一种新的分子标记方法－随机微卫星扩增多态DNA (RMAPD)

随机微卫星扩增多态DNA（RMAPD）是利用随机引物和微卫星的上游或下游引物一起作为该扩增的引物,在Taq DNA聚合酶、MgCl2、dNTPs和模板DNA等共同作用下进行PCR扩增的一种新型分子标记方法。其核心是RMAPD引物的有效性问题。通过对西农萨能奶山羊群体RMAPD电泳检测、数据统计分析及验证实验等证明RMAPD的引物是有效的。通过与微卫星和RAPD标记比较,发现RMAPD标记在扩增引物、扩增程序和重复性等方面区别于微卫星和RAPD标记;它是RAPD标记的一种广义的延伸,但又不完全等同于RAPD标记。因此,确定RMAPD是一种新的分子标记方法。该方法也具有DNA标记的特点,在群体遗传结构和亲缘关系分析以及标记辅助选择等遗传育种领域具有广阔的应用前景。     

RAPD技术在生物遗传多样性研究中的应用

遗传多样性的检测可在不同水平上进行 ,最初人们对遗传多样性的检测是从形态学开始的 ,进入 2 0世纪80年代 ,分子生物学与分子克隆技术的发展带来了一系列检测遗传多样性更为直接的方法。目前 DNA水平的分析方法已成为最有效的遗传分析方法 ,原则上可以做到对任何基因组中任何片段进行分析。本文对 RAPD技术及其在生物遗传多样性研究中的应用作一综述。1　 RAPD技术随机扩增多态 DNA标记 (Random amplified poly-morphic DNA,RAPD)技术是 Williams和 Welsh等于1990年同时建立的一项 DNA多态性检测新技术 ,是一项建立在 PCR基础…     

分子标记技术的发展及应用

介绍了几种应用前景较广的分子标记,如基于DNA杂交技术的分子标记:限制性片段长度多态性(RFLP)和DNA可变串联重复数标记(VNRT);基于PCR技术的分子标记:随机扩增多态性 DNA(RAPD)、酶切扩增多态性(CAPS)、扩增片段长度多态性(AFLP)、微卫星DNA(SSR)和DNA单链构象多态性(SSCP);以及新兴的第3代分子标记,即基于DNA芯片技术的分子标记:单核苷酸多态性(SNP)等。分别阐述了它们的原理、方法步骤与优缺点、应用注意事项和适用范围,同时概述了它们在生物学研究中的应用和进展。     

白鲢和鳙鱼的随机扩增多态DNA分析

根据鱼类外周血细胞都有核的特点,采用从冷冻和低渗双重处理分离的细胞核提取基因组DNA.以此法获得的白鲢和鳙鱼的基因组DNA为模板,和Operon公司生产的OPN和OPM两个组共40个随机引物,对这两种鱼进行了随机扩增多态DNA(RAPD)分析;确定了对这两种鱼基因组相关区域可进行随机PCR扩增的有效引物,特别是哪些可产生种群内或群体的RAPD遗传标记,即可产生个体特异性和群体特异性RAPD带谱的引物.讨论了RAPD遗传分子标记在鱼类遗传,特别是遗传多样性研究,和鱼类种质资源评估和管理中的应用前景问题.     

布氏田鼠封闭群遗传结构的随机扩增多态DNA分析

目的使用随机扩增多态DNA标记建立标准化的布氏田鼠封闭群遗传质量控制分子标记库。方法使用高盐沉淀法从鼠尾中提取布氏田鼠基因组DNA。采用40条PRAD引物对布氏田鼠封闭群进行PCR扩增,琼脂糖电泳分离条带,参考标准分子量标记计算条带大小,并使用多态位点数、单态位点数以及多态位点比率评价种群的遗传多样性。结果筛选出8个能获得清晰稳定扩增条带的RAPD标记。这8个RAPD标记检测到的多态位点数存在明显差异。8个引物得到的遗传多态位点的数据之和能揭示种群的遗传结构。结论本实验建立了检测布氏田鼠封闭群遗传结构的RAPD标记。     

