用RFLP和PCR—RFLP技术研究东北虎和华南虎线粒体DNA多态性

采用ｍｔＤＮＡＲＦＬＰ和ＰＣＲＲＦＬＰ技术研究了东北虎和华南虎的ｍｔＤＮＡ的多态性。在ｍｔＤＮＡＲＦＬＰ研究中,分离纯化了东北虎和华南虎肝、肾和心脏组织的ｍｔＤＮＡ,用２０种识别６碱基对的限制性内切酶消化,结果只有１种限制性内切酶（ＸｂａⅠ）检测到多态性片段,其余１９种限制性内切酶消化产生的限制性格局在东北虎和华南虎完全一致。在ＰＣＲＲＦＬＰ研究中,用ＰＣＲ技术分别扩增了东北虎和华南虎ｍｔＤＮＡ的控制区（ｃｏｎｔｒｏｌｒｅｇｉｏｎ）,用８种识别４碱基对的限制性内切酶分别对扩增产物进行消化,结果只有１种限制性内切酶（ＲｓａⅠ）检测到多态性片段。ｍｔＤＮＡＲＦＬＰ及ＰＣＲＲＦＬＰ的结果均提示东北虎和华南虎之间的遗传距离极小。这可能与下列因素有关：两者分布区间无天然隔离屏障;具有强扩散能力;近几百年才被相互隔离。     

植物病原真菌中DNA分子鉴定技术

就基因组ＤＮＡ中Ｇ＋Ｃ含量、分子杂交、聚合酶链式反应（ＰＣＲ）、限制性片段长度多态性（ＲＦＬＰ）、随机扩增多态性（ＲＡＰＤ）,以及扩增片段长度多态性（ＡＦＬＰ）等分子标记技术,在植物病原真菌鉴定工作中的应用情况和发展趋势作了介绍。     

中国4个民族mtDNA D环多态性研究

本文利用聚合酶链反应限制性片长度多态性（ＰＣＲ－ＲＦＬＰ）方法,检测了３６名汉族,３０名达斡尔族,３２名鄂伦春族,３０名鄂温克族随机选择正常个体ｍｔＤｎＡ Ｄ环４６５ｂｐＤＮＡ片多性并加以比较。结果表明,ｍｔＤＮＡ Ｄ环该片段的ＲＦＬＰ分析共产生２７种限制性类型,计算出４个民族的ｍｔＤＮＡ Ｄ环平均核苷酸歧异频率,并对４个民族的亲缘关系进行了聚类分析。     

AFLP标记在果树遗传育种研究中的应用

1993年 ,Ｚａｂｅａｕ等发明了一项专利技术 ,扩增片段长度多态性 (ａｍｐｌｉｆｉｅｄｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ ,ＡＦＬＰ) ,该技术结合了ＲＦＬＰ技术和ＰＣＲ技术的优点 ,以其高效性和高重复性等特点 ,被广泛应用于多种植物的遗传育种研究。1 .ＡＦＬＰ技术的原理和特点ＡＦＬＰ技术是基于对限制性片段的选择性扩增 ,基因组ＤＮＡ被限制性内切酶切割后 ,将限制性片段末端连上双链接头 ,根据接头序列和相邻的限制性位点序列设计引物对限制性片段进行扩增。扩增产物经电泳检测后再比较其谱带的差异。ＡＦＬＰ…     

扩增片段长度多态性(AFLP)——一种新的分子标记技术

ＡＦＬＰ（扩增性片段长度多态性）是一种新的ＤＮＡ分子标记。与ＲＦＬＰ、ＲＡＰＤ相比,ＡＦＬＰ具有在一次试验中可同时观察到大量的限制性片段的优点。本文阐述了ＡＦＬＰ的原理和方法,综述了ＡＦＬＰ目前在植物遗传育种研究中的应用进展,并对ＡＦＬＰ技术在植物遗传育种中的应用前景提出了初步设想。     

