福尔马林固定、石蜡包埋组织中提取DNA的实验研究

发布时间：2018-01-18 13:21

本文关键词：福尔马林固定、石蜡包埋组织中提取DNA的实验研究　出处：《吉林大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的探讨福尔马林固定、石蜡包埋组织中提取DNA的最佳方法。方法①根据石蜡切片厚度(2.5μm、5.0μm、10.0μm)、组织切片脱蜡时间(切片脱蜡2次,时间分别为2小时、2小时和2小时、12小时)、消化液中蛋白酶K浓度(125μg /ml、250μg /ml、500μg /ml、1000μg /ml)、组织消化时间(4小时、8小时、12小时、24小时)等参数的不同,排列组合出96种不同条件,分别用于提取DNA。②石蜡组织连续切片,切片在二甲苯恒温水浴中振荡脱蜡后无水乙醇漂洗,真空干燥,加入细胞裂解液(自制),恒温水浴中振荡消化组织,苯酚/氯仿/异戊醇反复抽提,醋酸钠沉淀DNA,离心去上清,保存DNA备用。③不同方法提取的DNA量的检验:稀释200倍的DNA溶液用分光光度计在260nm和280nm分别读出光密度值,即OD260值和OD280值,DNA含量为:OD260值×10(μg/μl)。④不同方法提取的DNA质的检验:所有标本的OD260/OD280值均应该在1.6-1.8范围内,说明DNA中无蛋白、RNA和苯酚污染。PCR反应扩增所提取的DNA中RET基因第11外显子,10μl PCR产物经2.0 %的琼脂糖凝胶电泳,初步判断提取的DNA是否可以用于PCR反应;从琼脂糖凝胶中纯化目的基因DNA后再次以分光光度计测量纯化后的DNA含量,比较提取的DNA经PCR扩增后目的基因的产量;纯化后的PCR产物经荧光标记后自动测序仪测定目的基因碱基序列,观察提取的DNA是否可以用于直接序列测定。结果所有标本提取的DNA之OD260/OD280值均在1.6-1.8范围内。各种石蜡切片厚度均可以提取一定数量的DNA,三组石蜡切片厚度之间比较无明显差异。但2.5μm组提取的DNA用于PCR扩增时效果远不如其它2组,即使PCR扩增后PCR产物电泳虽可见目的基因条带,条带也较淡,目的基因测序图混乱,难以读出碱基序列;2次脱蜡时间为2、2小时组脱蜡效果不如2次脱蜡时间为2、12小时组;随着蛋白酶K浓度的增加,提取的DNA数量也增加,最后行DNA测序时蛋白酶K浓度为500μg/ml和1000μg/ml组均可以得到满意的DNA测序图;组织消化8小时、12小时和24小时得到的DNA均适用于序列测定,但8小时组提取的DNA经PCR扩增后产物较12小时和24小时组少,12小时组与24小时组间比较无明显差别。目的基因DNA含量大于16ng/μl的标本测序时测序图较好,否则不适合于测序。结论从中性福尔马林固定-石蜡包埋组织中可以提取高质量DNA,在提取DNA过程中应注意剔除石蜡切片中坏死组织区域,高温降解核酸酶和蛋白酶K等步骤有利于下一步实验取得理想的结果。提取DNA的最佳条件是:石蜡切片厚度为10.0μm;脱蜡时间以2次脱蜡,时间分别为2、12小时,可以满足10.0μm厚切片的完全脱蜡;消化液中蛋白酶K浓度为500μg/ml,消化12小时组织可以完全被消化。
[Abstract]:Objective to study the best method for extracting DNA from formalin-fixed and paraffin-embedded tissues. Methods (1) according to the thickness of paraffin section, 2.5 渭 m ~ 5.0 渭 m ~ (5.0 渭 m) ~ 10.0 渭 m ~ (-1), the dewaxing time of tissue section (2 times) was 2 hours, 2 hours and 2 hours, respectively. The concentration of protease K in digestive fluid was 125 渭 g / ml ~ 250 渭 g / ml ~ 500 渭 g / ml ~ (-1) 渭 g / ml ~ (-1) 渭 g / ml ~ (-1) 渭 g / ml ~ (-1), and the time of tissue digestion was 4 hours. Different parameters such as 8 hours 12 hours and 24 hours were arranged and combined 96 different conditions were used to extract DNA.2 paraffin tissue serial sections. The slices were rinsed with anhydrous ethanol after dewaxing in a constant temperature water bath of xylene and then dried in vacuum. The cells were added into the cell lytic solution (self-made, oscillatory digestive tissue in constant temperature water bath, phenol / chloroform / isoamyl alcohol) extracted repeatedly. Sodium acetate precipitated DNA and centrifuged the supernatant. Examination of the DNA extracted by different methods for preserving DNA. 3. The values of optical density were read out by spectrophotometer at 260 nm and 280 nm respectively in diluted 200 times DNA solution. That is, the OD260 value and the OD280 value. The content of DNA is 0: OD260 脳 10 (渭 g / 渭 l). 4 the qualitative test of DNA extracted by different methods: the OD260/OD280 value of all specimens should be in the range of 1.6-1.8. The results showed that the RET gene exon 11 of the extracted DNA was amplified by the reaction of protein-free DNA and phenol-contaminated .PCR. The 10 渭 l PCR product was detected by 2.0% agarose gel electrophoresis to determine whether the extracted DNA could be used in the PCR reaction. After purification of DNA from agarose gel, the content of purified DNA was measured by spectrophotometer, and the output of DNA amplified by PCR was compared. The purified PCR products were labeled with fluorescence and sequenced by automatic sequencer to determine the base sequence of the target gene, and whether the extracted DNA could be used for direct sequencing. Results the OD260/OD280 values of DNA extracted from all specimens were in the range of 1.6-1.8. A certain number of DNA could be extracted from all kinds of paraffin sections. There was no significant difference in the thickness of paraffin sections among the three groups, but the effect of DNA extracted from 2.5 渭 m group for PCR amplification was not as good as that in the other two groups. Even if the PCR product electrophoresis after PCR amplification can be seen the target gene band, the band is also relatively light, the target gene sequencing map is confused, it is difficult to read the base sequence; The dewaxing effect of the two dewaxing time group was not as good as that of the second dewaxing time group of 2 hours and the second dewaxing time was 12 hours. The amount of DNA extracted increased with the increase of protease K concentration. At the end of DNA sequencing, a satisfactory DNA sequencing map was obtained when the concentration of protease K was 500 渭 g / ml and 1000 渭 g / ml, respectively. The DNA obtained from tissue digesting for 8 hours at 12 hours and 24 hours was suitable for sequencing, but the DNA extracted from 8 hours group was amplified by PCR less than that of 12 hours and 24 hours groups. There was no significant difference between the 12-hour group and the 24-hour group. Objective the sample with DNA content greater than 16ng / 渭 l was better in sequencing, otherwise it was not suitable for sequencing. Conclusion High-quality DNA can be extracted from neutral formalin-paraffin-embedded tissue, and the necrotic tissue should be removed from paraffin sections during the extraction of DNA. The high temperature degradation of nuclease and protease K is beneficial to the further experiment to obtain ideal results. The optimum conditions for extracting DNA are as follows: the thickness of paraffin section is 10.0 渭 m; The dewaxing time of two dewaxing times is 2 ~ 12 hours respectively, which can satisfy the complete dewaxing of 10.0 渭 m thick slice. The concentration of protease K in digestive fluid was 500 渭 g / ml, and the tissue could be completely digested 12 hours after digestion.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R361.2

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