福尔马林保存鱼类标本中大片段DNA的提取

从福尔马林保存的鱼类标本中获得高质量DNA是比较困难的。我们对前人的方法进行了如下改进：1）在标本的前处理过程中,通过长时间的缓冲液浸泡、短暂的加温、真空干燥来消除福尔马林对样品的影响;2）在样品消化过程中,加入相对过量的蛋白酶K和还原剂;3）提取DNA后立即进行PCR反应,并增加反应的循环次数和提高退火温度。通过这些改进,我们成功地从福尔马林保存的鱼类标本中提取出了高质量DNA;通过对比不同方法（福尔马林、酒精及冰冻）处理过的标本的DNA测序结果,表明该方法是值得信赖的;标本从死亡到用福尔马林处理之间的时间延搁可能是影响所提取的DNA质量的重要因素。     

保存方式和回软温度对蜜蜂标本DNA提取的影响北大核心CSCD

旨在探寻保存方式及制作干标本前回软温度对蜜蜂不同部位DNA的影响。采用酚-氯仿法对不同方式保存的蜜蜂以及不同温度回软后的蜜蜂干标本的总DNA进行提取,通过琼脂糖凝胶电泳和PCR扩增对DNA的提取结果进行鉴定。电泳结果显示,从新鲜标本、无水乙醇泡制或自然干燥保存半年的标本中均可提取到较高质量的总DNA,尤以头部与足部提取效果最佳。经不同水浴温度回软后保存半年的蜜蜂干标本总DNA提取结果显示,回软温度为65℃时对蜜蜂DNA的破坏性最小,DNA提取的最佳部位为蜜蜂足部。正交试验结果显示,55℃回软后的足部为蜜蜂干标本DNA提取的最优组合。PCR扩增结果显示,本实验提取的总DNA能成功地应用于蜜蜂线粒体基因16S rRNA和COI的扩增。从保存方式、提取部位和回软操作3个因素对蜜蜂标本DNA的提取进行了研究,提供了较好的选择方案。     

保存方式和回软温度对蜜蜂标本DNA提取的影响

旨在探寻保存方式及制作干标本前回软温度对蜜蜂不同部位DNA的影响。采用酚-氯仿法对不同方式保存的蜜蜂以及不同温度回软后的蜜蜂干标本的总DNA进行提取,通过琼脂糖凝胶电泳和PCR扩增对DNA的提取结果进行鉴定。电泳结果显示,从新鲜标本、无水乙醇泡制或自然干燥保存半年的标本中均可提取到较高质量的总DNA,尤以头部与足部提取效果最佳。经不同水浴温度回软后保存半年的蜜蜂干标本总DNA提取结果显示,回软温度为65℃时对蜜蜂DNA的破坏性最小,DNA提取的最佳部位为蜜蜂足部。正交试验结果显示,55℃回软后的足部为蜜蜂干标本DNA提取的最优组合。PCR扩增结果显示,本实验提取的总DNA能成功地应用于蜜蜂线粒体基因16S rRNA和COI的扩增。从保存方式、提取部位和回软操作3个因素对蜜蜂标本DNA的提取进行了研究,提供了较好的选择方案。     

福尔马林固定白鱀豚标本DNA提取及其遗传多样性的初步研究

福尔马林固定标本是宝贵的遗传资源,但是如何有效利用其中的遗传信息一直存在问题。本文尝试从标本预处理、消化、PCR扩增各方面综合考虑和优化改进,成功提取并扩增21头福尔马林固定白豚标本线粒体DNA控制区410bp片段。采用了3种预处理方法尽量去除固定标本中残存的甲醛,从试验结果来看,从酒精梯度 临界点干燥处理的标本中提取的DNA在扩增时具有明显优势。通过蛋白酶K消化过程中对于酶的浓度、温浴时间的比较试验,发现随着采用大幅提高酶浓度、延长消化时间等高强度的蛋白酶消化操作后,DNA的质量和产量均得到显著提高。针对标本DNA降解严重的特点,设计特异性好且长度合适的引物以及使用巢式引物扩增,均提高了标本DNA扩增的特异性和灵敏度。通过对所测得的21头白鱀豚线粒体DNA控制区部分序列的对比,发现全部个体在该片段上的序列完全一致,说明白豚遗传多样性极低。     

