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1.
扩增大段靶DNA的PCR方法 总被引:3,自引:0,他引:3
扩增大段靶DNA的PCR方法陈尚武,王章(中山大学生命科学学院,广州)PCR和分子克隆是扩增遗传物质的常用技术。热稳定Taq(Thermusaquaticus)DNA聚合酶的应用以及PCR技术所固有的迅速、简便、廉价等优点,使DNA片段的PCR扩增成... 相似文献
2.
免疫-PCR结合了抗原抗体反应的特异性和PCR的高敏感性,是一种极为敏感的抗原检测技术,并适合于各种微量抗原的检测。免疫-PCR的关键在于形成抗体-标记DNA偶联物,作为桥梁连接免疫反应的特异和PCR的高扩增能力。本文简述了-PCR的基流程以及与标DNA相偶联,PCR产物检测方法的进展。 相似文献
3.
人基因组YAC克隆DNA的Alu—PCR反应条件的系统研究 总被引:3,自引:0,他引:3
在人基因组YAC克隆的Alu-PCR指纹分析中要求DNA扩增带具有YAC特征性;在Alu-PCR方法对YAC克隆中的人类基因组DNA片段进行特异的同位素标记时则要求被增标记的DNA序列在插入片段中具有一定弥散性,我们建立了两种不同的Alu-PCR反应体系以满足这一不同要求,并已得到较为满意的结果,根据不同条件下的Alu-PCR结果,分析了多引发位点PCR中的一些现象,并作了解释。 相似文献
4.
人基因组YAC克隆DNA的Alu-PCR反应条件的系统研究 总被引:2,自引:0,他引:2
在人基因组YAC克隆的Alu-PCR指纹分析中要求DNA且扩增带具有YAC特征性;在用AlU-PCR方法对YAC克隆中的人类基因组DNA片段进行特异的同位素标记时则要求被扩增标记的DNA序列在插入片段中具有一定的弥散性。我们建立了两种不同的Alu-PCR反应体系以满足这一不同要求,并已得到较为满意的结果。根据不同条件下的Alu-PCR结果,分析了多引发位点PCR中的一些现象,并作出了解释。 相似文献
5.
用于扩增较大片段DNA的PCR方法方向东,陈淳(中科院上海生物化学研究所国家分子生物学实验室,上海200031)诸江(上海第二医科大学上海血液学研究所,上海200025)关键词DNA扩增,PCR自从Taq(Thermusaquaticus)DNA聚合... 相似文献
6.
用PCR法直接快速筛查重组阳性克隆 总被引:2,自引:0,他引:2
应用PCR法快速筛查插入有苯丙氨酸脱氨酶cDNA重组阳性克隆。方法:用于PCR扩增的引物是位于载体pET23b启动子处的T7启动子引物和位于目的基因PALcDNA3’端终止密码TAA处的引物。以灭菌吸头挑一单菌落加入PCR体系扩增。结果:在筛查的3个克隆中,有2个阳性克降,并且插入方向正确,经DNA序列测定得到进一步证实。结论:以PCR方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方面,不 相似文献
7.
超高忠实性PCR用DNA聚合酶 总被引:4,自引:0,他引:4
PCR(PolymeraseChainReaction)法已在生物工程领域得到了广泛的应用,而PCR法的推广完全得益于Taq耐热性DNA聚合酶。近年来,PCR法的应用除生物工程领域外,已应用至工业、农业、医学、制药等与人们生活息息相关的各个领域。随着PCR法的不断推广,人们对DNA聚合酶的忠实性(Fidelity)要求越来越高,一般来说,TaqDNA聚合酶在进行PCR延伸反应过程中的错配率为0.5%左右。TaKaRa公司独自研制成功的PyrobestDNA聚合酶是一种来源于Pyrococcuss… 相似文献
8.
斜茎黄芪根瘤菌的16S rDNA和23S rDNA PCR-RFLP比较分析 总被引:2,自引:0,他引:2
在表型性状数值分析和AFLP指纹图谱分析的基础上,选取54株斜茎黄芪根瘤菌的代表菌株及已知根瘤菌参比菌株,进行16SrDNA和23SrDNA的PCR-RFLP比较分析。结果表明斜茎黄芪根瘤菌具有极大的系统发育多样性,分别具有24个16SrDNA遗传图谱类型和22个23SrDNA遗传图谱类型,16SrDNA与23SrDNAPCR-RFLP聚类分析树状图谱有较好的一致性,但也存在一些差异。在对较大类群的划分上,它们的结果与表型性状数值分析结果有较好的一致性。将16SrDNA和23SrDNAPCR-RFLP分析 相似文献
9.
本文讨论了检测丙型肝炎病毒核糖核酸(HCVRNA)的样本来源和处理,引物的设计,合成和选择等问题;重点介绍了蛋白酶K消化法,聚乙二醇沉淀法,异硫氰酸胍一步法,硅凝胶提取法和直接捕获法提取HCV RNA的5种方法,以及一步PCR法,常规RT-巢式PCR,直接RT-巢式PCR,化学修饰的RT-巢式PCR,联合PCR,双温PCR和定量竞争性PCR等7种PCR方法检测HCV RNA,用PCR检测HCV RNA方法的标准化以及检测HCVRNA具有非常重要的意义。本文还介绍了一种新型的定量方法-bDNA信号放大技术检测HcVRNA。 相似文献
10.
多聚酶链反应技术诊断结核病的进展 总被引:1,自引:0,他引:1
多聚酶链反应(PCR)技术在结核病诊断上发展迅速。本文就①结核菌DNA提取技术;②引物的设计;③用于诊断结核的PCR方法;④PCR产物的检测方法;⑤PCR的敏感性和特异性;⑥PCR临床应用和注意事项等方面,简述该技术在结核病诊断中应用的进展和需要进一步研究的问题。 相似文献
11.
