scissile bond Arg(58)(P-1)-His(59)(P-1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Extraction, PCR amplification, and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda-specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA(Thr) gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA(Thr) gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. (C) 1998 Wiley-Liss, Inc.

Characterization of Sarcocystis species in domestic animals using a PCR-RFLP analysis of variation in the 18S rRNA gene: a cost-effective and simple technique for routine species identification

Thirteen restriction endonucleases were used to investigate nucleotide sequence variation in the 18S rRNA DNA of 88 individuals from ten Sarcocystis taxa collected as cysts from their intermediate hosts, swine, cattle and water buffalo. A DNA sequence of

A PCR-based RFLP analysis of Sarcocystis cruzi (Protozoa : Sarcocystidae) in Yunnan province, PR china, reveals the water buffalo (Bubalus bubalis) as a natural intermediate host

A polymerase chain reaction-based restriction fragment length polymorphism (RFLP) approach is used to examine Sarcocystis cruzi-like taxa from the atypical intermediate host, water buffalo, in Yunnan, People's Republic of China. The loci examined lie with

麂属动物陈旧皮张标本的DNA提取及PCR扩增

用改进的方法从保存于本单位标本中提取DNA, 所得DNA片段的分子量从100bp到1kb以上。利用线粒体DNA细胞色素b通用引物和PCR技术。从小麂、印度麂、贡山麂、费氏麂、黑麂DNA中扩增出307bp的 细胞色素b特异片段(加上两端引物后长度为364bp)。用28种限制性内切酶对 新鲜血样和从陈旧皮张标本中所得扩增片段进行酶切分析, 发现只有4个酶(DraⅠ、xbaⅠ、HaeⅢ、HpaⅡ)在这个片段上有切点, 其中HaeⅢ和HapⅡ的识别位点在各种麂中有所不同。 图3参10

CORRELATION OF ZONA-BINDING WITH OOCYTE MATURATION AND SPERM MOTILITY IN RHESUS-MONKEYS BY HEMIZONA ASSAY

The hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona-binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PB1). The mean numbers of sperm bound to hemizona for PBI, PVS, GV, and MC groups were 132.9 +/- 12.0, 71.5 +/- 10.1, 36.1 +/- 4.0, and 20.1 +/- 2.9 (Mean +/- SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona-binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 +/- 2.2 and 25.3 +/- 2.9 (Mean +/- SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1)The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the ''zona maturation'' hypothesis. (C) 1994 Wiley-Liss, Inc.

Characterization of acid-sensing ion channels in dorsal horn neurons of rat spinal cord

Acid-sensing ion channels (ASICs) are ligand-gated cation channels activated by extracellular protons. In periphery, they contribute to sensory transmission, including that of nociception and pain. Here we characterized ASIC-like currents in dorsal horn neurons of the rat spinal cord and their functional modulation in pathological conditions. Reverse transcriptase-nested PCR and Western blotting showed that three ASIC isoforms, ASIC1a, ASIC2a, and ASIC2b, are expressed at a high level in dorsal horn neurons. Electrophysiological and pharmacological properties of the proton-gated currents suggest that homomeric ASIC1a and/or heteromeric ASIC1a + 2b channels are responsible for the proton-induced currents in the majority of dorsal horn neurons. Acidification-induced action potentials in these neurons were compatible in a pH-dependent manner with the pH dependence of ASIC-like current. Furthermore, peripheral complete Freund's adjuvant-induced inflammation resulted in increased expression of both ASIC1a and ASIC2a in dorsal horn. These results support the idea that the ASICs of dorsal horn neurons participate in central sensory transmission/modulation under physiological conditions and may play important roles in inflammation-related persistent pain.

检测HIV-1载量的荧光实时定量PCR技术的建立及其应用

其它部委、高等院校基金;中国科学院基金

An improved microtiter assay for evaluating anti-HIV-I neutralizing antibodies from sera or plasma

Background: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. Methods: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor( s), the tested sera or plasma was removed by a washout procedure after initial 3 - 6 h culture in the assay. Result: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. Conclusion: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.