福尔马林固定白暨豚标本DNA提取及其遗传多样性的初步研究

福尔马林固定标本是宝贵的遗传资源,但是如何有效利用其中的遗传信息一直存在问题。本文尝试从标本预处理、消化、PCR扩增各方面综合考虑和优化改进,成功提取并扩增21头福尔马林固定白暨豚标本线粒体DNA控制区410bp片段。采用了3种预处理方法尽量去除固定标本中残存的甲醛,从试验结果来看,从酒精梯度 临界点干燥处理的标本中提取的DNA在扩增时具有明显优势。通过蛋白酶K消化过程中对于酶的浓度、温浴时间的比较试验,发现随着采用大幅提高酶浓度、延长消化时间等高强度的蛋白酶消化操作后,DNA的质量和产量均得到显著提高。针对标本DNA降解严重的特点,设计特异性好且长度合适的引物以及使用巢式引物扩增,均提高了标本DNA扩增的特异性和灵敏度。通过对所测得的2l头白暨豚线粒体DNA控制区部分序列的对比,发现全部个体在该片段上的序列完全一致,说明白暨豚遗传多样性极低。     

松墨天牛成虫标本保存及其DNA提取质量比较

【目的】探讨适合DNA提取的天牛成虫标本保存方法。【方法】采用SDS-蛋白酶K消化法对液氮中冷冻保存、无水乙醇-20℃冷冻保存、无水乙醇室温保存和干标本室温保存且保存时间在2年以上的松墨天牛Monochamus alternates Hope成虫标本基因组DNA进行提取,并对不同保存方式提取的DNA样本进行了质量比较和分析。【结果】在上述常见的松墨天牛成虫标本4种保存方式中,以液氮中冷冻保存效果最佳,其次为无水乙醇-20℃冷冻保存,插针干标本室温保藏效果最差。利用昆虫线粒体基因CO I和CO II的通用引物从上述DNA中均能够成功扩增出目的片段,测序结果证实扩增片段符合预期。【结论】液氮和无水乙醇-20℃冷冻保存适合松墨天牛成虫标本长期保存,且不影响后续的PCR扩增和测序。     

福尔马林固定铜鱼基因组DNA的提取与扩增

铜鱼（Coreius heterodon）作为长江中、上游的重要经济鱼类是研究三峡大坝阻隔效应的重要材料之一。然而,过去的铜鱼标本都保存在福尔马林溶液中,有必要探讨从福尔马林固定的铜鱼标本中有效提取基因组DNA的方法以及这些DNA用于微卫星和线粒体分析的可行性。本实验通过改进的酒精梯度浸泡法去除标本中的甲醛,然后用酚-氯仿抽提法成功地提取到了铜鱼标本的基因组DNA;设计引物后进行了线粒体和微卫星的PCR扩增,扩增产物经银染检测。微卫星扩增结果显示只有部分个体可以扩出目的带,而线粒体控制区部分区段在所有个体中均能稳定重复的扩出;mtDNA SSCP分析显示带型一致。结果表明,福尔马林固定的铜鱼标本可以被用来开展短片段的扩增和遗传变异分析等方面的相关研究。     

一种高效快速的鱼类标本基因组DNA提取方法

以95%酒精保存的黄鳝(M onopterus albus)和斑鳢(Channa maculates)标本为材料,采用先沉降DNA再去除杂质的方法从鱼类标本中提取基因组DNA。基因组DNA的琼脂糖凝胶电泳和紫外分光光度法检测以及PCR扩增结果显示,本方法提取的鱼类基因组DNA的电泳主带清晰明亮;A260/A280值在1.7830-2.0144之间;PCR扩增产物条带清晰明亮,且单一整齐没有拖带,表明本方法可从酒精保存的鱼类标本中提取比较纯净的DNA,能够满足一般分子生物学试验需要。与传统苯酚/氯仿法相比,本方法操作简单快速,避免了苯酚等物质对后续实验的影响,可作为一种常规动物组织DNA提取方法。     

