DNA content alterations in Tetrahymena pyriformis macronucleus after exposure to food preservatives

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人类基因组计划（HumanGenomeProject）

⼈类基因组计划（HumanGenomeProject）⼈类基因组计划（Human Genome Project,HGP）1．什么是⼈类基因组计划：⼈类基因组计划是由美国能源部和NIH联合做出的，⾃1990年开始，争取在15年内完成的⽬标。

即：鉴定⼈体DNA估计约8万个基因，测序构成⼈DNA的30亿个碱基，贮存这些信息于databases(数据库)并发展data analysis的⼯具。

（1）实际包括两部分⼯作，⼀是mapping，⼀是sequencing，故先前叫做“Mapping and Sequencing the human genome”.⽽Mapping⼜分为遗传连锁图谱和物理图谱。

（2）HGP是第⼀个庞⼤的科学事业，会引起⼀些由此计划暴发出来的伦理、法律、社会学上的诸多争论。

（DOE熟悉⼤科学模式；⽣物学家习惯⼩科学模式，应完美结合。

该计划会引发出许多商业和法律，社会学和论理学⽅⾯的问题。

）（3）为了有助于这些⽬标的实现，还要研究⼀些⾮⼈⽣物体的遗传图谱。

（包括E.coli、酵母、秀丽隐杆线⾍、果蝇、实验⽤⼩⿏等模式⽣物。

）（4）在植物⽅⾯，美国农业部集中研究⽟⽶和南芥菜（Arabidopsis）基因组，我国科学家提出了⽔稻基因组计划。

2．背景：早在1984年Utah州Alta城的专业会议（DOE环境与健康研究办公室，OHER 和国际环境诱变剂和致癌物防护委员会，ICPEMC协办）。

开始讨论HG DNA全序列测定的前景。

1985年5⽉由Sinsheimer组织专门会议提出测定HG全序的动议。

DOE为何操办：（1）DOE承担低⽔平辐射和其它环境因素引起的遗传性损伤的监测，即需要在108bp的DNA中检测出⼀个碱基的改变，此项任务与HG全序列测定有关并且任务同等艰巨；（2）DOE已在两个国家实验室对复杂基因开展了⼯作，即1988年的国家基因⽂库计划（NG Library Project），在Laurence Livermore国家实验室（LLNL）中纯化单种染⾊体并构建单个染⾊体⽂库。

棉花GhManA2基因纤维特异表达启动子的克隆与功能初步分析的开题报告

棉花GhManA2基因纤维特异表达启动子的克隆与功能初步分析的开题报告一、研究背景及意义棉花是世界上最重要的农作物之一，棉花纤维是人类最早使用的纺织原材料之一。

GhManA2基因编码的酵母甘露醇结合蛋白在棉花纤维合成过程中具有重要作用，因此对该基因的研究具有重要意义。

GhManA2基因表达模式的研究和启动子的克隆对于解析棉花纤维发生、发育及合成机制，以及开发新的棉花品种具有重要的参考意义。

二、研究目的本研究旨在克隆棉花GhManA2基因的启动子区域，并对其进行初步的功能分析，为深入研究该基因的表达调控机制提供基础。

三、研究内容1.克隆GhManA2基因的启动子区域。

采用PCR技术依据基因序列信息，设计引物克隆GhManA2基因的启动子区域，利用测序技术对其进行验证。

2.构建转录报告载体并进行表达分析。

利用启动子区域克隆的结果构建转录报告载体，利用荧光素酶检测系统对其进行表达分析，并评估其在棉花中的表达活性和特异性。

3.初步分析GhManA2基因启动子的功能。

使用转录报告载体在棉花中构建瞬时表达系统，分析GhManA2基因启动子的启动活性、响应外界刺激的能力以及不同部位的表达模式。

四、研究方法1.克隆GhManA2基因启动子区域利用PCR技术克隆GhManA2基因启动子区域，并对其进行测序验证。

2.构建转录报告载体并进行表达分析将克隆得到的启动子片段克隆至转录报告载体中，通过荧光素酶检测系统对其进行表达分析，检测载体在棉花细胞中的表达活性和特异性。

3.初步分析GhManA2基因启动子的功能利用转录报告载体在棉花中构建瞬时表达系统，通过检测荧光素酶活性、定量PCR等技术方法，分析GhManA2基因启动子的启动活性、响应外界刺激的能力以及不同部位的表达模式。

五、研究意义本研究可以为深入探究 cotton GhManA2基因的调控机制和棉纤维发生发育、合成机制提供重要的参考和基础，对促进棉花新品种的培育和棉花产业的发展具有重要意义。

南开大学细胞生物学综合复习题

第一章绪论1.细胞生物学的研究内容有哪几个方面、包含哪几个层次？2.简述细胞学说的主要内容。

3.纵观细胞生物学发展史，你认为该学科近百年来快速发展的主要原因是什么？第一章习题解析：1.简要说明细胞生物学的研究内容及其发展方向。

细胞生物学是研究细胞基本生命活动规律的学科，其研究的对象是各种细胞及其相关的各个方面。

它不仅研究细胞各个部分的结构和功能，而且研究细胞的增殖、分化、衰老与死亡、细胞信号传递等各个方面的生命活动，以及细胞的起源和进化等各种生命现象。

因此，理论上讲其研究内容涵盖了细胞的各种层次和各个方面。

目前，细胞生物学的发展方向有两个（1）是在分子水平上不断深入地解析细胞及其各个部分的结构和功能；（2）是在系统综合的向度上将细胞整体水平、亚细胞水平和分子水平三个方面的研究成果有机地结合起来，以动态的观点来考察细胞和细胞器的结构与功能，全面深入地解读细胞的各项生命活动。

深刻性与综合性是当代细胞生物学及其进一步发展的特点。

2.简述细胞学与细胞生物学的发展历史。

细胞学开始孕育于细胞的发现之时。

经过170多年的资料积累，特别是19世纪上半叶，随着显微镜质量的提高和切片机的发明，细胞学说于1838~1839年创立，这是细胞学的第一次飞跃。

到1892年，Hertwig的《细胞与组织》一书出版，使细胞学作为一个独立的学科正式诞生。

随后又经过几十年的发展，到20世纪40年代，细胞学已发展成为一门内容丰富、学科体系比较完整的学科。

然而，细胞学主要是一门描述性学科。

20世纪50年代开始，电子显微镜与超薄切片技术相结合，产生了细胞超微结构学，使对细胞结构的认识得到了很大程度的更新和拓展；生物化学与细胞学的相互渗透和结合，使细胞生化得到了快速发展；70年代以来，科学家们将分子生物学的概念与技术引进了细胞学，为细胞生物学的最后形成与建立奠定了坚实的基础。

