Silica-KI快速提取外周血基因组DNA的实验研究

目的　建立一种快速的外周血基因组DNA提取方法。方法　用硅胶 碘化钾 (Silica KI)吸附法直接从外周血提取基因组DNA。结果　提取的基因组DNA相对分子质量大 ,纯度较高 ,A2 60 / 2 80nm为 1.75～ 1.93。每毫升外周血可获得 2 5～ 35 μgDNA ,体外扩增和限制性内切酶酶切结果满意。 结论　Silica KI吸附法是一种简单、高效、快速、安全、适用于大量临床标本处理DNA的提取方法。     

Silica-KI快速提取外周血基因组DNA的实验研究

目的 建立一种快速的外周血基因组DNA提取方法。方法 用硅胶-碘化钾(Silica—KI)吸附法直接从外周血提取基因组DNA。结果 提取的基因组DNA相对分子质量大，纯度较高，A260／280nm为1．75～1．93。每毫升外周血可获得25～35μg DNA，体外扩增和限制性内切酶酶切结果满意。结论 Silica—KI吸附法是一种简单、高效、快速、安全、适用于大量临床标本处理DNA的提取方法。     

探讨从人血凝块中提取基因组DNA

目的从人血凝块中提取基因组DNA。方法室温自然解冻,人工磨碎,用Relax Gene Blood DNA System血液基因组DNA提取试剂盒提取DNA。结果用此试剂盒从人血凝块中成功提取基因组DNA,提取DNA浓度为16.1±4.7mg/L。结论用此试剂盒提取的基因组DNA的总量较高,提取效率高,PCR扩增效果好,可很好的用于血凝块基因组DNA的提取。     

改良碘化钾法提取冻存外周血基因组DNA的方法探讨

目的探讨用改良碘化钾法（改良法）提取长期冻存外周血基因组DNA的方法。方法对碘化钾（KI）法加以改良,包括：血量由100μL增加到200μL;使用0．9％NH4CL溶液代替无菌重蒸馏水裂解红细胞;增加裂解白细胞膜的5mol／LKI溶液的用量（为70μL）;低温（4℃）离心。然后用改良碘化钾法从82份．80℃冻存外周血标本中提取基因组DNA。同时使用碘化钾法提取其中的30份血标本,比对两种方法提取DNA的浓度和纯度,并进行CYP2C19相关基因PCR扩增。结果两种方法所提取的DNA浓度和纯度分别是碘化钾法为128．2±34．9μg／mL和A260／A280 1．74±0．08,改良法为220±91．29μg／mL和A260／A280 1．87±0．12。改良法获得的DNA纯度更高,差异有统计学意义（P〈0．01）,通过增加样本量及对方法进行改良获得的DNA量较多。对改良后方法提取的基因组DNA进行PCR扩增,结果稳定。结论改良碘化钾法从长期冻存外周血中所提取的基因组DNA质量较高,能够满足对临床冻存样本研究的需求。     

外周血DNA提取方法的比较

快速、经济地从外周血或组织中提取高产量、高纯度的DNA,对于疾病的分子水平研究非常重要。目前基因组DNA的提取方法较多,如酚/氯仿、尿素提取法、氯化锌     

全自动提取大量全血样本基因组DNA方法的建立及其在HLA基因分型中的应用

目的研究和建立从大量全血样本中提取基因组DNA的有效方法,以应用于Luminex HLA流式磁珠基因分型。方法使用自动工作站(瑞士TECAN公司)提取基因组DNA,提取的DNA样本用紫外分光光度仪测定其浓度和纯度,DNA的完整性用琼脂糖电泳检测,并统计分析每一DNA样本流式磁珠HLA-A、B和DRB1基因扩增产物经探针分子杂交后的荧光信号强度。结果从60μl全血中提取基因组DNA,产量平均为(1.584±0.824)μg,样本的A260/A280值平均为1.741±0.229。琼脂糖电泳法测得DNA的分子量约为21kb。结论本方法适用于从大量全血样本中快速提取基因组DNA,所得基因组DNA适用于高通量HLA流式磁珠基因分型等下游的分子生物学实验。     

外周血单个核细胞DNA提取方法的研究进展

DNA的提取是分子生物学研究的基础技术,提取的DNA的纯度及结构完整性是进行基因工程各项研究所必需的条件.外周血作为一种常用的临床检测材料,如何从外周血单个核细胞中快速、高效的分离提取基因组DNA,对于临床血液检验与分析非常重要.目前,DNA的抽提方法有很多,如酚-氯仿法、盐析法、离心吸附柱法等人工方法,以及磁珠法等半自动方法及试剂盒法.本文就从外周血血液中提取DNA方法的原理、具体操作步骤及方法评估等给予简要的综述.     

