稻米深加工产品基因组提取方法及其对PCR的影响

以4种米粉为原料,每个样品做3个梯度(120、800和2000mg),采用改进的经典酚/仿法、CTAB(十六烷基三乙基溴化铵)沉淀法以及盐酸胍/氯仿法提取基因组,经PCR扩增内标基因(SPS)检测方法的优劣。结果显示,120mg的样品经3种方法提取的基因组均不能扩增出内标基因;800和2000mg的样品只有用CTAB沉淀法提取的基因组(采用相同的模板量)能全部扩增出内标基因。结果表明,CTAB沉淀法提取基因组的效果最好,对PCR的抑制现象最少。     

LiCl沉淀法提取富含荚膜的细菌基因组DNA

结合提取基因组DNA的CTAB法(hexadecyltrimethylammoniumbromide,十六烷基三甲基溴化铵)和用于提取海藻基因组DNA的LiCl法,设计了针对产生较多荚膜多糖细菌的DNA提取方法—LiCl沉淀法。通过电泳、分光光度计及16SrRNA基因扩增检测,证明LiCl沉淀法可以有效地提取产荚膜细菌基因组DNA,其片断大小约为23kb,不需进一步纯化即可用于后续分子生物学实验。     

棉花根际促生菌基因组DNA的提取及其鉴定

植物根际微生物基因组的提取往往因为细胞壁不能完全裂解以及内含物的成分和含量受到影响,获得完整的基因组DNA一直是土壤微生物分子生物学研究的关键。本文以新疆盐碱地棉花根际促生菌为材料,比较了SDS法、CTAB法以及高盐结合的CTAB法提取棉花根际促生菌基因组DNA的效率,结果显示,SDS法和CTAB法在细胞裂解后,由于过多的多糖类物质混于上清液中使其过于粘稠而无法分层,没能得到DNA样品。高盐结合的CTAB法则有效弥补了上述方法的不足,能够获得A260/A280为1.82、A260/A230为2.43的高质量基因组DNA。同时实验还以细菌16SrDNA为内标进行PCR扩增,结果证明所获得的棉花根际促生菌基因组DNA可直接用于PCR等分子生物学进一步研究。     

DNA提取方法对一串红不同部位DNA提取的比较

本文以一串红的叶片、茎段、花为材料,分别采用高盐低pH法、十二烷基硫酸钠（SDS）法、改良十六烷基三乙基溴化铵（CTAB）法、核DNA法和十六烷基三乙基溴化铵（CTAB）区室法等5种不同方法对其进行了基因组DNA提取效果的比较分析。结果表明,除用高盐低pH法在一串红花中未提取出DNA外,其它均可提取出DNA,都能进行RAPD扩增。5种方法在一串红DNA提取中,提取效果由高到低依次为：核DNA法、改良CTAB法、SDS法、CTAB区室法、高盐低pH法;3个部位提取结果显示：叶片较容易得到高质量的DNA,茎段次之,花最差。     

RAPD分析用白花丹参叶片DNA的提取方法

本研究采用改良CTAB法和SDS法提取新鲜白花丹参叶片的基因组DNA,初步筛选RAPD扩增引物。结果表明改良CTAB法提取的DNA较SDS法质量好,随机引物p2扩增条带相对较为清晰。再利用p2随机引物对2种方法提取的DNA进行RAPD检测比较,结果显示仅改良CTAB法能扩增出有效条带,说明改良CTAB法更适合用于白花丹参基因组DNA的RAPD检测分析。本文将为白花丹参叶片DNA的提取提供方法学参考。     

乙肝表面抗原基因的表达载体构建及对百脉根的转化

摘要：本研究利用PCR克隆法成功的从乙肝患者血清中扩增出乙肝表面抗原基因，并构建了含有此基因的植物表达载体。用农杆菌侵染法将乙肝表面抗原基因转入百脉根，从转化植株中提取DNA，PCR检测初步验证了目的基因已整合到百脉根的基因组中。     

苹果基因组DNA的快速提取

提出了一种适用于苹果（Malus pumila Mill)RAPD分析的基因组DNA的快速提取方法。用本法提取的DNA质量高，简便，快速，经济，每天可提取200个DNA样品，每个DNA样品可供50－100次PCR反应，用10个碱基随机引物进行PCR扩增，95％以上的样品都才扩增出3－10条清晰的泳带。     

干制羊肉基因组DNA不同提取方法的比较

为了研究干制加工羊肉基因组DNA的最佳提取方法,本试验采用传统酚-氯仿法、磁珠法、改良CTAB法、离心柱法分别提取干制处理后的羊肉基因组DNA,并对4种方法提取的羊肉基因组DNA浓度、纯度、完整性以及提取所需时间、PCR扩增效果等进行比较。结果表明,采用磁珠法提取DNA的效果更好,DNA浓度为118.87 ng·μL^-1,A₂₆₀/A₂₈₀值为1.89,而且此方法具有提取时间短、效率高、污染小等特点。本研究结果为干制加工羊肉基因组DNA的大批量提取和检测提供了参考依据。     