目前细胞生物学与分子生物学在许多领域仍互相交汇和融合，其研究内容与范畴与生命科学的其它学科往往交错在一起，以致目前很难为细胞生物学划出一个明确范围。

DNA的粗提取与鉴定改进实验

人教和人教版高中生物选修1 订本必修》和人教版高中生物选修1 生物技术实践》都开设《DNA的粗提《生物技术实践》都开设《DNA的粗提取和鉴定》的实验教学。取和鉴定》的实验教学。这是一个较为复杂的生化实验，为复杂的生化实验，教材中的实验过程存在着：操作步骤繁琐，程存在着：操作步骤繁琐，操作要求实验所需时间长，所用药品多、高；实验所需时间长，所用药品多、量大；提取到的DNA的量少， DNA的量少量大；提取到的DNA的量少，观察效果不佳等不足之处。不佳等不足之处。为了更方便地开展实验课，我们对原实验进行了改进。实验课，我们对原实验进行了改进。
实验结果与结论
DNA的粗提取与鉴定改进实验 DNA的粗提取与鉴定改进实验
汕头林百欣中学刘雁芸
实验方法步骤
1、破碎细胞，释放DNA 破碎细胞，释放DNA 2、溶解细胞核内的DNA 溶解细胞核内的DNA 析出含DNA DNA的黏稠物 3、析出含DNA的黏稠物 ——提取含杂质 4、洗涤黏稠物——提取含杂质洗涤黏稠物—— 较少的DNA 较少的DNA DNA的鉴定 5、DNA的鉴定
实验自评：实验自评：
本实验虽然得到的DNA纯度不够， DNA纯度不够 ① 本实验虽然得到的DNA纯度不够，但快速而且也遵循实验原理。也遵循实验原理。实验材料、药品、仪器、设备用量减少， ②实验材料、药品、仪器、设备用量减少，降低了对环境的污染且降低成本。了对环境的污染且降低成本。实验材料具有广谱性，广东各地均易于取材， ③实验材料具有广谱性，广东各地均易于取材，价格便宜。一般的普通中学均能开设，价格便宜。一般的普通中学均能开设，从而有利于该实验的普及推广。于该实验的普及推广。步骤简单，操作易行， ④步骤简单，操作易行，学生很容易掌握操作过节省时间。程，节省时间。实验效果清晰，易于判断，达到了实验目的。 ⑤实验效果清晰，易于判断，达到了实验目的。

DNA测序中常见影响因素的研究

生物技术通报・研究报告・ BIOTECHNOLOGY　BULL ETIN 2005年第1期DNA测序中常见影响因素的研究宋艳斌1　马文丽1　郑文岭2(1南方医科大学分子生物学研究所,广州　510515;2广州军区肿瘤分子生物学研究所,广州　510010)摘　要:　对测序中的模板、引物、测序反应条件及测序反应纯化方法和仪器操作等进行研究。

结果显示测序模板的纯度影响测序的质量,浓度对测序的长度有影响。

引物设计时除符合一般设计原则外,T m值最好在50℃～60℃之间,且无成串的G、C。

改变变性、退火、延伸的时间和温度对特殊DNA模板的序列测定有较好的效果。

测序反应产物的纯化有几种方法,以70%乙醇沉淀法最经济、方便。

因此模板的纯度和浓度对测序成功与否起决定作用。

最佳反应条件可降低成本,提高测序成功率,乙醇沉淀法是首选的测序反应产物纯化方法。

仪器操作熟练、正确与否也会影响测序结果。

关键词:　DNA　循环测序　影响因素The I nfluence of Some F actors on DNA SequencingS ong Y anbin1　Ma Wenli1　Zheng Wenling2(1Department o f Biochemistry,the Fir st Military Medical Univer sity,Guangzhou　510515;2Institute o f Molecular Oncology,Liu Hua Qiao Hospital,Guangzhou　510010)Abstract:　The effects of DNA tem plates,primers,cycle sequencing reaction conditions,purification methods,operation of in2 strument were com paratively analyzed.The quality of sequencing was in fluenced by the purity of tem plates,the length of sequenc2 ing was in fluenced by the concentration of tem plates.The T m value of primers should be between50℃～60℃and there shouldn’t be too much G+C in the primers.The alteration of the tem perature and time of the denaturation,annealing,extension facilitated sequencing of s ome of the DNA tem plates.The purity and concentration of DNA tem plates were closely related to the quality se2 quence data.The optimized reaction conditions could decrease the cost of sequencing and im prove the ratio of success.The ethanol precipitation was the optimized method of products purification of the sequencing reactions.Instrument operation could als o in flu2 ence the sequencing results.K ey w ords:　DNA　Cycle sequencing　FactorDNA序列测定是分子生物学领域最常用的技术之一,是了解基因结构和功能的基础。

改进的SDS_CTAB法提取濒危植物连香树总DNA_黄绍辉

武汉植物学研究2007,25(1):98～101J ou rna l of W uhan B otan ica l R esea rch改进的S D S2CTAB法提取濒危植物连香树总D NA黄绍辉,方炎明3(南京林业大学,南京　210037)摘　要:对珍稀濒危植物连香树(Cercidiphyllum japonicum)的6种总DNA提取方法进行了对比试验,结果表明改进的S DS2CT AB法更适合于连香树总DNA提取。

该方法提取的DNA经紫外消光值检测,其A260/A280为1.8532,优于CT AB法(1.4872)、S DS法(1.3552)、P VP法(1.5079)、尿素法(1.1858)和高盐低pH法(1.4534)。

琼脂糖凝胶电泳和PCR扩增结果也得出同样的结论。

关键词:连香树(Cercidiphyllum japonicum);DNA提取;改进的S DS2CT AB中图分类号:Q946123234 文献标识码:A 文章编号:10002470X(2007)0120098204D NA Extracti on of the Endangered tree Spec i es C ercidiphyllum japon icumBa sed on the M od i f i ed M ethod of S D S2CTABHUANG Shao2Hui,F ANG Yan2M ing(N anjing Forestry U niversity,Nanjing　210037,China)Abstract:Six methods of DNA extracti on of endangered tree s pecies Cercid iphyllum japonicum are ana2 lyzed and compared in this article.The results showed that the modified method of S DS2CT AB is better than the others in ter m s of the quality of t otal DNA extracti on.The value of A260/A280is1.8532f or DNA extracted by this of modified S DS2CT AB method.It’s higher than the others,such as1.4872for CT AB method,1.3552f or S DS method,1.5079f or P VP method,1.1858f o1r urea method and1.4534f or l ow pH mediu m with high salt method.The results of electr ophoresis and PCR a mp lificati on als o indicated that the modified method of S DS2CT AB is better than the others.Key words:Cercidiphyllum japonicum;DNA extracti on;Modified S DS2CT AB method DNA的分离提取是进行植物分子生物学研究工作的基础,DNA样品质量是分子生物学实验成败的关键因素之一。