用PCR—RFLP方法研究藏族HLA0—DQA1和—DQB1基因多态性

应用目前ＨＬＡ研究领域中成熟的、有效的ＰＣＲ－ＲＦＬＰ基因分型技术,从ＤＮＡ水平对藏族健康群体进行了ＨＬＡ－ＤＱＡ１（４９人）和－ＤＱＢ１（４９人）基因分型,这在国内外属首次。所采用的ＰＣＲ－ＲＦＬＰ基因分型技术是在ＨＬＡ－ＤＱＡ１和－ＤＱＢ１各等位基因全部序列已知的情况下,对其第２个外显子碱基序列扩增进而进行ＲＦＬＰ分析的方法。这种方法得到的ＲＦＬＰ的所有片段都是已知序列,因而精确度很高,同时为     

用分子生物学技术对草菇进行菌株鉴别

利用ＡＰ－ＰＣＲ和ＲＡＰＤ技术对三个草菇菌株进行鉴别,其结果与用草菇菌株Ｖ３４基因文库中的中等重复序列为探针进行限制性内切酶长度多态性分析（ＲＦＬＰ）,及对编码核糖体５．８ＳｒＲＮＡ的ＤＮＡ（ｒＤＮＡ）进行ＰＣＲ扩增后的产物进行限制性内切酶长主多态性分析（ＰＣＲ－ＲＦＬＰ）的结果相一致。这一结果显示出用这四种方法对草菇菌株进行鉴别具有相似的效果。同时用这四种方法构建的分子生物学标记显示出这三个菌株     

AFLP分子标记及其在植物育种上的应用

扩增酶切片段多态性（ＡｍｐｌｉｆｉｅｄＲｓｔｒｉｃｉｔｏｎｆｒａｇｍｅｎｔｐｏｌｙｍｏｒｐｈｉｓｍ,简称ＡＦＬＰ）是由Ｚａｂｅａｕ等１９９２年发明的一项新的ＤＮＡ指纹技术,它结合了ＲＦＬＰ和ＰＣＲ技术的特点,具有ＲＦＬＰ技术的可靠性和ＰＣＲ技术高效性,其基本原理是对基因组ＤＮＡ酶切片段的选择性扩增,ＡＦＬＰ扩增片段的谱带数取决于采用的内切酶及引物３′端选择碱基的种类数目和所研究基因组的复杂性,实验     

植物总DNA样品的快速制备

利用Ｑｉａｇｅｎ微量植物ＤＮＡ提取试剂盒,在１小时内即可从植物组织获得总ＤＮＡ,提取过程中勿需酚／氯仿和ＳＤＳ抽提,操作简便、快捷。所得ＤＮＡ样品的ＯＤ２６０／ＯＤ２８０值在１．７－１．９之间。样品纯度高;该样品不含ＰＣＲ反应抑制剂及其他酶反应抑制剂,可被各种限制性内酶完全降解,适合于ＰＣＲ、印迹、ＲＡＰＤ、ＡＦＬＰ和ＲＦＬＰ分析等各种下游应用。     

AFLP标记及在植物中的应用

ＡＦＬＰ是 1 992年由荷兰Ｋｅｙｇｅｎｅ公司Ｚａｂｅａｕ、Ｖｏｓ在ＰＣＲ和ＲＦＬＰ的基础上发展起来的一种检测ＤＮＡ多态性的新方法[1] ,并于1 993年获得欧洲专利局专利。与ＲＦＬＰ类似 ,ＡＦＬＰ也是通过限制性内切酶片段的不同长度检测ＤＮＡ多态性的一种ＤＮＡ分子标记技术。由于ＲＦＬＰ是以传统的Ｓｏｕｔｈｅｒｎ杂交为基础的 ,操作繁琐 ,对ＤＮＡ多态性的检出的灵敏度不高 ,在连锁图上有很多大的空间区。随着ＰＣＲ技术广泛应用 ,对分子标记技术的发展产生了巨大的推动作用。除了ＲＦＬＰ(限制性内切酶酶切片段长度多态性标记 …     

A Preliminary Study on the Use of RFLP Analysis of the PCR Amplified Products in the ystematic Investigation of the Subtribe Astragalinae (Fabaceae)

The analysis of variation at the molecular level has been proven extremely useful in the reconstruction of plant phylogenetic relationships. A DNA fragment of 3100-base pairs (bp) in the chloroplast genes ndhF and psbA has been amplified from 9 species of 7 genera in the subtribe Astragalinae and 1 species in the subtribe Glycyrrhizinae. A proper procedure for specifically PCR-amplifing the 3.1 kb fragment was presented. By this procedure, the PCR products could be digested directly after amplification. The restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified products indicated that it had potential systematic significances in the phylogenetic studies of the subtribe Astragalinae. This approach could also reduce time, expense and the amount of DNA required, and furthermore, obtain reliable results.     

