DNA分子标记的发展及主要技术

DNA分子标记技术是一种新的比较理想的遗传标记,本文综述了DNA分子标记技术的发展概况、种类及特点,以及广泛应用于农作物分子标记基因定位的分子标记技术：限制性片段长度多态性（RFLP）,随机扩增多态性DNA（RAPD）,简单序列长度多态性标记（SSR）和扩增的限制性内切酶片段长度多态性（AFLP）的原理、特点及他们的优缺点。     

RAPD技术在绿脓杆菌DNA分析中的应用

RAPD技术是近几年发展起来的克隆识别技术,以一条含17个碱基的随机引物对10株绿脓杆菌的DNA进行PCR扩增,经电泳,得到了8种DNA指纹图谱,重复实验,结果稳定,证明该技术适用于DNA多态性分析及分子流行病学研究。     

RAPD标记及其在植物育种上的应用

RAPD标记是一种基于DNA多态性的遗传标记，文章对RAPD标记的基本原理及其在植物育种上的应用做了综述，讨论了RAPD标记的优点及局限性，展望了其应用前景。     

DNA分子标记与动物育种

论述了DNA分子标记的分类以及限制性酶切片段长度多态性、随机扩增多态性DNA、扩增片段长度多态性、简单重复序列、单核苷酸多态性等5种主要DNA分子标记的基本原理和优缺点。同时，还分析了它们在动物遗传育种中应用情况和发展前景。     

AFLP技术及其在动物遗传育种中的应用

扩增片段长度多态性(AFLP)被称为“下一代分子标记”。是检测DNA多态性的一种新技术．该技术可靠性强,多态性检出率高,因而被认为是最有效的DNA指纹分析技术,AFLP已广泛应用于分类学、种群遗传学、病理学、DNA指纹分析的研究和建立数量性状基因图谱,成为最主要的遗传标记,论述了AFLP的原理、特点、影响实验成功的关键因素及在动物遗传育种中的应用。     

应用RAPD技术对新疆布顿大麦遗传多样性的研究

利用RAPD分子标记，通过40条10碱基随机引流，对32份新疆布顿大麦进行了遗传多样性评价。在40个10碱基随机引物中，有22个引物（占55％）的扩增产物具有多态性，每条多态性引物能产生1－7条多态性DNA带纹。40个引物共扩增227条带，其中95条（占41．9％）具有多态性，平均每条引物能产生1．7条多态性DNA带纹。基于RAPD技术计算了32份布屯大麦间的遗传相似系数（GS）变化值为0．427－0．848，平均值为0．681，利用不完全加权算术平均数（UPGMA），采用1－GS矩阵对其进行遗传聚类，结果表明，RAPD标记将32份参试材料区分为3类（或亚类），来源于新疆北部的20份材料有17份材料（占85％）聚入Ib亚类，而第Ia亚类则主要来自于新疆中部的一些材料。研究表明，RAPD标记揭示的新疆布顿大麦间的遗传多样性与其地理来源紧密相关。     

分子标记及其在作物育种上的应用

分子标记是随着遗传学的发展而诞生的一种基于DNA多态性的遗传标记.主要综述了几种分子标记的基本原理及其在作物育种上的应用,实践证明,分子标记技术为作物育种提供了一种新的研究手段,必将在作物育种领域开拓广阔的应用前景.     

高粱抗丝黑穗病基因的分子标记RAPD分析

丝黑穗病是影响我国高粱生产发展的主要病害之一,在高粱产区每年都有发生,发病率有时高达70 %,给生产造成很大损失,而选育抗病品种是最为有效的抗病策略.分别以高粱2381恢复系(抗病)、矮四恢复系(感病)的叶片为试材,采用CTAB法提取高粱基因组DNA,并应用RAPD筛选技术对高粱基因进行分子标记.在最佳的RAPD反应体系中共筛选了100个RAPD随机引物,筛选出来27个适宜引物,获得了稳定的RAPD扩增结果及多态性较好的DNA谱带.其中有6个引物对DNA扩增产生了差异谱带.     

RAPD鉴定远缘杂交牧草蔗新品系94-42

应用RAPD技术对远缘杂交牧草蔗新品系94—42，父本PT43—52及母本甘蔗栽培良种C0419进行分子鉴定．67个引物共扩增出284条带，其中l16条为多态性带，占40．85％．计算相似性系数，并对3个品种进行亲缘关系的分析，结果分别为：新品系和母本：0．9138；新品系和父本：0．8009；父本和母本：0．7658．RAPD结果证明，牧草蔗94—42基因组出现多态性，具有父、母本特异DNA带，是两者远缘杂交的后代．提示RAPD技术是一种适合远缘杂交物种分子鉴定的有效方法．     

动物线粒体DNA

线粒全DNA作为分子标记已被广泛应用于家畜育种、生物进化、发病机理、临床诊断等方面的研究，并取得了许多有价值的结果．综述了动物线粒体DNA的结构特征、遗传特征，并阐明了线粒体DNA多态性研究方法、研究进展以及线粒体DNA多态性在动物遗传育种中的应用．     

西瓜种植物RAPD分析影响因子的研究

以西瓜种内的１２个品种（系）为模板ＤＮＡ,研究了影响ＲＡＰＤ扩增效果的因素。结果表明,模板ＤＮＡ、Ｔａｑ酶、随机引物、Ｍｇ２＋浓度以及ＰＣＲ反应程序在ＲＡＰＤ分析中都具有重要的作用。根据以上因子的研究结果,建立了适合西瓜种植物的ＲＡＰＤ分析的技术体系：１０μＬ反应液中,模板１０ｎｇ／１０μＬ,引物０５μｍｏｌ／Ｌ,ＴｒｉｓＨＣｌ（ｐＨ８３）５０ｍｍｏｌ／Ｌ,ＢＳＡ５００μｇ／ｍＬ,ＭｇＣｌ２２５ｍｍｏｌ／Ｌ,ＴａｑＤＮＡ０６Ｕ／１０μＬ,ｄＮＴＰｓ２００μｍｏｌ／Ｌ,１０％蔗糖４００和１ｍｍｏｌ／Ｌ甲酚红。     