用cDNA末端扩增法克隆柞蚕攻击素(attacin)基因及序列分析

ＡｂｔａｔＴｈｓｒｃｅｃＤＮＡｓｑｕｎｅＯｔｃｎｇｎ（ｎｎｃｅｉｎＮｏＡＹ６６０）ｒｍｉｅｅＯａｌ－ｅｅｃｆａａｔｉｅｅＧｅＢａｋａｃｓｏ９０８ｆａＯＣｈｎｓｋＳｉｋ
号：Ｙ６６０。柞蚕ｈ－Ａ９０８）Ａ素基因ｅＮＤＡ序列全长９２ｂ，中６９ｂｇ蛋白质编码区可编码２３个氨基酸。预测１ｐ其９ｐｊ３
蛋白质分子量为２Ｄ，５ｋ等电点（Ｉｐ）为７５。柞蚕攻击素基因中包含２个内含子。ＳｇａＰ３０Ｓｒｒ．４ｉｌ，ｅｅ程序分析结ｎｖ果显示，柞蚕ｈＥ素第ｌ７位氨基酸为信号肽序列。蛋白质功能区分析预测，－ —ｌ柞蚕攻击素在第４７一ｌ２氨基酸之１
ａｄｉｌａｕ５ｎｔｐｌｅｉ７．４．ＦＯｇｎｍｉｓｖｓｒｍｅｏｃＤＮＡ．ｔｅｅａｅｔｎｒｓａｄｔｒｅｅｏｓｗｉｉｔｅｏｅｅｄｎｈｒｒｗｏｉｔｏｎｎｈｅｘｎｔｎｈｐｎｒａｉｇｈｆｍｅ．Ａｍｉｏａｉｅｅｎｅａａｙｉｇｂｉｎｌ０ＳｒｅｈｗｅｈｔｈｒｓａｓｇｌｅｐｉｅｏｍｉｒａｎｃｄｓｑｕｃｎｌｓｎｙＳｇａ３．ｅｖｒｓｏｄｔａｅｅｉｉｎａｐｔｆ１ａ－Ｐｔｄ７ｎｃｄｌｌｅＮ．ｅｍｉａ．Ｐｅｃｉｆｐｏｅｎｆｕｃｉａ１ｏａｎｉｄｃｔｄｌａｈｔｒｎ１ｏｉｆＡｐｏａｉｓａｈｔｒｎ１ｒｄｉｔＯｒｔｉｏｎｔｏｎｏｎｍｉｎｉａｅｈｌｅＮ－ｅｍｉａｍａｎＯ－ｄｔｄａｔｃｎｉｆｍ７ｌ２ａｎｃｄａｄＣ．ｅｍｉａｏａｎｉｆＯ１３ｌ３３ａｉｏａｉｖＰａ．１ｄｄ— ｔｉＳｒ４Ｏ１ｍｉｏａｉｎｔｒｎ１ｍｉＳｒｍＯ２ｍｎｃｄｂｆｍａＯ１ｄ１ｎａｉ