适于变性梯度凝胶电泳（DGGE）分析的草莓根际土壤微生物的DNA模板制备

根际土壤中微生物DNA的有效提取是土壤微生物变性梯度凝胶电泳(DGGE)分析的基础.研究对比了十二烷基硫酸钠(SDS)高盐抽提法、十六烷基三甲基溴化铵(CTAB)法和预洗处理的聚乙烯吡咯烷酮(PVP)法3种DNA提取方法对4个栽培地块草莓(Fragaria ananassa Duch.)根际土壤微生物总DNA提取的效果.结果表明,SDS高盐抽提法和预洗处理的PVP法从不同土样中提取的微生物总DNA片段长度大于23 kb,DNA均量分别达到32.94和43.06 靏/g;其中预洗处理的PVP法提取DNA的A260/A280和A260/A230比值平均为1.1623和0.8135,比SDS高盐抽提法(1.1238和0.5901)分别高出0.0385和0.2234,对土壤样品中蛋白质和腐植酸的去除效果较好;CTAB法提取的总DNA非常少.纯化后的DNA,分别采用细菌F341/R534和真菌FR1/FF390引物进行PCR扩增,成功获得细菌16S rDNAV3区和真菌18S rDNA目的片段,对其PCR扩增产物进行DGGE分析,4个土壤样品均得到清晰的DGGE图谱.草莓根际土壤微生物DGGE分析中DNA模板制备采用预洗处理的PVP法和SDS高盐抽提法均可成功获得,预洗处理的PVP法效果最好,而CTAB法制备效果较差.     

一种适于PCR扩增的植物基因组快速提取新方法

在传统的碱裂解法提取基因组的基础上建立了一种新的基因组提取方法,这种方法对传统碱裂解制备基因组的方法进行优化,并且调整了中和剂的用量,引入异丙醇沉淀步骤对基因组进行纯化.实验证明这种方法扩大了碱裂解法制备基因组的适用范围,可直接从植物种子中快速提取出用于PCR(polymerasechain reaction)模板的基因组,对于600 bp以下的目的基因的检测效果显著,可以发现待测组分含量大于1%的样品.整个基因组提取时间控制在21 min,大大缩短了基因组提取时间,并且避免了酚/仿等对人体有害药品的使用.     

短序十大功劳基因组总DNA提取方法研究

以短序大功劳嫩叶为材料,采用CTAB法、CTAB改良法1、CTAB改良法2、SDS法和试剂盒法五种方法提取短序十大功劳基因组总DNA,用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的纯度和得率,用ISSR-PCR扩增的方法检测所得总DNA的质量。结果表明,五种方法均能从短序大功劳叶片中提取到基因组DNA,但不同方法提取得的基因组DNA的纯度、浓度和得率存在明显的差异。CTAB改良法2和试剂盒法提取的DNA纯度高,可直接用于下游分子生物学实验,CTAB法、CTAB改良法1和SDS法提取的总DNA质量较差,不利于下游的分子生物学实验;五种方法提取的总DNA的得率在10．836-451．709μg/g之间,呈CTAB法〉SDS法〉CTAB改良法1〉CTAB改良法2〉试剂盒法的现象。此实验获得的结果可以为短序十大功劳分子生物学研究提供基础。     

蔺草基因组DNA提取方法的比较研究

以16个蔺草品种实生苗和4个蔺草品种组培苗为材料,探讨了CTAB改良法与DNA试剂盒法对蔺草基因组DNA提取质量的影响。结果发现,对于同一品种而言,CTAB改良法提取获得的DNA A260nm和OD值远低于试剂盒提取法,前者的DNA浓度仅为0.65~4.90μg/ml,而后者则为5.10~32.80μg/ml,后者为前者的1.6~21.2倍。这表明试剂盒提取法更适合于蔺草基因组DNA的提取。试验还发现提取材料对基因组DNA的质量有显著影响,蔺草组培苗优于实生苗。     

Cultivar identification of rice (Oryza sativa L.) by polymerase chain reaction method and its application to processed rice products

As the cultivars of rice markedly affect eating quality, processing suitability, and price, identification or differentiation of rice cultivar is very important. We developed suitable 14 STS (sequence-tagged site) primers for PCR (polymerase chain reaction), and it became possible to differentiate 60 Japanese dominant rice cultivars from each other using template DNA extracted and purified from rice grains. A multiplex primer set was shown to be useful to effectively differentiate rice cultivars produced in various countries by PCR. A novel multiplex primer set for PCR has been developed to differentiate KoshihikariBL, which is closely related with the premium cultivar, Koshihikari, in Japan. The application of the cultivar identification method by PCR method to commercially processed rice products was investigated. We developed an enzyme treatment method, in which the gelatinized starch is decomposed by the heat-stable alpha-amylase at 80 degrees C, followed by the hydrolysis of proteins by proteinase K with sodium dodecyl sulfate and purification of extracted DNAs by phenol/chloroform/iso-amyl alcohol. It became possible to identify the material rice cultivars of the commercially processed rice products, such as cooked rice, rice cake, or rice cracker, by a PCR method using template DNA prepared by the enzyme treatment method and novel multiplex primer sets.     