不同保存方式下蝗虫组织DNA的提取及RAPD分析

为了开展蝗虫分子系统学研究,分别对冷冻、乙醇浸泡(100%、乙醇、70％乙醇)和干制蝗虫标本用饱和NaCl法进行了基因组DNA的提取,并用随机引物进行扩增,结果表明：70％乙醇固定的标本和部分干标本提取的总DNA得率较低,在琼脂糖凝胶电泳检测中大音琏分有明显降解,导致PCR扩增中信息缺失,甚至无扩增条带;而保存完好的干标本、-20℃冷冻标本和100%,乙醇浸泡标本提取的总DNA带型整齐,无拖尾,PCR扩增结果的稳定性好,成为蝗虫分子系统学研究中首选的三种保存方式。     

几种提取枣和酸枣DNA用于RAPD分析的方法比较

利用不同的方法提取枣和酸枣的总DNA,并分析了不同样品状况、抗氧化剂和纯化过程等对所提DNA质量及其RAPD扩增效果的影响。实验显示：用改良CTAB法优于SDS法,可有效去除多糖。加入的抗氧化剂PVP、抗坏血酸、β-巯基乙醇等可有效阻止多酚类物质褐化,但不同抗氧化剂间效果差别不大。分别将样品干燥处理、固定处理、液氮处理、冷藏,与新鲜材料相比,用液氮处理并保存于-80℃的材料与新鲜材料所提DNA相当,而用其他方法DNA都有不同程度降解。不同材料提取结果比较显示：幼叶所提DNA产量和质量优于老叶,并且不需要较多的纯化过程。在有RNA和少量蛋白质时,对扩增结果影响不大。     

Formalin fixing and paraffin embedding may lead to extra band development in PCR

The molecular biological analysis of infectious agents requires the availability of a reliable source of microorganisms to be used to recover DNA. Clinical samples can be obtained directly from infected patients or can be propagated using in vitro or in vivo systems. However, repeated sampling from patients is not always possible as the procedure may be invasive or unpleasant, or it is not possible to catch the same agent at the time of second sampling. Moreover, the techniques used may also produce false-positive and false-negative results. We therefore studied the impact of formalin-fixing and paraffin embedding on tissue sampling, and the methodologies such as DNA isolation and PCR amplification of DNAs from archival materials in the diagnosis of Mycobacterium tuberculosis. PCR analyses were done according to standard methods with some modifications. Demonstration of mycobacteria was successful both in tissue sections of the formalin-fixed lymph nodes and in stained fresh materials from patients. However, the results showed the presence of two extra bands in the gel. We accounted for extra band development due to the harshness of the methodology used to isolate nucleic acids from formalin-fixed and paraffin embedded tissue samples or the nature of the fixation procedure, or because of the time passed during storage in which alteration in the chromosomal DNA would take place. Thus, if disease- and tissue specific morphological features, such as sample size, type of fixation, and intralesional heterogeneity are ignored, errors because of sampling and methodologies used may lead to false-positive and false-negative results.     

Formalin removal from archival tissue by critical point drying

The extraction of high-quality nucleic acid may be problematic in formalin-fixed tissues because of cross-linking between proteins and DNA. Old fixed tissue specimens do produce fragmented DNA (<1.2 kb), which is only used for PCR amplification. Here we show that high molecular weight DNA (>194 kb) can be successfully extracted from fixed tissue samples (16-70 years old) by gradual dehydration and critical point drying. The reliability of extracted DNA was measured by its ability to serve as a template for the amplification of mtDNA fragments (403 and 1198 bp) and an nDNA fragment (1844 bp). In addition, fingerprinting analysis was performed using DNA from fixed human tissue to ensure the ability of extracted DNA to hybridize with the DNA probe. DNA derived by this method can be subject to amplification, complete digestion by restriction endonuclease, and hybridization.     

Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues

Fresh or frozen tissue samples will always be the best tissue source for the analysis of nucleic acids and proteins from tissues. However, their long-term storage is expensive and laborious. Much interest has therefore been focused on the question whether the almost infinite resources of formalin fixed and paraffin embedded tissue samples in the archives of pathology and histology departments can be used for research on biomarkers and molecular mechanisms of disease. In recent years the methods and protocols for the extraction of DNA, mRNA, miRNA and proteins from formalin-fixed and paraffin-embedded tissue samples have improved enormously. Especially, the possibilities of analysing DNA and miRNA in FFPE have reached a level that allows their application as a first line approach in the search for biomarkers. In contrast, many questions remain in terms of quantification of mRNA and protein expression levels in formalin-fixed and paraffin-embedded tissue samples. This review gives an overview on current potentials and limitations of the quantification of DNA, miRNA, mRNA and the proteome in FFPE tissue samples. The chemical events during formalin fixation and paraffin embedding and alternatives to formalin fixation are described. In addition, methods and general problems of DNA, miRNA, mRNA and protein extraction and the current knowledge on the feasibility and accuracy of quantitative gene expression analysis in FFPE tissues is summarized.     

Droplet digital PCR is an accurate method to assess methylation status on FFPE samples

Most tissue samples available for cancer research are archived as formalin-fixed paraffin-embedded (FFPE) samples. However, the fixation process and the long storage duration lead to DNA fragmentation and hinder epigenome analysis. The use of droplet digital PCR (ddPCR) to detect DNA methylation has recently emerged. In this study, we compare an optimized ddPCR assay with a conventional qPCR assay by targeting a dilution series of control DNA. In addition, we compare the ddPCR technology with results from Infinium arrays targeting two separate CpG sites on a set of colon adenoma FFPE samples. Our data demonstrate that qPCR and ddPCR assess methylation status equally well on dilution controls with a high DNA input. However, the methylation detection on low-input samples is more accurate using ddPCR. The proposed primer design (methylation-independent primers with amplification of solely the converted DNA target) will allow for methylation detection, independent of bisulfite conversion efficiency. Those data show that ddPCR can be used for methylation analysis on FFPE samples with a wide range of DNA input and that the precision of the assay depends largely on the total amount of amplifiable DNA fragments. Due to accessibility of the ddPCR technology and its accuracy on high- as well as low-DNA input samples, we propose the use of this approach for studies involving degraded FFPE samples.     

激光显微切割技术用于子宫内膜异位组织DNA的提取及其完整性分析

目的：探索一套激光显微切割（LCM）分离子宫内膜异位症腺体细胞后提取微量DNA并进行完整性分析的操作流程。方法：分别对20例石蜡标本及20例冰冻标本进行LCM,收集切割后的腺体细胞;2组标本各取10例提取微量DNA,检测DNA浓度并通过PCR扩增进行验证;余20例标本分别进行全基因组扩增,检测产物浓度并利用8种常见管家基因作为引物通过PCR扩增进行验证,对比分析其结果。结果：石蜡标本与冰冻标本在LCM获取腺体细胞及提取微量DNA两个环节中均可获得满意效果;但经全基因组扩增后,石蜡标本无法保留完整DNA信息。结论：LCM获取子宫内膜异位症腺体细胞提取微量DNA是一种操作简单、结果稳定的方法,可作为日后子宫内膜异位症基因组研究的常规方法;冰冻切片相对石蜡切片,更能保留完整的DNA信息。     

Detection of human papilloma virus DNA sequences by polymerase chain reaction

We have developed a polymerase chain reaction-based procedure for reproducible detection of the E6-E7 gene in human papilloma virus DNA sequences using formalin-fixed, paraffin-embedded tissue sections. This procedure is a simple one-step procedure which does not require any elaborate hybridization following polymerase chain reaction amplification. The protocol combines modified tissue treatment and proper primer selection for efficient amplification of target DNA in a highly specific manner allowing identification in ethidium bromide-stained gels. The procedure described here is useful for a variety of tissue preparations, particularly formalin-fixed, paraffin-embedded archival tissues.