Quantabio repliQa

Quantabio, 100 Cummings Center Suite 407J, Beverly, MA 01915IFU-115.1 Rev01repliQa™ HiFi Assembly MixCat. No. 95190-010 95190-050Size:10 reactions 50 reactionsStore at -25°C to -15°CDescriptionThe repliQa™ HiFi Assembly Mix simplifies the construction of recombinant DNA through the simultaneous and seamless assembly of multiple DNA fragments possessing terminal regions of sequence overlap in a single, isothermal reaction. Similar in principle to the Gibson Assembly ® Method 1, the high efficiency repliQa HiFi Assembly Mix is ideal for a range of genetic engineering applications including routine molecular cloning, site-directed mutagenesis, assembly of large constructs for synthetic biology applications, and the construction of diverse sequence libraries for directed evolution studies. The concentrated, two-component format allows flexibility in design of assembly reactions and compatibility with less concentrated DNAsamples. The repliQa Mix has been optimized for use with a total input quantity of DNA fragments in the range of 0.03 to 0.5 pmols. The assembly of up to six DNA fragments is recommended, though the repliQa Mix has been successfully used for more complex assemblies.Double stranded DNA fragments for assembly can be generated by PCR amplification, chemical synthesis, or isolation of restriction fragments. When working with fragments PCR amplified from plasmid vectors, the included DpnI restriction endonuclease can be used for selectively digesting methylated, residual plasmid DNA to reduce background transformants. The repliQa mix is directly compatible with most common E. coli cloning hosts and generally provides a high yield of accurately assembled product.The DNA assembly occurs through the actions of three enzymes:• A non-thermostable 5' to 3' exonuclease that partially eliminates one strand of a DNA duplex to expose complementary overlap regions forhybridization.• A high-fidelity thermostable polymerase that fills the gaps remaining between the hybridized fragments of the overlapping regions.• A thermostable DNA ligase that covalently seals the resulting nicks at fragment junctions, generating double-stranded, assembled DNA moleculessuitable for transformation of cells.ComponentsReagent Description95190-01095190-050 repliQa HiFi Assembly Enzyme Mix Optimized formulation of enzymes for 5’-endresection, high fidelity 3’-end extension, and nick sealing.1 x 0.02 mL1 x 0.10 mLrepliQa 10X Assembly Reaction Buffer 10X reaction buffer containing dNTPs, magnesium, and cofactors.1 x 0.1 mL 1 x 0.50 mLDpnI (20 U/µl)Restriction endonuclease for the (optional) post-PCR digestion of residual unamplified plasmid template.1 x 0.05 ml 1 x 0.25 mlStorage and StabilityStore kit components in a constant temperature freezer at -25°C to -15°C upon receipt. For long term buffer storage (> 30 days) store buffer at -70°C. Refer to the product label or lot-specific Product Specification Sheet (PSF) available at /resources for applicable expiration date.A general diagram of assembly cloning is shown below:Additional reagents and materials that are not supplied• PCR-Grade, nuclease-free water (do not use DEPC-treated water)• High Fidelity DNA Polymerase (Enzymatics VeraSeq TM 2.0, P7511L or equivalent)• A heat block, thermocycler, or water bath capable of holding a temperature of 50 ± 2°C for one hour. • PCR or microcentrifuge reaction tubes.• PCR product purification kit (QIAGEN ® QIAquick ® PCR Purification Kit, 28104 or equivalent). •Competent E. coli cells and accessories as recommended by manufacturer.Before you begin• Design the DNA fragment sequences and assembly strategy. Guidelines are given in Appendix 1.• (Optional) Treat PCR reaction with DpnI if plasmid DNA was used as template for generating DNA fragments to be assembled. (Appendix 2).•(Recommended) After determining PCR fragment or restriction endonuclease-digested fragment size and purity by agarose gel electrophoresis, purify using a spin column-based cleanup or other method. This step is not required but is highly recommended to achieve highest efficiency of fragment assembly.• Measure the concentration of each isolated DNA fragment by absorbance at A 260 or by using a fluorometric quantitation reagent. Agarose gel electrophoresis with mass-calibrated size standards can also be used to quantify fragment mass and quality simultaneously. • Calculate the number of picomoles of each fragment using the following formula:pmols = (weight in ng) x 1000/(bp x 662).• Determine the number of pmols of each fragment to add to the assembly reaction. For cloning, highest efficiencies are achieved with 0.02 to 0.04 pmols of linear vector fragment (50 to 100 ng of 4 kb vector) and 2 to 8-fold molar excess of inserts. • Prepare outgrowth medium and culture plates with appropriate antibiotics for plasmid selection.•Equilibrate the heat block, thermal cycler, or water bath to 50°C for incubation of the assembly reactions .Protocol1. Thaw the repliQa HiFi Assembly Kit components, briefly vortex to mix, and place on ice.2. For each assembly, add reaction components in the order listed in the table below to chilled reaction tubes.The optimal amount of enzyme mix to add per assembly reaction depends on the total quantity of DNA fragments present.ComponentRxn. component volumes (µl) for varying amounts of total DNA≤ 0.125 pmol> 0.125 pmol but ≤ 0.25 pmol > 0.25 pmol Nuclease-free water(17.5 – X) µl (17.0 – X) µl (16.0 – X) µl repliQa 10X Assembly Reaction Buffer 2.0 µl 2.0 µl 2.0 µl DNA fragmentsX µl X µl X µl repliQa HiFi Assembly Enzyme Mix 0.5 µl 1.0 µl 2.0 µl Total volume20 µl20 µl20 µl3. Incubate reactions at 50°C in heat block, thermal cycler with heated lid (set to ~60-80°C), or covered water bath for 1 hr. Hold assembled product mix at 4°C until ready to proceed with transformations. If transformations cannot be performed on the same day, reactions can be stored at -20°C for up to one month.4. Competent E. coli should be transformed, recovered, and plated as per manufacturer guidelines or standard lab practices. Note: If electroporation is to be used for transforming cells, we recommend first diluting the assembly reaction 1:5 in high purity water. There is no need to dilute the assembly reactions prior to transformation of chemically competent cells.5. (Optional) Analyze a portion of the remaining assembly reaction by agarose gel electrophoresis. If DNA fragment assembly occurs properly, a ladder of higher molecular weight DNA bands would be generated.Note: For reactions using three or fewer fragments the incubation time in step 3 can be shortened to 15 minutes.Appendix 1 – Guidelines for Designing DNA Fragments for Assembly1.When designing the DNA fragment sequences and assembly strategy, allow for a region of sequence homology between adjacent DNA fragments.Be sure to avoid regions of repeated bases or repeated short DNA motifs in the design of these overlaps where possible. Regions of secondary structure such as hairpins or stem loops should also be avoided.2.The kit is optimized for the assembly of fragments with overlap regions between 15 – 60 bp. It is recommended that the overlaps be at least 20bp with a minimum of 25% GC content, however overlaps of 30 bp or longer size will provide higher efficiency assembly reactions.3.For generating PCR fragments to be assembled, design primers with a 5’ segment of homology to the adjacent fragment or vector. If the adjacentfragment is also generated by PCR amplification, the overlap can be split between two primers if desired. The 3’ segment of primers should contain sequence specific to the DNA target of interest. Amplify targets using a high-fidelity thermostable DNA polymerase such as VeraSeq 2.0 (Enzymatics, P7511L) or equivalent per manufacturer instructions.4.When designing synthetic gene fragments for assembly, ensure that the 5’ and 3’ segments contain regions of homologous overlap sequencebetween adjacent gene blocks, PCR fragments, or isolated restriction fragments.5.For site-directed mutagenesis applications, the assembly strategy should be designed such that the mutation of interest is centered betweenadjacent PCR fragments. Design the PCR primers as with the standard fragments above, except that the mutation (substitution, insertion, or deletion) should be included within the 5’ segments for both of the adjacent fragments.6.When designing DNA fragments to be assembled with isolated restriction fragments, be aware that any 5’ overlaps from staggered restriction cutswill be eliminated because of the 5’-->3’ nuclease present in the assembly mix, and so should not be included in the measurement of overlap size. If desired, design the 5’ overlap segment of the adjacent fragment to either preserve or eliminate the restriction site.Appendix 2 – DpnI treatment to remove residual plasmid DNAWhen plasmid vector is used as PCR template to generate a fragment for assembly, it is recommended that the reaction be treated with DpnI to eliminate residual methylated plasmid prior to setting up the assembly reaction.1.Add 1 µl DpnI (20U) directly to the PCR reaction (50 µl) following amplification of fragment.2.Incubate at 37°C for 1 hr.3.Heat inactivate DpnI by incubation at 80°C for 20 min.4.(Recommended) Purify the fragment using a spin column-based PCR purification kit.Quality ControlThe repliQa HiFi Assembly Mix is functionally tested for assembly of three 1-kb PCR fragments into 2kb and 3 kb products.The individual components of the repliQa HiFi Assembly Mix are tested to be free of contaminating DNase and RNase.Limited Label LicensesThis product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.References1. Gibson, D.G., et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-5.。

兰花DNA的优化提取

兰花DNA的优化提取摘要：我国的兰花品种繁多，因而鉴别起来比较困难，而现在我们采用分子标记进行兰花品种鉴别是一种比较有效的方法，而在进行分子标记实验时，DNA的浓度和纯度的要求都是相当严格的。

因此，本实验采用SDS方法和CTAB方法分别对兰花的四个品种春兰(Cymbidium goe-ring)、蕙兰(Cymbidium)、寒兰(CyambidumMakion)、墨兰(Cymbidium sinense)进行DNA提取并进行比较，结果表明SDS方法是这几种兰花DNA的优化提取法。

关键词：兰花；DNA；SDS；CTAB；优化提取Abstract: orchid is kinds of variety in china,Thus more difficult to identify,Now using molecular markers to identify species is a more effective method,Conducted experiments marker, of the requirements are quite stringer DNA concentration and purity of the requirements are quite stringent,Therefore, the use of experimental methods and CTAB SDS respectively Chunlan(Cymbidium goe-ring)、Huilan (Cymbidium)、Hanlan(Cyambidum Makion)、Molan(Cymbidium sinense)orchid varieties of extracting DNA comparison,The results showed SDS is these types of orchids DNA optimization of extraction method.Key words：Orchlid,DNA,SDS,CTAB,Optimization of extraction兰花一般是指兰科植物(Orchlidaceae)，它是鲜花植物中最大科之一，全世界约有800 多属，25000多种，分布于世界各地，大多数种类分布于东南亚、澳大利亚、中南美洲、非洲和马达加斯加。

