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1.
目的 获得最佳模板提取方法用于随机扩增多态性DNA(RAPD)。方法 对 4种方法 (挑丝法、沉淀法、水清法和裂清法 )提取的模板DNA进行琼脂糖电泳、紫外线扫描及随机扩增多态性DNA ,比较其片段大小、纯度及RAPD指纹图差异。结果 挑丝法和沉淀法均可提取出大于 2 3kb且纯度较高的模板DNA ,二者指纹图完全一致且均有 2~ 4kb的较大片段。水清法和裂清法提取的模板有弥散、碎裂的DNA并纯度较差 ,指纹图上仅有 6 0 0~ 2 0 0 0bp的较小片段。 结论 用挑丝法或沉淀法提取的模板DNA最适于进行RAPD。  相似文献   

2.
两种全血基因组DNA提取方法的比较   总被引:1,自引:0,他引:1  
目的比较两种不同的DNA提取方法提取人全血基因组DNA,对DNA提取率、纯度以及PCR扩增的效果影响.方法分别用传统的酚、氯抽提法和改良法两种不同的方法,提取人全血基因组DNA.结果两种方法提取的DNA纯度用光吸收比值为指标检测A280/260值为:1.45和1.82,提取DNA总量分别为187μg/ml和260 μg/ml.用0.8%琼脂糖凝胶电泳鉴定改良法制备的DNA效果较好,PCR扩增效果有差异.结论用改良法提取的DNA总量明显较高,提取效率高,PGR扩增的效果好,是全血基因组DNA提取中首选考虑的方法.  相似文献   

3.
细菌随机扩增多态性DNA分析中模板提取方法的优化   总被引:21,自引:3,他引:18  
目的:筛选出最佳模板最方法用于随机扩增多态性DNA(RAPD),方法:对4种方法(挑丝法,沉淀法,加热裂解和裂解剂裂解法)提取的模板DNA进行琼脂糖凝胶电泳,紫外线扫描及随机扩增多态性DNA,比较其片段大小,纯度及RAPD指纹图差异,结果:挑丝法和沉淀法均可提取出大于23kb且纯度较高的模板NDA,二者指纹图完全一致且均有2-4kb的较大片段,加热裂解法和裂解法提取的模板有弥散,碎裂的DNA并纯度较差,指纹图上仅有600-2000bp的较小片段,结论:用挑丝示或沉淀法提取的模板NDA最适于进行PAPD。  相似文献   

4.
为了探讨微量组织核酸的更快速、简便、有效的提取方法,采用改良裂解法提取25 例微量胃粘膜组织标本的核酸,同时与经典的饱和酚抽提法和简便的消化煮沸法进行比较( 各25 例) ,用1 % 琼脂糖电泳观察结果,并用分光光度法检测提取核酸的量。结果显示该法的提取纯度接近酚抽提法,所获核酸的量较多,而所需的时间最短;由于该法的裂解液中含有 R N A 酶抑制剂,所以除了可以提取 D N A,还可以获得一定量的 R N A,用于反转录 P C R,对微量组织的 R N A 提取提供了一种有效而实用的方法。  相似文献   

5.
在 DNA 研究中,分子量较大的标准物比较缺乏,噬菌体 T_4的 DNA 为长度166kb 的双链线状分子,是一个很有实用价值的大分子量 DNA 标准物。提取 T_4DNA 的关键步骤在于分离 T_4颗粒,其经典方法主要有两种:①两相分离法(硫酸葡聚糖钠盐/聚乙二醇);②超速离心法。两种方法均耗时较长且价格昂贵。本文用 DEAE 纤维素处理代替上述两法,操作快速简便降低了成本,并获得较好结果。  相似文献   

6.
几种提取蓖麻RNA方法的比较   总被引:1,自引:0,他引:1  
本文比较了 CEtus、AGPC、Kevin 三种方法提取蓖麻子和蓖麻子芽的总RNA 的产量及纯度,并对 CEtus 法加以改进,建立了一个快速简便提取蓖麻总 RNA 方法。其中 AGPC 法的产量最高,纯度较差,Kevin 法纯度高但产量最低。改良的 CEtus 法虽然产量居中,但除蛋白和 DNA 效果好。每5g 蓖麻子可得总 RNA1.2mg,得 mRNA 18μg。没有 DNA 和蛋白质污染。对提取的蓖麻子、蓖麻子芽、根、茎、叶的总 RNA 和mRNA 进行了点杂交实验。各部均出现不同程度阳性杂交斑点。  相似文献   

