本文關鍵詞：福爾馬林固定、石蠟包埋組織中提取DNA的實驗研究　出處：《吉林大學》2007年碩士論文　論文類型：學位論文

  更多相關文章： 組織 石蠟 DNA 提取 基因測序

【摘要】： 目的探討福爾馬林固定、石蠟包埋組織中提取DNA的最佳方法。 方法①根據(jù)石蠟切片厚度(2.5μm、5.0μm、10.0μm)、組織切片脫蠟時間(切片脫蠟2次,時間分別為2小時、2小時和2小時、12小時)、消化液中蛋白酶K濃度(125μg /ml、250μg /ml、500μg /ml、1000μg /ml)、組織消化時間(4小時、8小時、12小時、24小時)等參數(shù)的不同,排列組合出96種不同條件,分別用于提取DNA。②石蠟組織連續(xù)切片,切片在二甲苯恒溫水浴中振蕩脫蠟后無水乙醇漂洗,真空干燥,加入細胞裂解液(自制),恒溫水浴中振蕩消化組織,苯酚/氯仿/異戊醇反復抽提,醋酸鈉沉淀DNA,離心去上清,保存DNA備用。③不同方法提取的DNA量的檢驗:稀釋200倍的DNA溶液用分光光度計在260nm和280nm分別讀出光密度值,即OD260值和OD280值,DNA含量為:OD260值×10(μg/μl)。④不同方法提取的DNA質的檢驗:所有標本的OD260/OD280值均應該在1.6-1.8范圍內,說明DNA中無蛋白、RNA和苯酚污染。PCR反應擴增所提取的DNA中RET基因第11外顯子,10μl PCR產物經2.0 %的瓊脂糖凝膠電泳,初步判斷提取的DNA是否可以用于PCR反應;從瓊脂糖凝膠中純化目的基因DNA后再次以分光光度計測量純化后的DNA含量,比較提取的DNA經PCR擴增后目的基因的產量;純化后的PCR產物經熒光標記后自動測序儀測定目的基因堿基序列,觀察提取的DNA是否可以用于直接序列測定。 結果所有標本提取的DNA之OD260/OD280值均在1.6-1.8范圍內。各種石蠟切片厚度均可以提取一定數(shù)量的DNA,三組石蠟切片厚度之間比較無明顯差異。但2.5μm組提取的DNA用于PCR擴增時效果遠不如其它2組,即使PCR擴增后PCR產物電泳雖可見目的基因條帶,條帶也較淡,目的基因測序圖混亂,難以讀出堿基序列;2次脫蠟時間為2、2小時組脫蠟效果不如2次脫蠟時間為2、12小時組;隨著蛋白酶K濃度的增加,提取的DNA數(shù)量也增加,最后行DNA測序時蛋白酶K濃度為500μg/ml和1000μg/ml組均可以得到滿意的DNA測序圖;組織消化8小時、12小時和24小時得到的DNA均適用于序列測定,但8小時組提取的DNA經PCR擴增后產物較12小時和24小時組少,12小時組與24小時組間比較無明顯差別。目的基因DNA含量大于16ng/μl的標本測序時測序圖較好,否則不適合于測序。 結論從中性福爾馬林固定-石蠟包埋組織中可以提取高質量DNA,在提取DNA過程中應注意剔除石蠟切片中壞死組織區(qū)域,高溫降解核酸酶和蛋白酶K等步驟有利于下一步實驗取得理想的結果。提取DNA的最佳條件是:石蠟切片厚度為10.0μm;脫蠟時間以2次脫蠟,時間分別為2、12小時,可以滿足10.0μm厚切片的完全脫蠟;消化液中蛋白酶K濃度為500μg/ml,消化12小時組織可以完全被消化。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R361.2

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本文編號：1441133