一种从石蜡包埋组织中快速提取DNA的方法

目的建立从甲醛固定石蜡包埋组织中高效、快速提取基因组DNA的方法。方法将一片10μm厚石蜡包埋大肠癌组织切片直接浸入150μl消化液中,56℃孵育0.5h、1h和2h,灭活蛋白酶K后离心取上清即为DNA提取物。琼脂糖凝胶电泳检测所提取DNA的片段分布,紫外分光光度法测定DNA的产量及纯度。以提取的DNA为模板,PCR扩增不同长度产物片段并进行产物的直接测序以评价提取方法。结果该提取方法能够获得较长片段的基因组DNA,从一片10μm厚石蜡切片中即可获得大约60μg的DNA,在0.5h消化条件下获取的模板DNA,即可实现对152bp、293bp、392bp和541bp等4种不同片段的100%扩增,1h消化组的PCR产物可直接用于DNA测序。结论建立了一种简单、快速地从甲醛固定石蜡包埋组织中提取DNA的方法,只需一步消化即可获得满足普通PCR及测序需要的基因组DNA。     

冷冻切片后甲醛固定石蜡包埋组织DNA质量分析

目的分析冷冻切片后再行甲醛固定、石蜡包埋组织的DNA质量,探讨其在分子病理检测中的应用价值。方法收集已行术中快速冷冻病理诊断的肺腺癌14例,分别提取同一病例冷冻切片后剩余组织蜡块及常规包埋组织蜡块DNA,检测DNA浓度及OD260/OD280值,行内参基因PCR扩增以检测DNA完整性,并以ARMS法行EGFR基因突变检测。结果冷冻切片后固定包埋组织提取DNA浓度及OD260/OD280均值,分别为157.33 ng/μL及1.96,均较常规包埋组织低;所有冷冻后包埋组织提取的DNA均具有100 bp及200 bp的PCR产物,但仅部分能扩增出300 bp及400 bp的PCR产物,DNA完整性稍逊于常规固定包埋组织;冷冻后包埋组织与常规包埋组织EGFR基因突变检测结果完全一致。结论组织冷冻后再行甲醛固定及石蜡包埋,会使得组织DNA进一步降解及片段化,但仍适用于ARMS法的分子病理学检测。     

石蜡包埋组织PCR检测中DNA提取方法的比较及改进

目前,PCR常用于新鲜组织或细胞提取标本中的核酸分子的检测,较少用于检测石蜡包埋的陈旧组织中的DNA,其主要原因是后者核酸的提取较为困难。针对这种情况,我们在对文献报道的几种石蜡包埋组织提取DNA的方法进行了比较的基础上,进行了改进,提高了石蜡包埋组织提取DNA进行PCR的阳性率,现介绍如下。     

石蜡包埋组织DNA的提取

多年存档的常规福尔马林固定石蜡包埋的病理标本,可用聚合酶链反应(PCR)技术进行回顾性研究或检测,但模板DNA的质量直接影响PCR的效果。本文以扩增c-Ki     

大鼠脑线粒体DNA提取方法

本研究用一种简便方法提取大鼠脑线粒体 DNA(m t DNA) ,所得的线粒体 DNA纯度为 1.8± 0 .0 1,得率为每克脑组织 0 .8～ 1.0 μg线粒体 DNA,以此为模板进行 PCR反应 ,扩增线粒体 DNA编码基因 ,产量丰富、特异性高。该方法操作简便、成本低 ,适用于一般实验室     

石蜡包埋组织DNA提取方法的比较及巢式PCR技术的应用

确定从福尔马林固定、石蜡包埋(FFPE)组织中提取DNA的最佳方法,增强长片段PCR产物的成功扩增率,便于分子生物学与临床医学的有机结合。本文选取正常甲状腺FFPE标本20份,分别应用一步法、酚/氯仿抽提法和试剂盒法三种不同的方法提取总DNA,并应用传统PCR法和巢式PCR法对线粒体DNA(mtDNA)的D环区进行扩增。结果酚/氯仿抽提法所得样本DNA的OD260/D280比值最高,为1.703±0.086。一步法、酚/氯仿抽提法和试剂盒法提取DNA的普通PCR长片段扩增成功率分别为0%、5%和10%,而巢式PCR长片段扩增成功率分别为0%、95%和85%。可见用蛋白酶K消化、酚/氯仿纯化法提取的FFPE组织标本DNA质量可靠,巢式PCR技术是从石蜡标本中扩增长片段DNA的有效方法。     