7.
目的通过测定半夏总生物碱的含量,比较不同地区栽培半夏、野生半夏的质量,为提高半夏的质量标准提 供一定的依据。方法采用氯仿提取,紫外-可见分光光度法在416 nm波长处测定总生物碱。结果盐酸麻黄碱浓度在 19.0-133μg·m^-1线性关系良好,标准曲线方程为:A =0.0733 C -0.0826, r =0.9996。测定结果显示不同产地半夏所含生 物碱不同,山东半夏含量较高,栽培品种高于野生半夏。结论氯仿提取法测定麻黄碱方法可靠、重复性好;可以用于半夏中 总生物碱的含量测定。  相似文献   

8.
本文报告一种适于普通实验室开展DNA探针工作的实验方法.我们用酚-氯仿抽提法制备探针供体菌重组质粒DNA,用透析袋电泳洗脱法从凝胶中回收酶切目的片段,用缺口翻译技术制备α-~(32)PdNTP标记探针,用低脂奶粉和6SSC(1×SSC:0.15M NaCl,0.015M柠檬酸三钠,pH7.0)代替Denhardt试剂等获得了满意的菌落原位杂交和Southern blot杂交结果.此外,我们将传统实验方法规定的时间相应缩短,从而有可能在15~24h内对痢疾腹泻患者标本作出明确诊断.这些方法可供基层实验室应用DNA探针开展传染病诊断和流行病学调查.  相似文献   

9.
目的 :建立金黄色葡萄球菌随机扩增DNA多态性 (RAPD)分型技术 ,以应用于医院感染流行病学调查。方法 :对临床分离的 85株金黄色葡萄球菌 ,用酚氯仿法提取DNA后进行RAPD分型。结果 :临床分离株大多数产生独特而稳定的RAPD带型 ,不同医院的基因型不同 ,同一医院基因型相似 ,在同一医院某些病区金黄葡萄球菌医院感染可能为交叉感染。结论 :RAPD基因分型技术不需预先知道核酸序列 ,分型率高 ,分辨力强 ,简便快捷 ,可为金黄色葡萄球菌临床分离株提供分型标志 ,是分子流行病学研究的有效方法。  相似文献   

10.
作者就随机扩增DNA多态性分析 (RAPD)技术用于结核暴发流行点传染源鉴别的价值进行了探讨 ,现报道如下。1 材料与方法1 1 痰菌分离培养 由全军结核病防疫队取自北京市某结核病暴发流行点共 13份痰标本 ,于次日用匡氏琼脂培养基做抗酸杆菌分离培养[1] 。1 2 菌型鉴定 采用多引物PCR和传统生化、培养分型 2种技术对抗酸杆菌阳性分离物分型鉴定[2 ] 。1 3 药敏试验 采用匡氏琼脂培养基鉴定结核杆菌分离株对 8种抗痨药物的抗性[3 ] 。1 4 DNA提取 用酚 氯仿法从受试结核菌提取DNA。为确保操作安全 ,用作DNA提取的菌…  相似文献   

11.
Gravesoil beneath decomposing cadavers undergoes substantial biochemical changes that have the potential to aid in PMI estimation and identification of clandestine gravesites. The use of DNA extraction methods is necessary for culture-independent downstream molecular applications such as PCR and next-generation sequencing. In this study, a comparison of four methods was performed for cadaver-soil collected beneath the heads and feet of 11 cadavers decaying in a natural setting. The four methods isolated 3.6–263 ng/μl of genomic DNA as determined by optical density analysis. The purity of the extracted DNA according to A260/280 and A260/230 ratios was determined. The A260/280 and A260/230 ratios were 1.24–1.97 and 0.27–2.12, respectively. The optical density at 320 nm was measured for humic acid quantification of the lysates from the method that provided the most efficient removal of humic acid. The results demonstrated that this method provided a 98% reduction of humic acid. PCR of 16S rRNA genes followed by gel electrophoresis was performed. The statistical analysis revealed a significant correlation between the yields and days on/in the soil using a phenol-chloroform method for soil collected at the head and feet. No earlier published work has extensively elucidated the efficacy of DNA extraction methods for DNA obtained from cadaver-soil.  相似文献   

12.
Recently, messenger RNA (mRNA), micro RNA (miRNA), and DNA methylation (DNAm) have been reported as novel markers for body fluid identification (BFID). Comprehensive analysis of these markers should be a flexible and reliable BFID method for various types of forensic samples. However, independent extraction of all targets can be difficult depending on the usable amounts of samples. In this study, the applicability of a co-extraction kit for these molecules, the AllPrep DNA/RNA/miRNA Universal Kit (APU), was evaluated by comparing RNA and DNA extracted from blood and saliva stains by the APU with those extracted by standard kits for each molecule and by previously reported methods for mRNA/DNA or miRNA/DNA co-extraction. Electrophoresis using the Bioanalyzer platform and real-time PCR analysis revealed that the APU performed almost equivalently to each standard kit in the quality of RNA or DNA extracted and extraction efficiency of mRNAs, miRNAs, and DNA. Moreover, the APU outperformed the co-extraction methods, especially in RNA integrity and miRNA extraction efficiency. In addition, pyrosequencing revealed that the methylation ratios of DNA extracted by the APU were not different from those extracted by standard DNA extraction kits. Overall, the APU is applicable to comprehensive analysis of mRNA/miRNA/DNAm markers for BFID analysis. Because the DNA eluate can also be used for DNA typing, the APU may be among the best choices for forensic examination of body fluid samples in terms of its flexibility and reliability in BFID and efficiency in sample consumption.  相似文献   