哺乳动物组织基因组DNA提取实验报告

二．实验报告
1、实验现象与结果
（1）、冰冷的生理盐水清洗肝脏后肝脏会泛白，洗涤液不再呈现血红色；（2）、玻璃匀浆器匀浆后肝脏被磨碎，呈现血红色；
（3）、离心后离心管底部出现沉淀，上层为淡黄色液体；
（4）、加入①和②提取液离心后都会分成有机相和水相，二者明显分层；（5）、放入超低温冰箱1到2小时后取出来时看到液体凝结；
（6）、加入75%冷乙醇离心后看到离心管底部有沉淀；
（7）、电泳后在紫外光下观察时看到明亮的条带，同时还有一些条带不太鲜亮，有拖尾现象。

2．对实验现象、实验结果的分析及其结论
（1）、玻璃匀浆器匀浆后，溶液呈现血红色，因为细胞破裂释放出了血红蛋白；
（2）、离心后的上清液含一些细胞碎片等废弃物；
（3）、加入提取液离心后，溶液分层，上层为水相，下层为有机相：水相含有我们所需要的DNA等物质，有机相为提取液酚类等物质；（4）、多次离心，移出水相，目的在于提取出DMA；
（5）、之后的分别加入NaAc、无水乙醇、冰乙醇目的在于沉淀DNA，并纯化，因为核酸与钠、钾、镁形成的盐在许多有机溶剂中不溶，也不会变性。

因此，离心后，溶液中出现沉淀，弃上清液，取沉淀；（6）、条带亮说明DNA浓度很高；一个泳道出现几个不同的条带说明DNA在过程中被降解了，降解成不同片段大小的片段，而在电泳过程中跑的速度不一样，所以呈现不同条带。

dna富含a的序列与金纳米团簇的关系_概述及解释说明

dna富含a的序列与金纳米团簇的关系概述及解释说明1. 引言1.1 概述在生物化学领域中，DNA的研究一直是一个重要的课题。

DNA是构成生物体的遗传信息分子，对于维持生命活动具有至关重要的作用。

而金纳米团簇则是一种微小且具有特殊性质的金纳米结构，近年来引起了广泛关注和研究。

本文将探讨DNA富含A的序列与金纳米团簇之间的关系。

DNA富含A的序列在生物体中普遍存在，并具有特殊的结构和功能。

金纳米团簇作为一种新兴材料，展现出了许多优异的物理、化学性质，并在生物医学、光电子学等领域中显示出潜力。

通过对DNA-A序列与金纳米团簇相互作用机制进行深入研究，可以揭示二者之间可能存在的相互影响和潜在应用价值。

同时，我们还将探讨影响这种相互作用过程的因素，以及进一步扩展该领域研究可能面临的局限性和未来发展方向。

1.2 文章结构本文将按照以下结构展开对DNA富含A的序列与金纳米团簇关系的探讨。

首先，在第2节中，我们将简要介绍DNA富含A的序列和金纳米团簇的特性和应用。

随后，第3节将提供具体实验方法与结果分析，以便更好地理解二者之间的相互作用机制。

接下来，第4节将重点探讨影响DNA-A序列与金纳米团簇相互作用的因素。

最后，在第5节中，我们将总结主要发现，并展望未来该领域可能的进一步研究方向。

1.3 目的本文旨在通过对DNA富含A的序列与金纳米团簇关系进行系统分析和阐述，为深入理解二者之间的相互作用提供更多科学支持。

同时，本文还为进一步探索这一领域潜在应用提供了思路和指导。

通过本文内容发布，期待能够推动相关科学工作者对于DNA和金纳米团簇交叉领域进行更广泛、深入的讨论和合作，并为生物医学、材料科学等领域带来新的突破与创新。

2. DNA富含A的序列与金纳米团簇的关系2.1 DNA富含A的序列介绍DNA（脱氧核糖核酸）是由四种不同的碱基组成的双螺旋结构分子，包括腺嘌呤（A）、胸腺嘧啶（T）、鸟嘌呤（G）和胞嘧啶（C）。

在DNA链中，A和T以双氢键相互配对，形成A-T碱基对；而G和C以三氢键相互配对，形成G-C碱基对。

QPCR检测盐胁迫下钝顶螺旋藻盐相关基因mRNA的表达差异的开题报告

QPCR检测盐胁迫下钝顶螺旋藻盐相关基因mRNA的表达差异的开题报告一、研究背景及意义钝顶螺旋藻（Spirulina platensis）是一种广泛应用于食品、药品和饲料等领域的蓝细菌藻类。

该藻类长期生长于盐碱地区，具有良好的耐盐性，能够适应极端的环境，因此其在应对盐胁迫方面具有独特的基因机制和代谢途径，是盐碱地区生态环境恢复及生物资源开发的理想模式生物。

盐胁迫是影响植物和藻类生长、发育和产量的重要环境因素，具有复杂的生理和分子生物学响应机制。

盐胁迫会引起细胞膜的损伤、离子和营养物质的平衡失调，激活一系列胁迫响应途径，导致大量的基因表达的改变和蛋白质的合成。

因此，对钝顶螺旋藻在盐胁迫下盐相关基因mRNA的表达差异进行研究，既可以深入了解其盐胁迫响应机制，又可为该藻类的适应性与耐受性提供参考依据，为实现藻类生产的高效化、可持续化和经济化提供科学依据。

二、研究内容及方法1. 研究内容本研究旨在利用QPCR技术检测盐胁迫下钝顶螺旋藻盐相关基因mRNA的表达差异，以验证其对盐胁迫的响应机制。

主要包括以下两个方面：（1）构建钝顶螺旋藻盐相关基因表达谱分别在高盐和低盐环境下培养钝顶螺旋藻，收集细胞样品并提取RNA，采用RT-PCR技术构建其盐相关基因表达谱，筛选感应表达量较大的基因作为后续实验的检测标志点。

（2）QPCR技术检测盐胁迫下钝顶螺旋藻盐相关基因mRNA的表达差异利用QPCR技术检测盐胁迫前后钝顶螺旋藻中感应表达量最大的盐相关基因的mRNA表达量，验证盐胁迫对该藻类盐相关基因表达的影响，为后续的基因功能研究提供一定的理论支持。

2. 方法（1）样品处理选取生长良好的钝顶螺旋藻菌液，分别加入不同浓度的NaCl溶液（如200mM、400mM、600mM等），培养48小时后，采用离心收集上清液，用RNAzol方法提取总RNA，并按照超微量RNA检测仪检测RNA的质量和纯度。

（2）cDNA合成按照RevertAid First Strand cDNA Synthesis Kit的说明书进行反转录反应，将总RNA转化为cDNA，并使用100ng的模板cDNA进行PCR放大。