土壤可培养细菌DNA的提取及RAPD条件的优化*

以改进的CTAB 溶菌酶 蛋白酶K裂解法抽提土壤可培养细菌总DNA ,直接进行随机扩增多态DNA (RAPD)分析。分别测试了不同浓度镁离子、dNTP、模板DNA、引物、DNA聚合酶及牛血清白蛋白对反应结果的影响 ,通过各因子的组合研究 ,确定了土壤可培养细菌遗传多样性分析的稳定的RAPD反应体系。     

AFLP和RAPD标记技术在栉孔扇贝遗传多样性研究中的应用比较

AFLP和RAPD标记技术是近年来发展最快的基于PCR基础上的两种DNA标记技术,本文比较了两种标记技术在我国栉孔扇贝群体遗传多样性研究中的应用。共筛选20个RAPD引物和7个AFLP引物组合,检测到AFLP标记的有效等位基因数和平均多态信息量稍低于RAPD标记,但AFLP标记在每单位分析中扩增到的野生和养殖群体的多态性条带数(23．8,24．8)分别高于RAPD标记(5．6,5．6),AFLP多态性检测效率显著高于RAPD标记。AFLP和RAPD两种标记技术所揭示的野生种群与养殖群体间的近交系数、遗传距离两项指标均表明,我国栉孔扇贝养殖群体和野生种群之间尚未出现明显的遗传分化。研究结果表明：RAPD和AFLP这两种标记技术均可用于栉孔扇贝遗传多样性的分析,其分析结果是一致的。     

用整合微流控芯片系统分析黄山松RAPD片段多态性

随机扩增多态DNA（randomly amplified polymorphic DNA,RAPD）标记可快速提供连锁信息,特别是在针叶树单倍体的大配子体中RAPD基因型也能得以确定［3］。在对多态性的RAPD片段进行分析时,传统的平板凝胶电泳分析法因人工操作,其有效的鉴别受到一定程度的限制,只能得到一些有关RAPD片段半定量信息。我们将整合微流控芯片系统作为一种新的工具运用于黄山松的多态性RAPD片段分析中。发现基于芯片的检测方法比琼脂糖凝胶电泳方法灵敏度更高,所需的样品量要少得多,所需的时间仅为其1/4。并且能自动对DNA片段进行定性、定量分析。它是一种高效、灵敏、迅速、重复性好的检测新技术。 Abstract:Randomly amplified polymorphic DNA (RAPD) markers quickly provide linkage information［1~2］,especially in conifers where haploid megagametophytes can be used for genotyping［3］.Traditionally use of slab gel electrophresis results in qualitative data that can be manually manipulated to gain semiquantitative information about the polymorphic RAPD fragments.We have proposed the use of an integrated microfluidic chip-based system as a new tool in the analysis of polymorphic RAPD fragments.The chip-based method was found to be very sensitive,requiring much less sample and only quarter the time compared to the agarose gel method.The automated data analysis sizes and quantitates the DNA fragments,thus yielding a more thorough,reproducible,sensitive,and rapid analysis.     

