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。     

穿山甲标本和甲片的DNA提取及PCR扩增

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定.结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA.以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作.     

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。     

基于线粒体细胞色素C氧化酶Ⅰ基因序列的细胞种属鉴别及细胞交叉污染分析

目的建立一种快速、简便、敏感和特异的基于线粒体细胞色素C氧化酶I(cytochrome C oxidase subunit I,CoI)基因序列的细胞基质检测方法,用于细胞种属鉴别和不同种属间的细胞交叉污染分析。方法①提取非洲绿猴肾细胞(Vero细胞)、狗肾细胞(MDCK细胞)和牛肾细胞(MDBK细胞)基因组DNA,以PCR扩增相应细胞的线粒体CoI基因保守区的基因条码序列,将扩增产物经琼脂糖凝胶电泳检测其基因片段的大小,并与文献报道的来源于ATCC相应细胞系线粒体CoI基因保守区的基因条码序列片段大小进行对比,以确定细胞的种属类别;②用Vero细胞、MDCK细胞和MDBK细胞的线粒体CoI基因保守区的基因的特异性引物分别与相应细胞和其他不同种属的细胞的基因组DNA进行PCR扩增,检测方法的特异性;用不同浓度的细胞基因组DNA与相应细胞的线粒体CoI基因保守区的基因的特异性引物进行PCR扩增,检测方法的灵敏度;③以不同种属细胞的线粒体CoI基因保守区的基因的特异性引物分别与多种细胞混合液提取的细胞基因组DNA进行PCR扩增,检测细胞间的交叉污染。结果 Vero细胞、MDCK细胞及MDBK细胞线粒体CoI基因保守序列中的基因条码序列片段大小,与来源于ATCC相应细胞系线粒体CoI基因保守序列中的基因条码序列片段大小相符。该方法具备较好的特异性,检测灵敏度为1.0 ng/μL,不同种属间细胞的交叉污染可被检出。结论该方法可有效地判断细胞的种属来源,亦可用于不同种属间细胞交叉污染的分析。     

福尔马林对固定标本DNA提取和扩增的影响

福尔马林被广泛应用于生物标本的长期保存。由于福尔马林可能影响标本DNA的质量,因此需要对福尔马林固定标本DNA的提取和扩增过程进行改进。影响从福尔马林保存标本中提取的DNA质量的主要因素包括福尔马林导致的DNA与蛋白质之间、蛋白质与蛋白质之间、DNA与DNA之间的交联,福尔马林溶液的化学成分、pH值及浓度,标本保存的时间和温度,标本保存部位等。本文总结了目前常用的对标本DNA提取和扩增过程的改进措施及其优点。     

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。     

复合PCR扩增人mtDNA HVR方法的初探

目的:建立一种高效的扩增线粒体DNA高可变区(mtDNA HVR)的方法.方法:本研究选取5例健康成人静脉血,用人血全基因组DNA试剂盒,提取全基因组DNA,设计引物,用复合PCR方式,对线粒体DNA中的高可变区进行扩增.复合扩增的方式为:用6对套叠引物分开进行两次独立的PCR,扩增mtDNA HVR.第一次扩增用3对引物,目标DNA片段基本涵盖整个线粒体DNA的高可变区,扩增后得到互不重叠的3个短片段,分别为113 bp,126 bp和131bp.第二次复合扩增用其余的3对引物,目标片段基本重叠在第一次扩增所得的目标片段的区域内,扩增得到3个互不重叠的片段为124 bp,133 bp和93bp.所有扩增产物经过纯化后测序.结果:复合PCR方式获得的mtDNA HVR基因序列完整,5个样本均出现特异性条带,电泳结果条带单一、清晰.结论:复合扩增PCR方法对mtDNA HVR区的扩增效率高,测序结果稳定,结合6对套叠引物,不但保证了序列的完整性,另外,两次独立的PCR也减少了PCR反应过程中错配的发生,此法也适用于保存时间较久的古代线粒体DNA短片段的研究.复合扩增PCR还展示出了潜在的高产量的特点,相对传统PCR显示了其更多的优势.     