13.
本文用斑疹伤寒群和斑点热群立克次体鸡胚培养的感染卵黄囊膜在1MKCl溶液中制成10%~20%悬液,以差速离心沉淀和乙醚处理法提取纯化立克次体.该法纯化的立克次体涂片经Giemsa、H_(33258).荧光素及免疫酶标染色,在光镜下观察,立克次体形态完整,纯度较好.其斑疹伤寒群的普氏立克次体(E株)平均蛋白回收量为0.88mg/g膜,斑点热群的西伯利亚立克次体(Barbash株)和国内分离鉴定的斑点热立克次体精河株、斑点热立克次体黑龙江株及斑点热立克次体内蒙株的平均蛋白回收量为0.34~0.40mg/g膜.纯化的立克次体在核酸提取、DNA碱基成份的测定和全立克次体蛋白组成及抗原多肽构成的分析中取得了满意结果.  相似文献   

14.
DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow® (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex® ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8 ng ± 1 to 900 ng ± 159 of DNA compared with the organic method ranging from 0.5 ng ± 0.9 to 855 ng ± 156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20 mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time.  相似文献   

15.
目的 探讨石蜡包埋的10%硝酸脱钙骨组织中简易有效的结核杆菌DNA提取方法并进行验证.方法 以20例石蜡包埋、10%硝酸脱钙的坏死性肉芽肿炎疑为骨结核的组织标本为研究对象,常规法直接采用德国QIAGEN公司的QIAamp DNA FFPE Tissue Kit方法提取DNA,而改良法中加入QIAamp DNA Micro Kit试剂盒中的carrier RNA,以两种方法分别提取20例标本中结核杆菌DNA,通过荧光定量PCR,比较常规试剂盒提取法和改良提取法对结核杆菌检测结果的影响.结果 改良法提取的DNA[(35.32±3.48)ng/μl]比常规法[(6.68±3.72)ng/μl]提取的DNA显著增多(P<0.05),改良法提取的DNA经荧光定量PCR检测结核杆菌的阳性率(55%)明显高于常规法(15%,P<0.01).结论 改良法提取石蜡包埋脱钙骨组织中结核杆菌DNA具有高效简便的特点,是较为理想的方法,值得在puncture和极微小石蜡样本中推广.  相似文献   

16.
A simple method is described for the extraction of high quality DNA for PCR amplification. The DNA was extracted by using Chelex-100 ion exchange resin or a special cell lysis buffer containing proteinase K. For further purification the DNA was bound to silica in the presence of a chaotrophic agent. Hence it is possible to unlimitedly wash the bound DNA and inhibitory substances are removed. By using diatoms as a source of silicates, this method is very economical and can therefore be used as a routine method.  相似文献   

17.
An optimized protocol based on the DNA IQ™ System has been tested for the extraction of DNA from envelope flaps. DNA is extracted directly without the need for opening and swabbing the flaps. The method is repeatable with <10% R.S.D. (relative standard deviation). The results of a systematic study show that it is an equilibrium extraction, and a small sample volume as well as high lysis buffer content in sample contribute to high extraction efficiency. The extracted DNA requires no further purification steps following its extraction with the DNA IQ™ System. Complete but skewed 15-locus short tandem repeat (STR) profiles, which is typical of degraded of DNA, have been generated from the DNA extracted from 6 to 9 years old casework envelope samples.  相似文献   

18.
DNA typing from human faeces   总被引:2,自引:0,他引:2  
A method has been developed for the forensic analysis of faeces by DNA amplification and direct sequencing of a polymorphic segment of mitochondrial DNA. Starting from as little as 10 mg wet weight of faeces, DNA was extracted by a variety of protocols and amplified using primers specific to hypervariable region I of the mitochondrial control region. The resulting amplification products were sequenced in solid phase using an automated DNA sequencer. In total, mtDNA sequences were generated from the faeces of nine Caucasians and compared with sequences generated from their respective blood samples. Sequences of faeces and blood samples from the same individual were identical in every case, but a range of 1–10 nucleotide differences was observed between individuals, with an average sequence variation of approximately 4.88 per 400 bp. Of the various extraction protocols assessed in this study, greatest success rates were achieved using magnetisable beads to bind and purify the DNA. STR analysis of DNA extracted from faeces was not routinely possible.  相似文献   

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