四种石蜡包埋组织DNA提取方法的比较

背景:由于在甲醛固定石蜡包埋组织的制作及保存过程中对DNA造成的损害,影响了后续的聚合酶链反应等研究。选择一种简便有效且经济实用的石蜡包埋组织DNA提取方法成为研究者们关注和亟待解决的问题。目的:比较甲醛固定石蜡包埋组织中4种提取DNA的方法对DNA质量的影响,探讨一种操作简便、污染少、经济实用的石蜡包埋组织中提取DNA的方法。方法:取手术切除的普通甲醛固定石蜡包埋的非小细胞肺癌组织标本20例,分别以二甲苯脱蜡-酚氯仿法、改良TES水浴脱蜡-酚氯仿法、试剂盒法和改良TES水浴脱蜡-试剂盒抽提DNA法提取其DNA,然后进行电泳分析、紫外分光光度计测定A260/A280比值及PCR扩增。结果与结论:改良TES水浴脱蜡-酚氯仿法和改良TES水浴脱蜡-试剂盒抽提DNA法可获得较大的DNA片段,且两种方法与试剂盒法所提取DNA的A260/A280值相比较,均有显著性意义(P0.05),4种方法的提取效率差异无显著性意义(P0.1),以改良TES水浴脱蜡-酚氯仿法所得DNA为模板,扩增的目的条带亮度与阳性对照相当。结果证实改良TES水浴脱蜡-酚氯仿法简便有效,所用试剂价格低廉,是一种经济实用的石蜡包埋组织DNA提取方法。     

聚合酶链反应检测心肌炎石蜡包埋组织柯萨奇病毒B3RNA

报道从心肌炎石蜡包埋组织单一切片(6～8μm)中提取RNA,使用定位于柯萨奇病毒B_3特异编码区(VP_1)的一对引物、通过聚合酶链反应(PCR)进行基因扩增。对PCR扩增产物,采用3′末端生物素标记的柯萨奇病毒B_3的特异寡核苷酸探针进行斑点杂交。结果表明,两例检查的心肌炎标本都是柯萨奇病毒B_3感染引起的病毒性心肌炎。本实验说明,从石蜡包埋的组织标本中提取RNA,进行PCR检测是可行的,为临床病理进行分子生物学研究开辟了一条新途径。     

胎鼠性别决定区蛋白基因快速检测（英文）

目的：为了将雄性胎鼠组织细胞移植进入雌性受体鼠，Y染色体特异的性别决定区蛋白基因（sex determining region protein, Sry）作为常用的遗传标志需要在细胞分离前快速检测|为了检测孕14 d胎鼠的Sry基因，我们建立了一种快速的PCR方法。基于C57BL/6J小鼠Sry基因序列，我们设计了特异检测引物，并优化了PCR的反应条件包括引物和循环参数等。方法和结果：模板的制备时间约30 min。取约1 mg胚胎组织，以10 mmol/L Tris 10 mmol/L EDTA pH 8.0 缓冲液洗涤2次，然后以200 μL裂解液（20 mg/L proteinase K, 0.5% NP-40, and 0.05% Tween 40）悬浮，再在60℃孵育15 min以消化蛋白，95℃以上5 min灭活蛋白酶K。取5-10 μL作为模板。PCR反应总体积为50 μL，使用两套引物同时扩增，一套扩增Sry目的基因片段，另一套扩增IL-3基因片段作为内对照。扩增时间为1.5 h。扩增产物10-15 μL采用2.5%-3%的琼脂糖凝胶在12-15 V/cm的电压下电泳。10 min即可观察电泳结果。根据引物设计判断结果，Sry目的基因片段为444 bp、IL-3基因片段为649 bp。操作约10个胚胎总时间小于3.5 h。PCR扩增的特异性与特异探针荧光原位杂交结果一致。结论：该PCR方法为检测胎鼠性别决定区蛋白基因的快速、简单、敏感及准确的方法。     

甲醛固定石蜡包埋组织中DNA的三种提取方法比较

为了寻找最佳的自普通10％甲醛固定石蜡包埋组织中提取DNA的方法,取我院2000年手术切除的10％甲醛固定石蜡包埋的食管癌标本10例,分别以蛋白酶K消化酚氯仿抽提法,蛋白酶K消化氯化钠盐析法和不同pH值下加热提取法提取其DNA,然后进行电泳和PCR扩增分析。结果显示,蛋白酶K消化酚氯仿抽提法,蛋白酶K消化氯化钠盐析法所得DNA产量和质量均高于不同pH值下加热提取法。从而认为蛋白酶K消化氯化钠盐析法简便快捷,不用接触有毒的化学药品,是理想的DNA提纯方法。     