碘化钾法抽提冻存外周血DNA的方法初探

目的:探讨碘化钾法从冻存外周血中抽提基因组DNA的产量及质量.方法:对400μL冻存血标本(-20℃保存半年)及200 μL新鲜血标本(4℃保存不超过1周)分别采用KI法提取基因组DNA,比较其浓度和纯度.并进行CYP2C19相关基因的PCR扩增.结果:通过增加处理标本量,可以相应提高从冻存外周血中提取的基因组DNA产量,但获取的DNA纯度低于新鲜血标本.结论:KI法提取DNA时,需要根据实验目的和要求,选择适当的保存方法.     

全自动提取大量全血样本基因组DNA方法的建立及其在HLA基因分型中的应用

目的 研究和建立从大量全血样本中提取基因组DNA的有效方法,以应用于Luminex HLA 流式磁珠基因分型.方法 使用自动工作站(瑞士TECAN公司)提取基因组DNA,提取的DNA样本用紫外分光光度仪测定其浓度和纯度,DNA的完整性用琼脂糖电泳检测,并统计分析每一DNA样本流式磁珠HLA-A、B和DRB1基因扩增产物经探针分子杂交后的荧光信号强度.结果 从60μl全血中提取基因组DNA,产量平均为(1.584±0.824)μg,样本的A260/A280值平均为1.741±0.229.琼脂糖电泳法测得DNA的分子量约为21kb.结论 本方法适用于从大量全血样本中快速提取基因组DNA,所得基因组DNA适用于高通量HLA流式磁珠基因分型等下游的分子生物学实验.     

四种DNA提取法在遗传性眼病DNA库中的比较

目的通过对低渗溶血法、盐酸胍法、氯化钠法、NP-40法提取人类基因组DNA的比较，以得到一种最稳定、可靠、实用的DNA提取方法，应用于遗传性眼病DNA库的建立。方法用四种方法提取眼遗传性疾病患者的血液基因组DNA，紫外分光光度测定，用琼脂糖凝胶电泳和PCR扩增鉴定提取的基因组DNA。结果低渗溶血法提取基因组DNA量最多，纯度最高，PCR扩增效果最好，有利于遗传性眼病DNA库的建立。结论低渗溶血法是一种实用、可靠、稳定的提取方法，在眼遗传性疾病家系DNA库建立上值得推广应用。     

改良碘化钠法提取人类微量全血基因组DNA的探索

目的通过改良碘化钠(NaI)法,建立一种简单、快速、经济的从人微量全血中提取基因组DNA的方法。方法采用经典NaI法和改良NaI法分别从人微量全血中提取基因组DNA,并进行常规和荧光定量聚合酶链反应(PCR),比较两种方法DNA提取的效果。结果采用改良NaI法从人外周全血中提取的DNA浓度和纯度与经典NaI法相比较,差异无统计学意义(P0.05),并且可以在较短时间内获得满意的常规和荧光定量PCR结果,且耗时较短。结论改良NaI法是一种简便、快速的提取人微量全血基因组DNA的方法,在临床和基础研究中具有较大的应用价值。     

三种简易提取全血基因组DNA方法的比较

目的比较兰种快速、简便、适用、低成本的从全血中提取基因组DNA的方法。方法选用快速氯仿-异戊醇法、简化盐析法、改良碘化钾法直接从全血中提取基因组DNA。结果改良碘化钾法提取的DNA纯度及含量最高，快速氯仿-异戊醇法次之，简化盐析法则相对较低。结论改良碘化钾法为较好的提取基因组DNA的方法。     