0#柴油对斑马鱼基因组DNA和环境DNA遗传毒性的RAPD比较

研究利用随机扩增多态性DNA (Random Amplified Polymorphic DNA, RAPD)技术, 以斑马鱼基因组DNA和其养殖水体中的环境DNA (environmental DNA, eDNA)为模板, 检测0#柴油可溶性组分对斑马鱼(Danio rerio)遗传毒性的影响。结果显示, 通过基因组DNA和eDNA扩增的RAPD图谱均可检测到0#柴油对斑马鱼的遗传毒性。在未受到柴油暴露时, 斑马鱼基因组DNA和水环境中eDNA在96h内的RAPD图谱均无明显变化; 在不同浓度的柴油暴露下, 随着暴露时间(0、24h、48h、72h、96h)延长, 基因组DNA和eDNA的多态性位点减少, 模板稳定性降低; 随着柴油浓度(15%、50%、100%)的增加, 基因组DNA和eDNA的多态性位点也减少, 模板稳定性降低。这表明0#柴油对斑马鱼基因组DNA和eDNA的遗传毒性均呈现时间-效应和浓度-效应关系, 并且无论以斑马鱼基因组DNA还是eDNA为模板, 柴油暴露组和未进行暴露的对照组的RAPD扩增图谱条带变化趋势一致。研究结果为通过RAPD技术检测柴油对水生生物的遗传毒性提供了新的研究思路和技术手段。     

土壤可培养细菌DNA的提取及RAPD条件的优化

以改进的CTAB-溶菌酶-蛋白酶K裂解法抽提土壤可培养细菌总DNA,直接进行随机扩增多态DNA(RAPD)分析。分别测试了不同浓度镁离子、dNTP、模板DNA、引物、DNA聚合酶及牛血清白蛋白对反应结果的影响,通过各因子的组合研究,确定了土壤可培养细菌遗传多样性分析的稳定的RAPD反应体系。     

Start Codon Targeted (SCoT) Polymorphism: A Simple,Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.     

RAPD分子标记及其在作物遗传育种中的应用

ＲＡＰＤ分子标记是一种新型的分了遗传标记,其原理是采用１０个碱基的随机引物,以基因组ＤＮＡ为模板进行ＰＣＲ随机扩增。其优越性在于其方法简单;分析中的花费少,用时短;分析所需的ＤＮＡ用量也不多等。本文着重介绍以电泳技术和ＰＣＲ扩增技术为核心的分子标记以及在农作物遗传育种中的应用。     

Taenia solium and Taenia saginata: identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies.     

A review of random amplified polymorphic DNA (RAPD) markers in fish research

During the last 15 years, molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The ease and simplicity of RAPD (randomly amplified polymorphic DNA) techniques make it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics. This review summarizes the use of RAPD markers in fish research. The advantages and disadvantages of the RAPD approach will be discussed.     

Genetic relationships among ten endod types as revealed by a combination of morphological,RAPD and AFLP markers

The genetic relationships among ten types of endod (Phytolacca dodecandra) cultivated by the Institute of Pathobiology of the Addis Ababa University to combat the disease bilharzia in Ethiopia were studied using morphology and molecular markers. A total of 18 morphological characters, 194 amplified fragment length polymorphism (AFLP) and 42 random amplified polymorphic DNA (RAPD) markers were used to determine genetic proximity between types. Genetic distance and cluster analysis of the AFLP data revealed the lack of genetic difference between E47 and E48 but relatively wider genetic difference among the other endod types. Cluster and principal component analyses performed on the AFLP and RAPD markers demonstrated the presence of distinct separation of E56 but not that of E44 from the others. The AFLP and RAPD data, thcrefore, did not support the hypothesis that the superiority of E44 in agronomic traits and molluscicidal potency is linked to its distinct genetic difference from the other endod types. Matrices correspondence tests demonstrated the presence of greater correspondence between AFLP and RAPD data (r = 0.842) but not between the morphology and that of AFLP and RAPD. This indicates the correspondence more between the two DNA markers systems than either of them with morphological traits. The cophenetic correlation coefficients also revealed poor fit for morphology (r = 0.716), good fit for RAPD (r = 0.872) and very good fit for AFLP (r = 0.975), reflecting the hyper-variability and higher resolving power of AFLP.     