PCR artifacts in LOH and MSI analysis of microdissected tumor cells

Polymerase chain reaction (PCR) analysis to study loss of heterozygosity (LOH) and microsatellite instability (MSI) in tumors is widely used. Microdissection techniques are applied to obtain tumor-specific tissue cells. By microdissection, however, the amount of template DNA extracted may vary considerably and interfere with optimal PCR amplification. To circumvent LOH and MSI misinterpretations due to low DNA input, we have assessed the critical level of DNA input for reliable PCR analysis. PCR analysis was performed by using 18 polymorphic markers (mono-, di-, tri-, and tetranucleotide) on DNA derived from both paraffin-embedded, formalin-fixed, and fresh frozen tumor specimens at template input levels ranging from 0.05 to 25.0 ng. We show a highly significant relation between DNA input and the occurrence of LOH and MSI artifacts. Furthermore, for DNA extracted from paraffin-embedded material, the percentage of LOH artifacts is significantly higher compared with DNA extracted from frozen tissue. For reliable PCR analyses using a mono-, di-, tri-, or tetranucleotide marker, a minimum of 10.0 ng DNA is required when DNA is isolated from formalin-fixed, paraffin-embedded tissue and 5.0 ng when isolated from fresh frozen tissue. HUM PATHOL 31:1414-1419.     

QIAamp及Biomed粪便细菌基因组DNA提取试剂盒提取人肠道细菌基因组DNA质量的差异*

背景：有研究表明,不同试剂盒提取的粪便细菌基因组DNA质量有差别,选择一种操作简便、质量优良的粪便细菌基因组DNA提取试剂盒成为研究者们关注和急需解决的问题。 目的：比较QIAamp及Biomed粪便细菌基因组DNA提取试剂盒提取的人肠道细菌基因组DNA质量的差异。 方法：收集健康成年人新鲜粪便标本30例,用两种试剂盒分别提取细菌基因组DNA,检测其浓度、吸光度A260/280 nm值及提取率,设计乳酸菌属16S rDNA基因特异性引物,以各自所提DNA为模板,进行常规聚合酶链反应,凝胶电泳后比较条带数量、明暗度及密度,并进行实时荧光定量聚合酶链反应定量检测各自所含乳酸菌属的数量。 结果与结论：QIAamp试剂盒提取的正常人肠道细菌基因组DNA的浓度、提取率、表达量及乳酸菌属的数量均高于Biomed试剂盒(P < 0.05或P < 0.01)。说明QIAamp试剂盒提取的粪便细菌基因组DNA质量优于Biomed试剂盒。     

Improved PCR amplification for molecular analysis using DNA from long-term preserved formalin-fixed,paraffin-embedded lung cancer tissue specimens

Archival tissue specimens are valuable resources of materials for molecular biological analyses in retrospective studies, especially for rare diseases or those associated with exposure to uncommon environmental events. Although successful amplification with PCR is essential for analysis of DNA extracted from archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens, we have often encountered problems with poor PCR amplification of target fragments. To overcome this, we examined whether heat treatment in alkaline solution could efficiently restore the PCR template activity of DNA that had already been extracted from FFPE lung cancer tissue specimens. The effect of the heat treatment was assessed by PCR for the TP53 gene and other lung cancer-related gene loci. The heat treatment of DNA samples in borate buffer resulted in successful PCR amplification of DNA fragments ranging from 91 to 152 bp. This technique for restoration of template activity of DNA for PCR amplification is very simple and economical, and requires no special apparatus, so it may be applicable for molecular analysis of DNA samples from FFPE tissue specimens at various laboratories.     

Clonal Ig-gene rearrangement in some cases of gastric RLH detected by PCR method.

Clonal immunoglobulin (Ig) heavy chain gene rearrangement in gastric reactive lymphoid hyperplasia (RLH) cases was investigated by means of the 'double' polymerase chain reaction (PCR) using formalin-fixed and paraffin-embedded tissue. Rearranged DNA sequences, formed by combinations of variable (VH) and joining (JH) regions, were amplified with oligomeric primers. One microgram of DNA extracted from formalin-fixed and paraffin-embedded tissue was applied as the 'first PCR' template and one ten-thousandth of the first PCR product was used as the 'second PCR' template. As a control study for the double PCR method, DNA isolated from frank B cell gastric malignant lymphomas was assessed. Clear single bands between 100 and 150 base pair markers in length were evident on agarose gel electrophoresis in 10 out of 13 cases (76.9%) of malignant lymphomas while 2 out of 22 cases (9%) of RLHs revealed clear single bands of the same length, suggesting malignant lymphomas; however, no histologic features of malignant lymphomas were present. It is concluded that even gastric RLH cases satisfying histopathologic criteria for benign lymphoid hyperplasia may contain occult monoclonal B cell populations suggesting a continuous and progressive spectrum of lesions contributing to B cell neoplasia.     

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: An analysis of resected gastric carcinoma

Although several factors affecting the sensitivity of potymer-ase chain reaction (PCR) amplification from formalin-fixed tissues have been Investigated mostly by experiments, the feasibility of archival formalln-flxed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2–3 days and 14% (4/28) of the samples fixed for 4–6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified pS3 products from archival tissues using a non-isotopic method, temperature gradient gel elec-trophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding Just after 1 day or less fixation for subsequent use in PCR amplification.     