Proboscidean DNA from Museum and Fossil Specimens: An Assessment of Ancient DNA Extraction and Amplification Techniques 总被引:3,自引:0,他引:3
Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification. 相似文献
12.
Daniela Woide Albert Zink Stefan Thalhammer 《American journal of physical anthropology》2010,142(2):321-327
The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low‐volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and β‐actin gene specific fragments were amplified via low‐volume PCR in a total reaction volume of 1 μl. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low‐volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
13.
Ancient DNA extraction from bones and teeth 总被引:1,自引:0,他引:1
This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20-23 degrees C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days. 相似文献
14.
Kim K Kim KY Jeon E Togloom A Cho YO Lee MS Lkhagvasuren G Choi JH Tumen D Ja Park A Kim KC Park KW Kim JH Noh M Yoo KJ Lee KH 《American journal of physical anthropology》2008,136(1):114-121
A novel method of ancient DNA (aDNA) purification was developed using ion-exchange columns to improve PCR-amplifiable DNA extraction from ancient bone samples. Thirteen PCR-resistant ancient bone samples aged 500-3,300 years were tested to extract aDNA using a recently reported, silica-based aDNA extraction method and an ion-exchange column method for the further purification. The PCR success rates of the aDNA extracts were evaluated for the amplification ability of the fragments of mitochondrial DNA, a high-copy DNA, and amelogenin, a low-copy DNA. The results demonstrate that the further purification of silica-based aDNA extracts using ion-exchange columns considerably improved PCR amplification. We suggest that the ion-exchange column-based method will be useful for the improvement of PCR-amplifiable aDNA extraction, particularly from the poorly preserved, PCR-resistant, ancient samples. 相似文献
15.
Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood. 相似文献
16.
A simple and efficient method for PCR amplifiable DNA extraction from ancient bones 总被引:2,自引:0,他引:2
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A simple and effective modified ethanol precipitation-based protocol is described for the preparation of DNA from ancient human bones. This method is fast and requires neither hazardous chemicals nor special devices. After the powdering and incubating of the bone samples Dextran Blue was added as a carrier for removing the PCR inhibitors with selective ethanol precipitation. This method could eliminate the time-consuming separate decalcification step, dialysis, application of centrifugation-driven microconcentrators and the second consecutive PCR amplification. The efficiency of this procedure was demonstrated on ten 500–1200-year-old human bones from four different Hungarian burial sites. A mitochondrial specific primer pair was used to obtain sequence information from the purified ancient DNA. The PCR amplification, after our DNA extraction protocol, was successful from each of the 10 bone samples investigated. The results demonstrate that extraction of DNA from ancient bone samples with this new approach increases the success rate of PCR amplification. 相似文献
17.
Comparison and optimization of ancient DNA extraction 总被引:8,自引:0,他引:8
Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success. 相似文献
18.
《微体古生物学报》2022,(1)
海洋沉积物柱状样有孔虫古DNA是近年来国际新兴的研究技术,对于解释海洋全球变化可以获取到传统形态学检获不到的遗传信息,并对其进行比较和补充。目前国际上有孔虫古DNA的研究主要开展于深海和极地等有利于DNA保存的环境,但对于近海陆架海域尚无相关有效的技术研究。为了探索适合提取和扩增陆架浅海环境沉积物柱状样中的有孔虫古DNA的方法,本实验以采自山东半岛附近的黄海沉积物柱状样为研究对象,通过改进DNA提取过程的涡旋震荡时间和洗脱液体积、比较不同的PCR扩增条件和引物对,对陆架浅海地区沉积物柱状样中有孔虫古DNA提取和PCR扩增的方法进行了探索。借助ImageJ软件对PCR产物的凝胶电泳图像进行了定量分析与比较。研究结果显示,延长涡旋震荡时间和减少洗脱液体积可以提高对海洋沉积物环境总古DNA的提取效能,使用引物对s14F0和s15以及优化后的PCR条件能成功扩增陆架浅海环境沉积物中的有孔虫古DNA。本文探索了适用于陆架浅海环境沉积物柱状样的有孔虫古DNA的研究技术,可为古海洋和古环境研究提供新的研究思路。 相似文献
19.
Wenhao Xu Yu Lin Keliang Zhao Haimeng Li Yinping Tian Jacob Njaramba Ngatia Yue Ma Sunil Kumar Sahu Huabing Guo Xiaosen Guo Yan Chun Xu Huan Liu Karsten Kristiansen Tianming Lan Xinying Zhou 《Ecology and evolution》2021,11(1):390-401
Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA. 相似文献
20.
古代DNA序列信息能够为物种演化研究提供最直接的分子证据,但获取古代DNA的技术仍存在诸多瓶颈,尤其是扩增中存在受损伤DNA模板的干扰、获取成本高和实验周期长等问题.改进了异丙醇沉淀提取法,并采用了尿嘧啶糖苷酶(UNG)去除受损伤DNA模板后进行扩增的方法,最终可以高效地获取真实的古代DNA序列.实验利用距今4 300~3 900年前的猪牙样本,将改进的古 DNA 获取方法与常规方法进行比较研究,结果表明,改进的异丙醇沉淀法提取结合UNG处理后进行PCR扩增的方法,可以在保证古代DNA获取成功率并提高获得的DNA序列可靠性的前提下,将经费投入和实验周期都各减少至常规方法的50%以下.这可以为开展大规模古代样本检测提供一种切实可行的 DNA 获取方法. 相似文献