RAPID TECHNIQUES FOR DNA EXTRACTION FROM ROUTINELY PROCESSED ARCHIVAL TISSUE FOR USE IN PCR
RAPID TECHNIQUES FOR DNA EXTRACTION FROM ROUTINELY PROCESSED ARCHIVAL TISSUE FOR USE IN PCR
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DOI：
10.1136/jcp.47.4.318
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发表时间：
1994-04-01
影响因子：
3.4
通讯作者：
SAKAMOTO, H
中科院分区：
文献类型：
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作者：
SEPP, R;SZABO, I;SAKAMOTO, H
目的：评价常规固定石蜡包埋档案组织的四种快速DNA提取方法为聚合酶链反应（PCR）提供DNA的能力。方法：18块不同组织标本，18块宫颈癌标本，9块B细胞淋巴瘤标本。研究了正常和活检标本大小的组织。提取DNA的方法有四种：蒸馏水煮沸20分钟；在5% Chelex-100树脂溶液中煮沸20分钟；蛋白酶K消化3小时；蛋白酶K消化3小时，然后在5% Chelex-100中煮沸。从提取物中扩增出p53基因的不同外显子、人乳头瘤病毒16型（HPV 16）序列和免疫球蛋白重链（IgH）基因重排。结果：45份样品采用Chelex煮沸法、蛋白酶K消化法和蛋白酶K消化-Chelex煮沸法均可获得适于扩增的DNA。45例中有3例（7%）在水中煮沸后产生的PCR模板不足，42例阳性病例中有6例（14%）观察到更微弱的条带，主要是当处理的材料是活检标本大小或B细胞淋巴瘤样本时。p53基因片段在水煮提取物中扩增到408个碱基对，在Chelex水煮制备物中扩增到647个碱基对，在蛋白酶K消化和蛋白酶K消化-Chelex水煮样品中扩增到984个碱基对，尽管最后一种情况下敏感性降低。在- 20℃条件下保存3个月后，所有模板均可重复使用。结论：Chelex煮沸、蛋白酶K消化和蛋白酶K消化后Chelex煮沸可从多种石蜡包埋组织中获得合适的PCR模板。由于在5% Chelex-100溶液中煮沸20分钟的简单方法需要最少的操作和时间，因此它可能是有用的，特别是在大量材料的常规处理中。
Aims-To evaluate the ability of four rapid DNA extraction methods tee provide DNA for the polymerase chain reaction (PCR) from routinely fixed, paraffin wax embedded archival tissues.Methods-Eighteen blocks of various tissues, 18 blocks of cervical cancer specimens, and nine blocks of B cell lymphomas were investigated. Both normal and biopsy specimen sized tissues were studied. DNA was extracted using four methods: boiling for 20 minutes in distilled water; boiling for 20 minutes in 5% Chelex-100 resin solution; 3-hour proteinase K digestion; and 3-hour proteinase K digestion, followed by boiling in 5% Chelex-100. Different exons of the p53 gene, human papillomavirus type 16 (HPV 16) sequence, and immunoglobulin heavy chain (IgH) gene rearrangement were amplified from the extracts.Results-The Chelex boiling, proteinase K digestion, and proteinase K digestion-Chelex boiling methods produced DNA suitable for amplification in all of the 45 samples. Boiling in water yielded insufficient template for the PCR in three of the 45 cases (7%), and in six of 42 positive cases (14%) much fainter bands were observed, mostly when the processed material was either biopsy specimen sized or a B cell lymphoma sample. Fragments of the p53 gene were successfully amplified up to 408 base pairs in water boiled extracts, up to 647 in Chelex boiled preparates, and up to 984 in proteinase K digested and proteinase K digested-Chelex boiled samples, although with decreased sensitivity in the last case. All of the templates were reusable after 3 months of storage at - 20 degrees C.Conclusions-Chelex boiling, proteinase K digestion, and proteinase K digestion followed by Chelex boiling produce suitable templates for the PCR from a large variety of paraffin wax embedded tissues. As the simple 20 minute boiling method in 5% Chelex-100 solution requires minimal manipulation and time, it could be useful, especially in the routine processing of large amounts of material.