12S rRNA和Cyt b基因序列测定在獐乳制品鉴定中的应用

采用改良的蛋白酶K消化和酚/氯仿抽提的方法从脂肪含量很高的动物乳制品及胃组织中提取出基因组总DNA,利用特异引物扩增了线粒体12S rRNA基因和Cyt b基因的部分片段,测定了12S rRNA和Cyt b基因的PCR扩增产物序列,使用BLAST搜索软件将其序列与GenBank中的同源序列进行比对,并利用DNAMAN分析软件分析序列同源性。结果表明3件检材均来源于獐Hydropotes inermis。本研究所用方法在野生动物乳制品鉴定中具有较高的应用价值。     

两种获取鱼类线粒体DNA的方法比较

直接从鱼类组织提取线粒体DNA和先提取基因组DNA再用相应引物PCR扩增所需mtDNA某一区域序列片段是获取鱼类mtDNA切实可行的两种方法。对这两种方法在使用过程中的注意事项、各自的优缺点及应用范围进行了较为细致的比较,为更好地利用mtDNA这一重要分子标记研究鱼类mtDNA的遗传多态提供参考。     

不同预处理方法影响昆虫干标本DNA提取效果

为从昆虫干标本中获取高质量DNA,以利于后期PCR扩增目的基因,本研究对DNA提取前昆虫干标本的预处理方法进行探讨,分别测定其纯度和质量,并用不同引物进行了扩增效果对比。结果显示:经过预处理的样品DNA平均含量均高于CK的52.283 ng/滋L,最高为0.9%Na Cl处理3 h,平均含量达122.632 ng/滋L;米象4种预处理方法提取的DNA利用引物Lco1490/Hco2198及UEA7/UEA10均能扩增出650 bp长度条带,仅有0.9%Na Cl溶液浸泡3 h处理的米象可通过引物J173/J1331扩增出1 000 bp的长片段条带。研究表明,经过预处理后,一定程度上减少了基因组DNA的断链,主要对样品DNA的质量浓度影响显著;以9%Na Cl溶液3 h预处理后的昆虫干标本,利用磁珠法所提DNA的质量及PCR扩增效果最佳。     

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。     

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.     

An efficient method for the extraction of DNA from formalin-fixed, paraffin-embedded tissue by sonication

A method was developed for fast and efficient isolation of DNA from formalin-fixed, paraffin-embedded tissue sections for subsequent use in PCRs and DNA hybridization assays. The method relies on the use of a sonicating water bath to disrupt tissue samples to which a small amount of micro-sized glass beads have been added. The sonicating glass beads provide fast and efficient physical shearing of fixed tissue sections, allowing for quick release and solubilization of the DNA. The extraction process from paraffin section to amplifiable target DNA takes 30 minutes. The method eliminates the need for repetitive solvent extractions and exhaustive proteinase K digestion. PCR amplification of human genomic and viral target sequences was successfully carried out on DNA isolated from a number of different types of normal and infected tissues.     

Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.     

Molecular identification of Taenia specimens after long-term preservation in formalin

The majority of Taenia tapeworm specimens in the museum collections are usually kept in a formalin fixative for permanent preservation mainly for use in morphological examinations. This study aims to improve Taenia tapeworm identification even of one preserved in formalin for a maximum of 81 years. Taenia tapeworms were collected by the parasite collection unit of the Swiss Natural History Museum and from units in Indonesia, Japan and Korea. A small amount of formalin-fixed tissue (100 mg) was crushed in liquid nitrogen and then soaked in a Tris-EDTA buffer for 3-5h. The sample was then digested in SDS and proteinase K (20 mg/ml) for 3-5h at 56 °C. After the addition of proteinase K (20mg/ml), SDS and hexadecyl-trimethyl-ammonium bromide (CTAB), incubation was continued for another 3h at 65 °C. A maximum yield of genomic DNA was obtained from this additional step and the quality of genomic DNA obtained with this extraction method seemed to be independent of the duration of storage time in the formalin fixative. The molecular identification of Taenia tapeworms was performed by using PCR and DNA sequences corresponding to position 80-428 of cox1 gene. T. asiatica was detected in the isolates of Indonesia, Japan and Korea. Improvements in the genomic DNA extraction method from formalin fixed museum collections will help in the molecular identification of parasites.     

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。     

Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.

Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.     

Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants

Medicinal plant species has a valuable economic importance because of its usage as pharmaceuticals, nutritional, as well as its use in popular medication. For DNA-based techniques, nanogram quantities of the purified DNA are requisite to amplify and yield sufficient amounts of PCR products. SDS-based DNA isolation method was used to extract DNA from 11 species of different aromatic and medicinal plants collected from Saudi Arabia. Three hundred milligrams of fresh shredded plant material was necessary. The DNA purity was further confirmed by agarose gel, restriction endonuclease digestion and microsatellite primed-polymerase chain reaction (MP-PCR). DNA yields ranged from 10-20 μg (in 100-μL elution volumes) from all plant material evaluated. The DNA obtained was free of any contaminating proteins, polysaccharides and colored pigments. The extracted genomic DNA was found suitable for restriction digestion and PCR amplification. Our experimental procedure provides an easy, suitable, non-toxic, cheap, and quick process for the amplification of DNA from medical plant tissue.