紫色水稻土水稳性团聚体土壤微生物基因组DNA提取方法探讨

为了找到提取土壤微生物总DNA的最佳方法,通过OD值检验、凝胶电泳、PCR和DGGE分析,比较了Reddy法、基于DNAout kit试剂盒改进的实验方法、以及Kuske修订法、Edgcomb改进法、SDS高盐提取法、Eichner调整法等常用的不同土壤微生物基因组的DNA提取方法在亚热带地区长期免耕紫色水稻土水稳性团聚体0.25～2.0 mm粒径上的提取效果.结果表明,6种方法都可以从团聚体中提取到长度大于23.1 kb的DNA片段,但不同方法提取的DNA的产量存在明显差异,土壤总DNA均不需纯化就可以用于PCR扩增,使用细菌16S rDNA基因V3区的通用引物可扩增得到相应的片段.研究表明,改进的DNAout kit试剂盒法是长期免耕紫色水稻土水稳性团聚体中微生物基因组DNA的最佳提取方法.     

落羽杉属树木基因组总DNA的提取及SRAP反应体系的优化

本文针对落羽杉属植物组织中多糖、多酚等次生物质含量高的特点,对其基因组DNA提取方法进行研究,比较了SDS法、CTAB法提取落羽杉属植物基因组DNA的效果,结果表明：CTAB法提取效果较佳。在此基础上,利用正交设计法,对SRAP反应体系中的各个主要影响因子Mg^2＋．dNTP、引物、TaqDNA聚合酶进行了优化筛选,确立了适合落羽杉属植物SRAP-PCR反应的最佳体系,即10μL体系中含有1μL 10×PCR bufier,Mg^2＋2．0mmol／L,dNTP100μmol／L,引物0-3μmol／L,彻DNA聚合酶0．5U和50ng模板DNA。利用该优化体系,通过48对SRAP引物组合对2个落羽杉属植物（落羽杉和墨杉）及4个杂交后代进行SRAP扩增,结果发现,SRAP引物及优化后的反应体系能够有效地用于落羽杉属植物种质资源鉴定及遗传多样性分析等研究。     

Validated method for quantification of genetically modified organisms in samples of maize flour

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.     

Novel preparation method of template DNAs from wine for PCR to differentiate grape (Vitis vinifera L.) cultivar

Template DNAs were extracted from wine and purified for use as samples for PCR to differentiate grape cultivars. It has been pointed out that the authentication of grape material by PCR using wine as a material is very difficult. The problems are (1) decomposition of DNAs during fermentation; (2) contamination of DNAs from microorganisms such as yeast; (3) interference of DNA extraction by polysaccharides and polypeptides in the beverages; and (4) coexistence of PCR inhibitors, such as polyphenols. For this study was developed a novel preparation method of template DNA from wine to differentiate grape cultivars using PCR by (1) lyophilizing and pulverizing the fermented beverage to concentrate the DNAs; (2) decomposition of polysaccharides and proteins so as not to inhibit DNA extraction using heat-resistant amylase and proteinase K without DNA damage by endogenous DNase; and (3) separation of the template DNAs for PCR from PCR inhibitors, such as polyphenols, by purification using 70% EtOH extraction and isopropyl alcohol precipitation. To prevent the amplification of microorganisms' DNAs during PCR, suitable PCR primers closely related to the specific plant DNAs, such as chloroplast DNA and mitochondrial DNA, were selected. The sequences of the amplified DNAs by PCR were ascertained to be the same as those of grape materials.     

不同保存方法和提取方法对扁桃基因组DNA质量的影响

以扁桃（Amygdalus conmmuis L.）幼叶为试材,通过7种保存方法并采用改良2×CTAB法、3×CTAB法和SDS法对其DNA进行提取,用琼脂糖凝胶电泳检测其DNA完整性,分光光度计测其产量和纯度,以探讨不同保存方法和不同提取方法对扁桃DNA提取质量的影响。结果表明,扁桃新鲜幼叶、压干幼叶后在-75℃保存、45℃烘干幼叶后在-75℃保存以及硅胶干燥幼叶后在-75℃保存三个月均可以用三种提取方法可获得完整的DNA,纯度和产量均高,其保存和提取方法简便易行且获得高质量DNA。扁桃鲜叶直接放在-20℃保存比-75℃保存更易获得DNA,而压干后的扁桃叶片-20℃保存三个月并采用SDS法提取,获得高产量、高纯度的DNA,不影响提取效果。SDS法和改良3×CTAB法均可获得扁桃DNA,但SDS法优于3×CTAB法,其操作简单、耗时少;改良2×CTAB法提取扁桃成功率较低,不稳定,产量比其它两种方法低。