全自动工作站与两种人工抽提全血基因组 DNA 方法的比较

目的比较全自动工作站与两种人工法抽提全血基因组DNA的质和量,总结三种方法的特点及提供一些建议。方法三种方法均选取同样36份样本抽提基因组DNA,并比较其浓度和纯度,并计算三种方法所需的费用。结果三种方法提取的基因组DNA均能达到相关分子生物学实验的要求,但在操作程序、价格和全血需要量等方面都存在较大差异。结论全自动工作站用全血量少,价格比较便宜,适合大批量全血样本抽提基因组DNA。Gentra法和胍盐酸法均适用于新鲜全血和冻融过的全血,胍盐酸法还适用于有血凝块的全血。     

Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele-specific oligonucleotide-primed polymerase chain reaction assay: development and validation.

A polymerase chain reaction (PCR) assay has been developed and validated by using allele-specific oligonucleotide (ASO) primers to specifically amplify E3, E2, and E4 polymorphic sequences of the human apolipoprotein E (apo E) genes. Degenerate ASOs containing one or two additional 3' mismatches provided greater specificity than did ASOs containing a single mid-sequence or 3' allele-specific mismatch with plasmid pEB4 or genomic DNA as template. Optimal specificity and efficiency of amplification did not correlate with primer annealing conditions, whether determined theoretically or via oligo-melting experiments. Pre-cycling denaturation times and high cycling denaturation temperatures were also required for optimal amplification, presumably because of the high G:C content (75-85%) of apo E gene sequences. Conditions permissive for amplification and discrimination with plasmid DNA did not transpose favorably to amplification from human genomic DNA from peripheral blood leukocytes; the latter required nested primer reactions. These data may be valuable in predicting PCR assay conditions for other G:C-rich sequences containing polymorphic sequence differences. The assay described is both more accurate and rapid (24 h) than previously described methods for phenotyping or genotyping human apo E from blood specimens.     

广西瑶族与汉族人多瘤病毒感染率的调查研究

目的研究广西瑶族与汉族健康成人外周血白细胞（PBLs）中人多瘤病毒（BKV）感染的发生率。方法采集广西瑶族及汉族健康人外周血标本各500份，提取淋巴细胞基因组DNA，用巢式PCR方法扩增BKV的保守区编码序列，统计分析不同民族、年龄、性别组BKV—DNA检出率。结果1000份健康人PBLs样品中BKV感染的发生率为61．3％，不同民族、性别、年龄组之间无显著性差异（P〉0．05）。结论BKV在广西瑶族及汉族健康人PBLs中存在较高的感染率，本研究证实了PBLs是BKV在体内的潜伏细胞及传播载体，应加强对BKV条件致病性的认识，积极防治、减少BKV相关的移植肾病（BKVN）的发生率。     

一种改良的人DNA微量快速检验技术

目的 探索一种以口腔粘膜脱落细胞为材料的安全、高效、低成本、敏感性高的人DNA微量快速检测方法。方法 采用从漱口液或棉签擦拭口腔粘膜获取脱落细胞,应用混合树脂（Chelex100）煮沸沉淀法提取DNA;用PCR分别对线粒体特异性DNA片断和基因组中的特定基因进行扩增检测。结果 通过对线粒体DNA中440 bp的非编码片段和染色体基因组中220 bp的乙醛脱氢酶DNA片段进行扩增,结果显示,从漱口液脱落细胞提取的DNA中可以稳定地扩增出上述两种DNA片断。结论 建立了一种改良的人口腔粘膜脱落细胞DNA微量快速检验技术。该法取样方便,DNA样品获取量较大,一次取样可同时进行多项线粒体和基因组标志DNA片段的快速PCR检测。     

Effect of foscarnet induction treatment on quantitation of human cytomegalovirus (HCMV) DNA in peripheral blood polymorphonuclear leukocytes and aqueous humor of AIDS patients with HCMV retinitis. The Italian Foscarnet Study Group.