Extraction of genomic DNA from paraffin-embedded tissue sections of human fetuses fixed and stored in formalin for long periods

The advent of polymerase chain reaction (PCR) technology has increased the interest in fetal specimens housed in anatomy museums, as they may represent a unique source of genetic material for the study of uncommon or rare pathological conditions such as congenital malformations, neoplastic processes and parasitic as well as other infectious diseases. The aim of this study is to evaluate the quality of genomic DNA extracted from paraffin-embedded tissue sections of human fetuses that have been maintained in formalin for several years. Fetal tissues were embedded in paraffin, and tissue sections were submitted to ethanol/xylene dewaxing, followed by DNA extraction with ammonium acetate. DNA fragments were amplified from DNA extracted from formalin-fixed tissue sections, but not from Bouin-fixed tissues (average yield of 13.7mug/ml from 10 umbilical cord sections of 10mum; A(260):A(280)=1.55,). The addition of bovine serum albumim increased the yield of PCR amplification. Genomic DNA can be reliably amplified from paraffin-embedded human fetal tissues that had been fixed in formalin during 19 years and used for microdissection studies. This simple, cost-effective, and non-laborious method should facilitate the molecular analysis of a large number of specimens fixed for long periods of time.     

Comparative studies on the detection of hepatitis B virus DNA in frozen and paraffin sections by the polymerase chain reaction.

The polymerase chain reaction (PCR) is an extremely sensitive technique that has been used for detection of DNA sequences in formalin-fixed, paraffin-embedded tissues. In order to verify that hepatitis B virus (HBV) DNA sequences are adequately preserved in routinely processed liver tissues, we performed PCR with five different primer pairs for HBV sequences on DNA extracted by two different methods from paraffin and frozen liver sections. The amount of PCR products obtained with DNA templates extracted by the proteinase K-SDS method from frozen sections was significantly larger than that from paraffin sections. However, boiling of deparaffinized sections in water containing Chelex-100 resulted in ample amounts of PCR products irrespective of the primers used. On Southern blots, the location of the bands of amplified DNA obtained by the different methods was consistent with the predicted size, suggesting that the viral sequences had not been altered by processing. Although freezing of fresh tissue yields quantitatively more HBV DNA, formalin fixation qualitatively preserves the viral DNA sequences adequately for detection by PCR. Therefore, formalin-fixed, paraffin-embedded tissues may be used for the detection of viral DNA sequences by PCR. Application of the described procedure to routinely processed tissues significantly broadens the applicability of this powerful diagnostic and investigative method.     

DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays.

DNA extraction from fixed tissues can be the most laborious and complex step in amplifying DNA by the polymerase chain reaction (PCR). We have previously reported a rapid and efficient method for extracting DNA by the use of sonication and glass beads. We have extended our experiences with this technique using fresh, frozen, and formalin-fixed paraffin-embedded tissues with and without the use of glass beads and report their results. Multiple tissue types were obtained at autopsy or as part of a surgical specimen. DNA was extracted from identical tissue when the sample was fresh, frozen, or formalin-fixed paraffin-embedded. Our results indicate that in most instances the sonication technique, which takes only 30 min from start to finish, can rapidly extract fresh, frozen, or formalin-fixed paraffin-embedded tissue and is superior to other rapid extraction techniques in terms of quality and quantity of DNA. It is much more rapid than those techniques that use long digestion periods. This technique will be of great value to those investigators extracting DNA for polymerase chain reaction assays.     

Pretreatment with Restriction Enzyme or Bovine Serum Albumin for Effective PCR Amplification of Epstein-Barr Virus DNA in DNA Extracted from Paraffin-Embedded Gastric Carcinoma Tissue

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.     

早期注射丹参酮ⅡA磺酸钠可抑制兔耳增生性瘢痕

背景：丹参酮ⅡA磺酸钠具有抗氧化、抑制纤维化等作用。 目的：观察丹参酮ⅡA磺酸钠早期局部注射对兔耳增生性瘢痕的影响。 方法：于兔耳腹侧面切除全层皮肤及软骨膜建立瘢痕模型,造模后28 d,分别在创面瘢痕内注射0.05,0.1,0.2 mg的丹参酮ⅡA磺酸钠或生理盐水,每周注射1次,共3周。 结果与结论：丹参酮ⅡA磺酸钠注射3周,兔耳瘢痕增生较轻,瘢痕厚度变薄;苏木精-伊红染色见瘢痕组织中胶原纤维减少,血管数目减少;Masson染色检测见瘢痕组织胶原纤维分布面积减少;电镜下瘢痕组织成纤维细胞数量减少,体积相对变小。说明早期注射丹参酮ⅡA磺酸钠对兔耳瘢痕增生有抑制作用。