小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性(英文)

应用一对寡核苷酸引物ITS1与ITS4对核DNAG＋C百分数小于30％的小克银汉霉（Cunninghamella）属的核糖体DNA（rDNA）内转录间区（YTS）进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为;第一组4种1变种,小于764碱基对（bp）;第二组2种,765～824bp;第三组2种1变种,大于825bp。所研究的许多种中,特别是第三组,单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了RsaI,TaqI;Tru9I,和HinfI4种限制性内切酶,对所有扩增产物进行了限制性片段长度多型性（RFLPs）的分析。在雅致小克银汉霉（Celegans）、巴西玉蕊小克银汉霉（Cbertholletiae）、和布拉氏霉（Cblakesleeana）各种的限制性酶切图谱,种内非常一致而种与种之间有差别。反之,在某些种其限制性酶切图谱不仅种间互不相同,在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下,只能综合各种资料才能将它们区分开来。本研究肯定了PCR－RFLP在区分小克银汉霉种和变种上的意义,并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。     

澳洲宝石鲈线粒体DNA COI基因的克隆与序列分析

对澳洲宝石鲈线粒体DNA细胞色素氧化酶Ⅰ亚基(cytochrome oxidase subunit Ⅰ,COI)基因进行了扩增、克隆和测序,并对COI序列进行了分析.结果显示,澳洲宝石鲈COI基因序列长度为631 bp,其中A、T、G和C 4种碱基的含量分别为27.7%,23.6%,29.8%和18.9%.与从GenBank中查到的其他4种蜊科鱼类的同源序列比对,邻接法(neighbor-joining)构建系统进化树.结果显示,5种蜊科鱼类聚在一起,分为两个大的支系,其中花身蜊与条纹蜊首先聚成一支,然后与詹氏弱棘鯻和银锯眶鯻聚成的一支共同组成一个支系;而高体革鯻单独聚为一个支系,所得的聚类结果与传统的分类结果基本一致.     

非损伤性方法的建立及其在亚洲黑熊分子遗传学研究中的应用

哺乳动物野生群体和濒危物种的分子遗传学研究长期以来受到组织样品来源的限制。因为传统的分析方法,如蛋白质电泳、ＤＮＡ限制性片段长度多态及ＤＮＡ序列分析等,需要采集诸如血液、肌肉、肝脏、心脏、肾脏和脾脏等组织材料以提取足量的蛋白质和ＤＮＡ。而这又常常涉及捕捉、损伤甚至杀死动物。本工作探索建立一种非损伤性的途径以解决这一困难。我们采用Ｂｉｏ—Ｒａｄ公司“ＩｎｓｔａＧｅｎｅＰｕｒｉｆｉｃａｔｉｏｎＭａｔｒｉｘ”从遗落于地上的亚洲黑熊单根毛发样品中提取出基因组ＤＮＡ。这种方法简便、快速,仅需一个步骤──在该试剂中以１００℃裂解细胞。不必再经酚和氯仿抽提。我们用ＰＣＲ技术从这种毛发ＤＮＡ中扩增了线粒体细胞色素ｂ基因片段,并测定了该片段的ＤＮＡ序列。我们的工作为开展亚洲黑熊及其他哺乳动物野生群体的分子遗传学研究奠定了基础。     

A Molecular Method for Detection of <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis> in Coffee Beans

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin that has been detected in a variety of food products, including green coffee beans. About 80% of Aspergillus carbonarius strains collected from coffee beans are able to produce OTA on this substrate. The rapid identification of this fungal species would be desirable. RAPD assays were applied to identify amplification products specific for A. carbonarius. One selected fragment, denoted OPX7₈₀₉, was cloned and sequenced. Based on the nucleotide sequence obtained, specific oligonucleotides (OPX7F₈₀₉ and OPX7R₈₀₉) were designed and used as primers for DNA amplification. One amplified band of 809 bp was obtained from A. carbonarius genomic DNA, whereas no amplified fragment from DNA of other Aspergillus species was detected. This PCR analysis was also successfully employed to detect A. carbonarius in coffee beans. This PCR assay could contribute to the early and rapid detection of the potential presence of OTA in coffee samples.     