Analysis of length distribution of short DNA fragments induced by ^7Li ions using the random-breakage model

Deoxyribonucleic acid (DNA) is an important bio-macromolecule. DNA double strand breaks (DSBs) are considered to be the most important initial damage responsible for all biological effects induced by ionizing radiation. In this paper the length distribution of DNA fragments induced by ^7Li ionizing radiation is fitted with the random breakage model. In this model, the parameter u is the average number of DSBs on every DNA molecule induced by ionizing radiation. The fitting result shows that the random breakage model cannot describe the distribution of DNA fragments in lower doses, while the random breakage model is in better accordance with the experimental data in higher doses. It is shown that the length distribution of DNA fragments has random statistical feature in higher doses. In this situation, the random breakage model looks like a model without any parameter since the u has specific physical meaning and can directly be obtained from experimental data.     

一氧化氮对食管癌细胞核DNA的损伤作用

以硝普钠 ( Sodium Nitroprusside,SNP)作为 NO( Nitric oxide)的体外供体 ,以食管癌细胞系 EC1 0 9作为细胞模型 ,采用单细胞凝胶电泳法检测不同浓度的 NO作用不同时间后 ,食管癌细胞核 DNA被损伤的情况 .检测结果表明 ,在一定的条件下 ,NO对食管癌细胞核 DNA的损伤作用具有明显的浓度和作用时间依赖性特征 ,总的来讲 ,NO的浓度越大 ,NO对细胞作用的时间越长 ,细胞核 DNA所遭受的损伤越重 .     

不同纬度野生大豆种群间的遗传变异

选用了经初步筛选的２０个短随机序列引物和２个长随机序列引物对５个不同纯度来源的野生大豆进行了ＤＮＡ随机扩增,其中有１９个引物扩增出多态性片段,区获得７０个ＲＡＰＤ多态性片段,平均每个引物３．６８条,运用自编的计算机程序对扩增进行聚类分析。     

纳米谷氨酸壳聚糖制备及其体外质粒转染研究

采用表面修饰方法制备出谷氨酸修饰的壳聚糖纳米基因载体。对样品进行红外分析、粒度分析、zeta电位分析、生物相容性、凝胶阻滞分析、DNA保护性试验、体外细胞转染研究。结果显示所制得的谷氨酸修饰的壳聚糖纳米颗粒平均粒径为170nm,其zeta电位为 4.7mV。红外分析显示谷氨酸已通过酰胺键结合在壳聚糖上。MTT实验结果显示纳米颗粒与细胞有良好的生物相容性。凝胶阻滞分析和DNA保护试验结果表明纳米载体可与DNA通过电性结合作用而结合,并可以有效保护DNA,防止核酸酶对其的降解作用。而体外细胞转染的结果表明,谷氨酸修饰的纳米粒能介导pEGFP-N1质粒转染HepG2细胞并在细胞中表达绿色荧光蛋白。因此,谷氨酸修饰的壳聚糖纳米颗粒可作为一种新型非病毒基因载体介导核酸类生物大分子进入细胞内。     

Reassembly of shattered chromosomes in Deinococcus radiodurans

Dehydration or desiccation is one of the most frequent and severe challenges to living cells. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called 'extended synthesis-dependent strand annealing' (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become 'sticky ends' that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.     

Inhibitory Effect of Lithium Chloride on Mouse Thymocyte Apoptosis

This study investigates the effect of lithium chloride (LiCI) on mouse thymocyte apoptosis. A primary culture of mouse thymocytes was preincubated with LiCI (from 5 to 500μmol/L) before exposure to dexamethasone (DEX), the apoptosis inducer. With 100μmol/L of LiCI, apoptotic cell death induced by DEX was almost completely prevented as determined by flow cytometric analysis, terminal deoxynudeotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and DNA laddering assay. The results show that the DEX-induced increment of caspase-3 activity in thymocytes is completelye liminated by LiCI preincubation. The results suggest that LiCI may protect Balb/c mouse thymocytes from apoptosis induced by glucocorticoid in a dose-dependent matter.     

一种基于构件重组建立超大容量DNA文库的方法

设计固定长度的随机序列,并在随机序列的末端加上共用接头作为标准构件,利用共用接头之间的互补连接,用重聚PCR的方法将标准构件进行随机组合,得到1个序列多样化的DNA文库。重聚优化的条件为:随机序列质量浓度18 ng/μL,重聚PCR退火温度27.8℃,终止引物浓度为随机序列浓度的1/40。文中方法的建立克服了目前突变体文库建立方法中强烈依赖起始基因的弊端,可以开拓更为广阔的序列空间。     

应用单细胞凝胶电泳技术测定农药对蚯蚓的DNA损伤

探讨采用当前国际上较先进的ＤＮＡ损伤检测技术－单细胞凝胶电泳技术,来检测农药对蚯蚓ＤＮＡ的损伤,由于蚯蚓是陆生生物与土壤生物之间传递污染物的桥梁,能很好地指示土壤环境的变化,是评价农药对生态环境安全性的一项重要指标。尝试建立蚯蚓的单细胞凝胶电泳来评价农药对生态环境的影响,结果表明两种新型农药对蚯蚓的ＤＮＡ均有损伤作用。认为蚯蚓的单细胞凝胶电泳技术能敏感地农药对细胞ＤＮＡ的损伤,并可作为农药对土壤污     

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.