RAPD技术及其在动物遗传育种中的应用

RAPD技术是在PCR基础上发展起来的一种DNA多态性检测技术,已广泛应用于基因组研究的各个领域。本文概述了RAPD反应的原理、特点,总结了其在遗传多样性检测、亲缘关系鉴定、遗传连锁分析和数量性状的辅助标记选择等方面的应用,并肯定了RAPD在动物遗传育种领域的应用前景。     

Rapid RAPD screening of plant DNA using dot blot hybridization

Scoring of the results of RAPD analysis using gel electrophoresis imposes a constraint on throughput. To circumvent this barrier, dot-blot hybridization was substituted for electrophoresis. Arbitrarily amplified fragments from barley and wheat genomic DNA were labelled and used as probes for the identification of identical fragments in subsequent amplification reactions. None of the twelve fragments used as probes exhibited significant levels of croos-hybridization to other fragments amplified by the same arbitrary primer. The strength of the hybridization signal facilitates more accurate and more sensitive detection of diagnostic fragments than gel electrophoresis. In addition, the defined spatial orientation (microtitre dish format) of the ± results provide an excellent format for automated data collection. The use of dot blot hybridization to analyse PCR products well decrease the cost and time requirements of marker-assisted selection. This technique will also facilitate the rapid application of PCR-based maps.     

Construction of a genetic linkage map of shiitake mushroom Lentinula edodes strain L-54

From fruiting bodies of L. edodes strain L-54, single-spore isolates (SSIs) were collected. Two parental types of L-54 were regenerated via monokaryotization. By means of random-amplified polymorphic DNA (RAPD), DNA samples from L-54, its two parental types, and 32 SSIs were amplified with arbitrary primers. Dedikaryotization was demonstrated, and 91 RAPD-based molecular markers were generated. RAPD markers that were segregated at a 1:1 ratio were used to construct a linkage map of L. edodes. This RAPD-linkage map greatly enhanced the mapping of other inheritable and stable markers [such as those that are linked to a phenotype (the mating type), a known gene (priA) and a sequenced DNA fragment (MAT)] with the aid of mating tests, bulked-segregant analysis, and PCR-single-strand conformational polymorphism. These markers comprised a genetic map of L. edodes with 14 linkage groups and a total length of 622.4 cM.     

WHBE 兔、日本大耳白兔和新西兰兔遗传多样性的 RAPD 分析

目的：应用随机引物扩增多态性DNA技术（ random amplified polymorphic DNA , RAPD）对大耳白黑眼兔（ white hair black eyes rabbit , WHBE rabbit ）、日本大耳白兔（ Japanese white rabbit , JW rabbit ）和新西兰兔（New Zealand white rabbit, NZW rabbit）3个实验兔品系进行遗传分析。方法选用90只实验兔的皮肤组织样品提取基因组DNA,用60个随机引物对实验兔基因组DNA进行PCR扩增,根据电泳结果筛选出多态性较高的引物进行RAPD-PCR分析,再利用Popgene 3．2统计软件对3个品系的扩增条带进行遗传分析,获得实验数据。结果分析结果表明：（1）60个随机引物中筛选出25个多态性较高的引物,3个品系实验兔共检测到493个扩增片段,长度在100～1800 bp之间,筛选的25个引物中,其中16个引物既可扩增出3个品系共同的DNA条带,也可扩增出WHBE兔特有的特征条带;（2） WHBE兔位点数为234个,其中多态位点数166个,多态位点比为70．94％,JW兔位点数为228个,其中多态位点数122个,多态位点比为53．51％,NZW兔位点数为231个,其中多态位点数94个,多态位点比为40．69％;（3）三个群体的Shannon多样性指数分别为0．3385,0．2222和0．1905;（4） JW兔和NZW兔的遗传相似系数最高,为0．8443,其次为WHBE兔和JW兔的遗传相似系数,为0．8204,WHBE兔和NZW兔的遗传相似系数最低,为0．7862。结论结果表明WHBE兔与JW兔和NZW兔之间有遗传的相似性,也存在着遗传差异,应用RAPD技术可以很好地检测实验兔不同品系之间以及同一品系不同个体之间的亲缘关系。