用RAPD技术检测不同生态类型蜘蛛代表品种的基因组DNA多态性

蜘蛛有3种生态类型,即洞穴型、结网型和游猎型。利用RAPD技术检测3种生态类型蜘蛛的代表品种,即白额巨蟹蛛(游猎型)、漏斗蛛(结网型)、虎纹捕鸟蛛和七纺器蛛(洞穴型)的基因组DNA的多态性。用11个随机引物对3种生态类型的代表蜘蛛的基因组DNA进行扩增,平均每个品种观察到约22.5个标记,单个引物获得的标记在4～13个之间。实验结果经统计学分析表明,L纺器蛛和虎纹捕鸟蛛的随机扩增多态DNA共享度(F)高,在分子水平上进一步证实了同生态类型蜘蛛的亲缘天系最近,聚类结果表明,3种类型蜘蛛的总体演化趋势为:洞穴型 结网型—游猎型,与以往的形态学研究相符。     

FINS (forensically informative nucleotide sequencing): a procedure for identifying the animal origin of biological specimens.

The keys to identifying different species normally rely heavily on morphological characteristics. However, when an animal has been killed for food or sport, these markers are often destroyed or intentionally removed from the animal. This presents a problem for government agencies who are involved in determining the species origin of an animal or products derived from it in order to enforce conservation and/or health-related regulations. The problem is compounded if the meat of the animal has been processed in any way. We have developed a procedure called FINS (Forensically Informative Nucleotide Sequencing) that overcomes these problems. FINS has four components. First, methods have been developed that can isolate DNA from a wide range of biological samples including processed foods (e.g., canned, partially cooked, pickled, salted or smoked). Second, a specific segment of DNA is amplified using PCR. Third, the nucleotide sequence of the amplified segment of DNA is determined. Fourth, this nucleotide sequence is subjected to a phylogenetic analysis using a database, and the most closely related species is identified. FINS is a rapid, reliable and reproducible procedure that is based on established techniques. This procedure fills the need for an accurate method of determining the species identity of a specimen when this is not possible by conventional means.     

Universal TA cloning

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.     

Phylogenetic relationships among Malagasy lemurs as revealed by mitochondrial DNA sequence analysis

Systematics and evolution of Malagasy lemurs has been analyzed using morphological characters, fossil evidence, ecological/ethological data, and chromosomal banding patterns. Recent developments in DNA technology have provided evolutionary biologists with additional and powerful tools for making phylogenetic inference. In the last years several studies concerning highly repeated DNA sequences (hrDNA) provided new insights about the systematic relationships among the different species of Lemuridae and Cheirogaleidae. Here, a reconstruction of molecular phylogeny of extant Malagasy lemurs based on the comparison of cytochrome-b mitochondrial DNA sequences is presented. With the Polymerase Chain Reaction (PCR) and direct sequencing of amplified DNA fragments, both the phylogenetic range and resolving power of comparative analysis can be extended. These techniques allow to gather sequence data useful to evaluate the pattern of molecular evolution offering opportunities for phylogenetic purposes. A 290-bp fragment of cytochrome-b gene has been amplified and sequenced from the following species:Tupaia glis, Galago alleni, Daubentonia madagascariensis, Indri indri, Varecia variegata, Eulemur fulvus, Eulemur coronatus, Eulemur rubriventer, Eulemur mongoz, Eulemur macaco, Lemur catta, andHapalemur griseus griseus. The phylogenetic trees obtained show the relationships among the Eulemur species and confirm the karyological and hrDNA results of a separated clade forL. catta/Hapalemur. The separation ofVarecia variegata from the other genus of the family Lemuridae is discussed.     

Methacarn fixation for genomic DNA analysis in microdissected, paraffin-embedded tissue specimens.

We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 x 1-mm area of cerebral cortex of a 10-microm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.