DNAMAN的使用方法

DNAMAN的使用方法第五章 DNAMAN 的使用方法DNAMAN是一种常用的核酸序列分析软件。

由于它功能强大，使用方便，已成为一种普遍使用的DNA序列分析工具。

本文以DNAMAN 5.2.9 Demo version为例，简单介绍其使用方法。

打开DNAMAN，可以看到如下界面:第一栏为主菜单栏。

除了帮助菜单外，有十个常用主菜单，如下所示:第二栏为工具栏:如下所示:第三栏为浏览器栏:如下所示:在浏览器栏下方的工作区左侧，可见Channel工具条，DNAMAN提供20个Channel，如左所示:点击Channel工具条上相应的数字，即可击活相应的Channel。

每个Channel 可以装入一个序列。

将要分析的序列(DNA 序列或氨基酸序列)放入Channel中可以节约存取序列时间，加快分析速度。

此版本DNAMAN提供自动载入功能，用户只需激活某个Channel，然后打开一个序列文件，则打开的序列自动载入被激活的Channel中。

本文以具体使用DNAMAN的过程为例来说明如何使用DNAMAN分析序列。

1(将待分析序列装入Channel(,)通过File|Open命令打开待分析序列文件，则打开的序列自动装入默认Channel。

(初始为 channel1)可以通过激活不同的channel(例如:channel5)来改变序列默认装入的Channel。

(,)通过Sequence|Load Sequence菜单的子菜单打开文件或将选定的部分序列装入Channel。

如果只想分析序列的一部分，或者给待分析的序列添加批注，点击激活该序列所在的Channel，通过Sequence|Current Sequence|Analysis Defination命令打开一个对话框，如下所示:对话框选项说明如下:Name 序列名称Circular DNA 环形DNAStart pos 待分析序列起点End pos 待分析序列终点根据需要，可以设定上述选项的内容，如果要分析整个序列，可以点击按钮，如果当前序列不是DNA，则可点击按钮，如果要添加或修改批注，可以使用(添加)，(移除)，和(清除全部)三个按钮进行操作。

四膜虫大核基质中含有NuMA类似蛋白

四膜虫大核基质中含有NuMA类似蛋白
孙英丽;陈丹英;高崇明
【期刊名称】《动物学报（英文版）》
【年(卷),期】2000(046)001
【摘要】@@NuMA (Nuclear mitotic apparatus protein)是细胞核基质中的一种高分子量蛋白质，具有一个特殊的细胞周期依赖性(cell cycle- dependent distribution)。

在多细胞动物中有关NuMA的研究已经很深入，但是目前尚未见原生动物中NuMA蛋白的报道。

四膜虫(Tetrahymena)是一种单细胞真核生物，属原生动物(Protozoa)纤毛虫纲(Ciliata)，具有较特殊的分类地位和分子生物学研究背景。

本实验结果表明，四膜虫大核基质中含有NuMA类似蛋白，不仅进一步验证了NuMA蛋白存在的普遍性，而且是对四膜虫中大核基质成分研究的补充。

【总页数】4页(P104-107)
【作者】孙英丽;陈丹英;高崇明
【作者单位】北京大学生命科学学院，北京 100871;北京大学生命科学学院，北京 100871;北京大学生命科学学院，北京 100871
【正文语种】中文
【中图分类】Q959.117
因版权原因，仅展示原文概要，查看原文内容请购买。

半滑舌鳎线粒体 DNA 含量测定方法的建立与优化

中国水产科学 2014年9月, 21(5): 920-928 Journal of Fishery Sciences of China研究论文收稿日期: 2013-12-07; 修订日期: 2014-03-11.基金项目: 国家自然科学基金项目(31172411, 31201981); 国家国际科技合作专项项目(2013DFA31410). 作者简介: 冯文荣(1986−), 女, 博士研究生, 从事渔业资源分子生态学研究. E-mail: fengwenrong1@ 通信作者: 庄志猛, 研究员, 从事渔业资源分子生态学研究. Tel: 86-532-85836340; E-mail: zhuangzm@DOI: 10.3724/SP.J.1118.2014.00920半滑舌鳎线粒体DNA 含量测定方法的建立与优化冯文荣1, 2, 柳淑芳2, 庄志猛2, 马骞2, 苏永全1, 唐启升1, 21. 厦门大学海洋与地球学院, 福建厦门 361005;2. 山东省渔业资源与生态环境重点实验室, 中国水产科学研究院黄海水产研究所, 山东青岛 266071摘要: 本研究旨在应用实时荧光定量PCR 技术, 建立半滑舌鳎(Cynoglossus semilaevis )线粒体DNA(mtDNA)含量测定方法并进行优化。

通过质粒标准品的线性化处理、模板DNA 的酶切和超声处理、mtDNA 和核DNA(nDNA)基因引物的筛选实验, 建立半滑舌鳎mtDNA 含量测定方法。

结果表明, 质粒标准品的构象对荧光定量PCR 标准曲线影响较大, 线性化的质粒更适合用作标准品; 酚-氯仿方法提取的模板DNA 适用于mtDNA 含量测定, 无需进行预处理; 用D-loop 和ND1这2对引物所得的拷贝数较小且结果一致, 适用于mtDNA 拷贝数的测定; 以不同核基因为参照所得的mtDNA 含量可能存在差异, 当以单拷贝核基因ENC1和MYH6为参照时, 可以计算出单个细胞中mtDNA 含量, 若以多拷贝基因GAPDH 为参照, mtDNA 含量测定值则较小。

多聚酶链式反应扩增日本血吸虫基因组DNA重复序列

多聚酶链式反应扩增日本血吸虫基因组DNA重复序列何纳;姜庆五;赵根明;刘建翔;韦建国;赵春琳;Joseph Hamburger【期刊名称】《中国公共卫生》【年(卷),期】2002(18)11【摘要】目的探索日本血吸虫基因组内是否存在DNA重复序列。

方法分别根据曼氏血吸虫基因组特异的DNA重复序列单位Sm1- 7和埃及血吸虫基因组特异的DNA重复序列单位TheDraIRepeat设计并合成了寡核苷酸引物 ,然后以日本血吸虫基因组DNA为模板 ,用自备的多达 12种的反应缓冲液进行多聚酶链式反应 (PCR)优化扩增。

结果分别成功地从日本血吸虫基因组中扩增出与曼氏血吸虫基因组“特异的”DNA重复序列单位Sm1- 7和埃及血吸虫基因组“特异的”DNA重复序列单位TheDraIRepeat同源的DNA重复序列。