The aim of this study was to investigate peripheral blood polymorphonuclear leukocytes and, whenever possible, aqueous humor from 65 AIDS patients with ophthalmoscopically diagnosed human cytomegalovirus (HCMV) retinitis to determine (i) whether patients consistently carry viral DNA and (ii) to what extent foscarnet induction treatment decreases viral DNA levels. HCMV DNA was quantified by PCR using densitometric analysis of hybridization products obtained from external standards and a standard curve from which the number of genome equivalents of test samples, normalized by using an internal amplification control, was interpolated. Results showed that 56 of 65 patients (86.1%) were positive for HCMV DNA prior to induction treatment. Of 41 of the 56 patients (73.2%) whose blood had become DNA negative after induction, only 5 had a high viral load (> 5,000 genome equivalents per 2 x 10(5) polymorphonuclear leukocytes) prior to induction, whereas as many as 13 of the 15 (26.8%) patients remaining DNA positive after induction had a high viral load prior to induction. Finally, of the nine patients (13.8%) with DNA-negative blood prior to induction treatment, three were shifted to foscarnet from ganciclovir, while six were erroneously enrolled in the study. Pre- and postinduction aqueous humor samples were obtained from 12 patients; all of these were DNA positive prior to induction, whereas after induction, 4 became negative, 6 showed a marked decrease in viral DNA, and 2 had nearly stable low DNA levels. In conclusion, PCR is a valuable tool for etiologic diagnosis and monitoring of HCMV retinitis treatment in AIDS patients. HCMV DNA is consistently present in the blood and aqueous humor of all patients with HCMV retinitis. Foscarnet induction treatment is highly effective in suppressing or reducing DNA levels in both blood leukocytes and aqueous humor.     

Failure to detect human cytomegalovirus DNA in peripheral blood leukocytes of healthy blood donors by the polymerase chain reaction

The transmission of cytomegalovirus (CMV) by blood transfusion may have a major effect on certain immunocompromised patients. To protect susceptible blood recipients from infection, it is advisable to use blood components from CMV-seronegative donors. However, serologic tests are not capable of indicating which blood component actually harbors infectious virus and can transfer it to the recipient. Therefore, a sensitive method is needed for the detection of the virus itself. There have been three reports on the detection of CMV in healthy volunteer blood donors by the polymerase chain reaction (PCR). CMV DNA was found in all seropositive and most seronegative blood donors. However, many other authors have failed to confirm these data. A highly sensitive and specific PCR assay was developed for the detection of CMV DNA in peripheral blood leukocytes. With this protocol, blood samples from 116 volunteer blood donors were investigated. None of these samples proved to be positive for CMV DNA. In contrast, CMV DNA was detected in 10 of 10 renal transplant patients early in the course of active CMV infection. It can be concluded that the CMV genome copy number in the peripheral blood leukocytes of healthy individuals is beyond the detection limit of current PCR technology.     

纳米磁珠法与试剂盒法分析载脂蛋白E基因的多态性

背景：相对于血液标本,口腔拭子标本更利于大规模载脂蛋白E基因多态性分析的研究,但目前对口腔拭子标本基因组DNA的提取尚无统一方法。 目的：探索合适的口腔拭子标本基因组提取方法以分析载脂蛋白E基因的多态性。 方法：收集50例散发性阿尔茨海默病患者口腔拭子标本,每份标本分别应用纳米磁珠法与PicoDNA微量核酸提取试剂盒法提取基因组DNA,对比分析两种方法获得的基因组DNA纯度和浓度,后续进行PCR反应,应用DNA电泳确认有无成功扩增出目的条带,通过DNA测序方法分析载脂蛋白E基因的多态性。 结果与结论：两种方法提取的基因组DNA纯度均较好,纳米磁珠法提取的基因组DNA浓度要高于PcioDNA微量核酸提取试剂盒法所得浓度（P〈0.05）。两组获取的基因组DNA均可成功进行PCR扩增,但电泳结果示纳米磁珠法扩增的目的条带更清楚。两组PCR产物DNA测序结果一致,载脂蛋白E基因ε2、ε3、ε4基因型的比例分别是6%,71%,23%。表明纳米磁珠法相对于PcioDNA微量核酸提取试剂盒法提取口腔拭子标本基因组DNA更适合用于大样本载脂蛋白E基因多态性的研究。