稻米深加工产品基因组提取方法的研究及其对PCR的影响

本实验以四种米粉为原料，每个样品做三个梯度：120mg, 800mg, 2000mg，采用改进的经典酚/仿法、CTAB沉淀法以及盐酸胍/氯仿法提取基因组，并通过聚合酶链式反应(PCR)通过扩增内标基因(SPS)来检测提取方法的优劣。结果显示：120mg的样品经三种方法提取的基因组均不能扩增出内标基因；800mg的样品和2000mg的样品只有用CTAB沉淀法提取的基因组(采用相同的膜板量)能全部扩增出内标基因。结论：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。     

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。     

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.     

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。     

大青杨基因组DNA提取方法的比较

本研究采用改良SDS法、常规CTAB法和改良CTAB法(1.5×CTAB,2×CTAB,3×CTAB)提取大青杨基因组DNA,并用紫外光普分析、凝胶电泳、限制性内切酶消化和RAPD方法进行鉴定。结果表明:5种方法中,改良SDS法DNA提取率最高,但CTAB法比改良SDS法提取获得的DNA纯度高,OD260/OD280为1.73～1.81。与常规CTAB法比较,改良SDS法和改良CTAB法能有效去除蛋白、多糖、酚类及次生代谢物质。综合分析确定改良2×CTAB法为大青杨基因组DNA的最佳提取方法。     

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。     

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。     

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.     

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.     

Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes

With the development of transgenic crops, many countries have issued regulations to label the genetically modified organisms (GMOs) and their derived products. Polymerase Chain Reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods generally need to amplify the transgene and compare the amplified result with that of the corresponding reference gene to obtain reliable results. In this article, we reported the development of specific primers and probe for the rice (Oryza sativa) sucrose phosphate synthase (SPS) gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 13 different rice varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other species, such as wheat, maize, barley, tobacco, soybean, rapeseed, tomato, sunflower, carrot, pepper, eggplant, lupine, mung bean, plum, and Arabidopsis thaliana, were used as templates, which demonstrated that this system was specific for rice. In addition, the results of the Southern blot analysis confirmed that the SPS gene was a single copy in the tested rice varieties. In qualitative and quantitative PCR analyses, the detection sensitivities were 0.05 and 0.005 ng of rice genomic DNA, respectively. To test the practical use of this SPS gene as an endogenous reference gene, we have also quantified the beta-glucuronidase (GUS) gene in transgenic rice using this reference gene. These results indicated that the SPS gene was species specific, had one copy number, and had a low heterogeneity among the tested cultivars. Therefore, this gene could be used as an endogenous reference gene of rice and the optimized PCR systems could be used for practical qualitative and quantitative detection of transgenic rice.     

Comparison of three DNA extraction methods for feed products and four amplification methods for the 5'-junction fragment of Roundup Ready soybean

Three methods of DNA extraction from feed products and four detection methods for the 5'-junction fragment of genetically modified (GM) Roundup Ready soybean (RRS) were compared and evaluated. The DNA extraction methods, including cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and guanidine hydrochloride (Kit), were assessed for their yields and purity of DNA, extraction time, and reagent cost. The DNA yields of CTAB, SDS, and Kit were 52-694, 164-1750 and 23-105 ng/mg sample, and their extraction time was 2.5-3, 2-2.5, and 1.5-2 h with reagent cost about US dollar 0.24, 0.13, and 1.9 per extraction, respectively. The SDS method was generally well suited to all kinds of feed matrices tested. The limits of detection for the four amplification protocols, including loop-mediated isothermal amplification (LAMP), hyperbranched rolling circle amplification (HRCA), conventional polymerase chain reaction (PCR), and real-time PCR, were 48.5, 4.85, 485, and 9 copies of the pTLH10 plasmid, respectively. The ranked results of the four detection methods were based on multiattribute utility theory as follows (from best to worse): HRCA, LAMP, PCR, and real-time PCR. This comparative evaluation was specifically useful for selection of a highly efficient DNA extraction or amplification method for detecting different GM ingredients.     

Comparison of several methods for the extraction of DNA from potatoes and potato-derived products

Eight methods were compared for the extraction of DNA from raw potato tubers, and nine methods were evaluated for the extraction of DNA from dehydrated potato slices, potato flakes, potato flour, potato starch, and two ready-to-eat potato snack foods. Extracts were assessed for yield using a fluorescence-based DNA quantification assay. Real-time amplification of an endogenous gene, sucrose synthase (sus), was used to assess extract and template quality. A CTAB-based method extracted the highest DNA yields from the tuber material. An in-house method, which utilized the Kingfisher magnetic particle processor, yielded the highest template quality from the tubers. For most of the tuber samples, the Kingfisher and CTAB methods recovered the highest levels of amplifiable sus. DNA yields for potato-derived foods generally decreased with the extent that the product had been processed. The methods that utilized the magnetic particle processor delivered the highest template quality from one of the snack products that was particularly high in fat. For most of the remaining processed products, the levels of amplifiable target DNA recovered were roughly correlated with total DNA recovery, indicating that overall yield had greater influence over sus amplification than template quality. The Wizard method was generally the best method for the extraction of DNA from most of the potato-derived foods.     

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.     

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。