PCR反应提示该两种重复序列在日本血吸虫基因组内的拷贝数均较低。

同时 ,日本血吸虫与曼氏血吸虫表现出较好的同源性。

结论日本血吸虫基因组内可能存在着长度为 12 1bp的DNA重复序列。

【总页数】2页(P1284-1285)【关键词】多聚酶链式反应;DNA重复序列;血吸虫;基因组【作者】何纳;姜庆五;赵根明;刘建翔;韦建国;赵春琳;Joseph Hamburger【作者单位】复旦大学公共卫生学院流行病学教研室;以色列希伯来大学Kuvin传染病与热带病研究中心【正文语种】中文【中图分类】R383.24;Q78【相关文献】1.多聚酶链式反应随机引物扩增的DNA多态指模技术在幽门螺杆菌菌株鉴别中的应用 [J], 陈旻湖;曾志荣;胡品津2.关于多聚酶链式反应扩增DNA片段结果分析方法的探究 [J], 赵奂;杨潘云;昌姝3.谈谈高中"多聚酶链式反应扩增DNA片段"实验 [J], 黄群声4.多聚酶链式反应扩增高度多态性区域在骨髓移植中的应用 [J], 刘变玲;曹履先5.基于生物学核心素养的“四驱教学法”--以“多聚酶链式反应扩增DNA片段”为例 [J], 姜珊;李艳秀因版权原因，仅展示原文概要，查看原文内容请购买。

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Acta Biologica Hungarica 63(4), pp. 483–489 (2012)DOI: 10.1556/ABiol.63.2012.4.70236-5383/$ 20.00 © 2012 Akadémiai Kiadó, BudapestDNA coNteNt AlterAtioNsiN TeTrAHymenA pyrIfOrmIs mAcroNucleus After exposure to fooD preserv Atives soDiumNitrAte AND soDium beNzoAteA riAdni C. L outsidou ,1 V AsiLiki i. H Atzi ,2 C. t. C HAsApis ,1G eorGiA i. t erzoudi ,2 C HArA A. s piLiopouLou 3 and M AriA e. s tefAnidou 3 *1 Department of pharmacy, university of patras, Gr-26504, patras, Greece2 institute of Nuclear & radiological sciences & technology, energy & safety, National centre forScientific Research, NCSR “Demokritos”, Ag. Paraskevi Attikis, Athens, Greece3 Department of forensic medicine and toxicology, medical school, university of Athens,75 M. Asias str., Goudi 115.27, Athens, Greece(received: November 29, 2011; accepted: January 23, 2012)the toxicity, in terms of changes in the DNA content, of two food preservatives, sodium nitrate and sodium benzoate was studied on the protozoan Tetrahymena pyriformis using DNA image analysis tech-nology. For this purpose, selected doses of both food additives were administered for 2 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the Mean Optical Density which represents the cellular DNA content. The results have shown that after exposure of the protozoan cultures to doses equivalent to ADI, a statistically significant increase in the macronuclear DNA content compared to the unexposed control samples was observed. The observed increase in the macronuclear DNA content is indicative of the stimulation of the mitotic process and the observed increase in moD, accompanied by a stimulation of the protozoan proliferation activity is in consistence with this assumption . since alterations at the DNA level such as DNA content and uncontrolled mitogenic stimulation have been linked with chemical carcinogenesis, the results of the present study add information on the toxicogenomic profile of the selected chemicals and may poten -tially lead to reconsideration of the excessive use of nitrates aiming to protect public health.Keywords: Tetrahymena pyriformis – sodium nitrate – sodium benzoate – DNA content – mean optical DensityiNtroDuctioNpreservatives are widely used in food, beverages, drugs and cosmetic industry during processing or storage to preserve flavour, to prolong the shelf-life of food and protect it against deterioration caused by microorganisms, to ensure its nutritional adequacy and safety or to enhance its taste and appearance. Among them, two of the most com -monly used food preservatives are sodium nitrate and sodium benzoate [11, 19, 27].sodium nitrate (NaNo 3, E251) due to its antimicrobial properties against C. botu-linum growth is widely used in the processing of red meat [11]. A small amount of* C orresponding author; e-mail: mstefan@med.uoa.gr484A riAdni C. L outsidou et al.Acta Biologica Hungarica 63, 2012the nitrate added to processed meat as a preservative is reduced to nitrite, in addition to any nitrite that may also be added. The nitrite then reacts with secondary amines to produce the potent carcinogens N-nitrosamines [33]. About 5% of nitrates con -sumed by healthy adults are reduced to nitrites by bacteria in saliva and alimentary tract. Sodium nitrate has been associated with disease conditions including reproduc -tive toxicity, anaemia and thyroid hypertrophy, while according to World cancer research fund uK, the addition of nitrates in the processed meat, which can be fur-ther metabolized to nitrites, has been linked with increased risk of colon cancer [6].sodium benzoate (Nac 6H 5co 2, e211) due to its bacteriostatic and fungistatic activities [2, 14, 26] is widely used as a food preservative in acidic foods, such as salad dressings, carbonated drinks, jams and fruit juices, pickles, purees and pie fill -ings [27]. It is also found in cough syrups and in alcohol-based mouthwash. Although the international program on chemical safety (ipcf) found no adverse effects in humans (at doses of 647–825 mg/kg of body weight per day) [3, 20], some studies have linked exposure to sodium benzoate with mild hypersensitivity reactions in human and experimental animals, such as allergic reactions [4], immune responses suppression [17], allergic contact dermatitis [32] inhibition of gluconeogenesis, ure-agenesis [7] and fatty acid oxidation [13].since the widely used food preservatives sodium nitrate and sodium benzoate have been accused for various toxic reactions such as hypersensitivity reactions (allergic reactions, immune responses suppression, allergic contact dermatitis), asthma, decreased lung function (chronic obstructory pulmonary disease) [1, 17, 20, 21], the aim of this study was to test their in vivo toxicity in terms of DNA content changes of the protozoan, Tetrahymena pyriformis (T. pyriformis ) by means of the computer-ized DNA image analysis system (CIAS), [22–24, 27–31, 38].Protozoa are eukaryotic cells widespread in the aquatic and terrestrial environ -ment. Their normal behaviour in nature may be related to the presence of pollutants and to air, soil and water quality. Among the various species of protozoa, ciliates are the most frequently used as experimental organisms [25, 36]. The protozoan T. pyri-formis is a ciliate monocellular organism and it is established as an alternative experimental model for functional, pharmacological, toxicological or immunotoxic studies [22, 28]. Our previous laboratory experience with Tetrahymena has led us to consider the DNA content (measured as mean optical Density), as a valuable param-eter to test responses to various toxicants. Tetrahymena contains one macronucleus, and many strains contain a micronucleus as well. The macronucleus of the protozoan contains 12–24 timesfold DNA in comparison to micronucleus, and practically repre-sents the total DNA content of the protozoan. Macro- and micronuclei both give strongly positive cytochemical reactions for DNA with Feulgen reagent [37]. Based on previous studies where DNA content has been proposed as a cytogenetic marker of toxicity for several chemical substances (butylated hydroxytoluene, tartrazine) and drugs (cocaine, crack) [29, 31], the aim of this study was to estimate the possible T. pyriformis macronuclear DNA content changes after exposure to selected doses of the food additives sodium nitrate and sodium benzoate by means of the DNA image technology.food additive toxicity on T. pyriformis macronuclei485Acta Biologica Hungarica 63, 2012mAteriAls AND metHoDs Culture conditions and chemical exposureT. pyriformis protozoa were grown axenically in a proteose-peptone broth at 25 °c, until reaching late log phase. The selected food preservatives (sodium nitrate and sodium benzoate) (sigma-Aldrich, Germany) were added separately to a 100-ml cul-ture as aqueous solutions. The selected doses were 4 × 10–4 M, 8 × 10–4 m (ADi) and 40 × 10–4 M for sodium nitrate and 2.5 × 10–4 M, 5 × 10–4 M (ADI) and 25 × 10–4 m for sodium benzoate. These doses were selected based on previous studies carried out in our lab, with the use of human lymphocytes [16], where the ADi doses for both food additives were the minimum effective doses. According to this study a spontaneous and stimulated DNA synthesis was observed. For each tested substance, an experi -mental series consisting of six cultures of the protozoan was used and another experimental series served as control. The cultures were allowed to stand for 2 hours after administration of food additives. Then, samples were obtained from each group of protozoan cultures and were observed under the microscope to check the viability, morphology and motility of the protozoa. All these parameters were normal. Afterwards, the protozoan population was counted using the hematocytometer under the microscope at a magnification of ×80 and Feulgen staining followed. Unexposed cultures of T. pyriformis were used as control samples.feulgen stainingOne drop (50 μl) of each protozoa culture, was pipetted onto a microscope slide, impregnated with the adhesive agent poly-l-lysine hydrobromide (sigma-Aldrich, Germany) 0.1% in water for injection, and was allowed to stand until drying [12]. The DNA of the protozoa nuclei was stained red to purple with the feulgen reagent to stain DNA specifically and quantitatively, according to the static cytophotometric procedure [29, 31].DnA image analysisDNA image analysis technique is based on a conversion of optical images of cells into arbitrary numerical values (digitization) based on light absorbance or transmis-sion and has proved to be an effective approach to examine and classify stained cells spread on a glass slide in the form of smears [29]. The counting of macronuclei and the mean optical density [18] of T. pyriformis macronuclei was determined by means of the digital DNA image analysis system (CIAS, SAMBA 2000) [16]. A total number of 50 T. pyriformis macronuclei from each culture were selected randomly from each sample (i.e. microscopic slide) and the quantification of MOD was performed at the wavelength of 560 nm. The statistical analysis of the results was carried out using the486A riAdni C. L outsidou et al.Acta Biologica Hungarica 63, 2012student’s t -test and mean values and standard deviations were calculated from six independent experiments. All p -values were considered statistically significant at p < 0.05.results AND DiscussioNin the present study, using DNA image analysis system, the DNA content (expressed as mean optical Density, moD) of T. pyriformis macronuclei after exposure to sodium nitrate and sodium benzoate has been estimated. The results have shown that both sodium nitrate and sodium benzoate (at the ADi value) affect the DNA content of T. pyriformis macronuclei when compared to the control samples (Table 1). In particular, the observed statistically significant increase in MOD after exposure to sodium benzoate and sodium nitrate was from 29.69 ± 8.49 (control value) to 33.99 ± 7.31 and 40.43 ± 8.43 (t = 6.34, p < 0.001) (t = 2.54, p < 0.01) respectively. Smaller doses from ADI have shown no statistically significant difference of the two parameters (moD and the population of the protozoa) in comparison with the control samples, while the larger doses selected had influenced significantly the protozoan viability and caused protozoan cell death.the observed increase in the macronuclear DNA content as a result of ADi values of both sodium nitrate and sodium benzoate is probably the consequence of the pre-servative-induced stimulation of the macronuclear DNA synthesis which leads to an increase in the mitogenic activity of protozoa. In particular, the preservative thatTable 1mean optical Density of the DNA content of 50 macronuclei of the protozoan T. pyriformis after 2 hoursexposure to sodium nitrate and sodium benzoatepreservativeconcentrationprotozoa/mlMOD ± SDcontrol – 2.4 × 10529.69 ± 8.49sodium nitrate4 × 10–4 m 2.6 × 10531.63 ± 7.01(p < 0.05)8 × 10–4 m (ADi) 4.7 × 10540.43 ± 8.43(t = 6.35, p < 0.001)40 × 10–4 m––sodium benzoate 2.5 × 10–4 m 2.1 × 10528.21 ± 9.54(p < 0.05)5 × 10–4 m (ADi) 3.3 × 10533.99 ± 7.31(t = 2.54, p < 0.01)25 × 10–4 m––the statistical analysis of the results was carried out using the student’s t -test and mean values and standard deviations were calculated from six independent experiments. All p -values were considered statistically significant at p < 0.05.food additive toxicity on T. pyriformis macronuclei487Acta Biologica Hungarica 63, 2012induces the highest increase in the protozoan moD value after exposure is also expected to induce the higher increase in the proliferative activity. Indeed, the observed statistically significant increase in the proliferative activity after exposure of T. pyriformis to sodium nitrate and sodium benzoate, with respect to controls, sup-ports this hypothesis. Specifically, a higher increase in cell division was observed in cultures treated with sodium nitrate [from 2.4 × 105 protozoa per ml (control value) to 4.7 × 105 cells/ml], than with sodium benzoate (3.3 × 105 cells/ml). Furthermore, sodium nitrate induces a higher increase in the moD value compared to sodium benzoate.in the present study, the increase in the macronuclear DNA content after exposure of T. pyriformis to sodium nitrate and sodium benzoate, results from a stimulation of the mitotic process which is expressed here as an increase in cell division. In general, it is well known that cytogenetic changes at the DNA level [8, 35], as well as uncon -trolled cellular proliferation [5, 9, 10, 15] are causative factors of genetic instability and carcinogenesis, while DNA content (DNA index) has been widely used in many studies as a significant prognostic tool of aneuploid tumors [34, 35, 38]. Therefore, the observed above-normal increase of the DNA content and cellular division pro-cesses after exposure to both food additives might indicate their potential carcino-genic profile. Moreover, similar cellular alterations might act antagonistically in other cellular processes of the living cells. For example, the energy deposit of the proto -zoan cell is consumed in the chemically-induced mitotic processes induction and other life-supporting mechanisms, such as phagocytosis, that is highly suppressed [28, 29]. However, further studies on the impact of both food preservative in other morphonuclear and densitometric parameters (e.g. area, ploidy balance, degree of aneuploidy), will elucidate whether the observed chemically induced increase in DNA content may lead to disturbances in the T. pyriformis chromosomal segregation during cellular division that would potentially lead to aneuploidy.In conclusion, the findings of the present work support a toxicogenomic effect of both sodium nitrate and sodium benzoate in the selected experimental model, T. pyri-formis which is expressed as an increase in the DNA content and protozoan prolif-erative activity. 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