【摘要】： 福爾馬林固定石蠟包埋組織(formalin-fixed and paraffin-embedded tissue,FFPET)因其能夠較長時間地保存組織或制備檢驗所需的組織標本,因此在臨床病理檢驗、法醫(yī)病理學鑒定和醫(yī)學科學研究過程中常被用到。這一處理過程雖然能夠使DNA免受內(nèi)源性核酸酶的破壞,但是甲醛不僅會導致DNA鏈的斷裂和降解、DNA發(fā)生脫嘌呤以及DNA-蛋白質(zhì)的交聯(lián),造成被固定組織DNA質(zhì)量的改變,而且其在模板DNA中的殘存也嚴重影響著PCR擴增。 研究表明,影響FFPET DNA提取的因素有很多。如固定液的種類、固定時間、切片厚度、切片存儲時間以及不同的提取處理方法等均可影響FFPET提取DNA的質(zhì)量,從而影響其基因分型。通過優(yōu)化蛋白酶消化條件、增加循環(huán)次數(shù)或采用巢式PCR等,雖然可以提高擴增產(chǎn)物量或擴增特異性,但并未明顯改善其檢出率；通過縮短擴增產(chǎn)物片段長度,如開發(fā)擴增片段較小的miniSTR (mini-short tandem repeat)系統(tǒng)或SNP (single nucleotide polymorphism)系統(tǒng),雖然提高了對降解DNA檢材的分型成功率,但仍然普遍存在諸如等位基因或位點丟失、不平衡峰、偽峰等問題。此外miniSTR系統(tǒng)擴增片段長度不可能無限的減小(基本在50-200bp之間)；SNP分析技術(shù)要達到與目前所用STR試劑盒一樣的識別率,所需的位點數(shù)較多等缺點,也限制了其在實際檢案中的應用。 本研究基于法醫(yī)學檢案的實際需要,結(jié)合前人的研究成果,通過對醋酸銨鹽析法進行改進,并結(jié)合PCR前修復技術(shù)提高模板DNA質(zhì)量,以期為法醫(yī)學實際檢案中FFPET DNA的STR分型提供一種新的方法。 材料與方法 1、死后24h內(nèi)尸體腎臟組織(1cm×1cm×0.5cm)及心血(15ml),均由中國醫(yī)科大學法醫(yī)病理學教研室提供。 2、經(jīng)10%中性福爾馬林溶液固定1d、3d、5d和7d后,進行石蠟包埋,常規(guī)切片備檢。 3、血液DNA提取,新鮮組織DNA提取,FFPET切片DNA提取,對醋酸銨鹽析法影響因素進行探討,并將其與酚／氯仿法、Chelex-100法進行比較,同時探討法醫(yī)學可以實現(xiàn)成功分型的最小FFPET檢材量。 4、對改良醋酸銨鹽析法所提基因組DNA采用修復酶進行模板DNA的修復。 結(jié)果 1、不同脫蠟方法提取基因組DNA的PCR-PAGE結(jié)果顯示未脫蠟和脫蠟均可得到清晰譜帶；但是從譜帶亮度上看95℃水浴脫蠟譜帶最亮,65℃水浴脫蠟次之,后依次為二甲苯脫蠟、微波脫蠟、不脫蠟。 2、不同消化緩沖液消化提取DNA的PCR-PAGE結(jié)果顯示0.5%Tween 20消化譜帶最亮,1%Triton X-100消化次之,后依次為0.5%SDS消化,2%CTAB消化。 3、不同濃度醋酸銨溶液沉淀所得基因組DNA的瓊脂糖凝膠電泳結(jié)果顯示三種濃度醋酸銨溶液提取的DNA量無顯著性差異。PCR-PAGE結(jié)果顯示三種濃度醋酸銨溶液提取的基因組DNA擴增產(chǎn)物均可得到清晰譜帶,譜帶亮度上三者之間無顯著性差異。 4、不同固定時間FFPET所提基因組DNA進行瓊脂糖凝膠電泳結(jié)果顯示不同固定時間切片組織所提取的基因組DNA的電泳譜帶均成彌散狀,且隨著固定時間的延長,譜帶彌散越明顯,譜帶最亮區(qū)域逐漸下移。PCR-PAGE結(jié)果顯示不同固定時間切片組織均可得到清晰譜帶,但隨著固定時間的延長,譜帶亮度逐漸減弱。 5、不同提取方法所得基因組DNA的瓊脂糖凝膠電泳結(jié)果顯示Chelex-100法所得DNA的電泳譜帶最亮,改良醋酸銨鹽析法次之,酚／氯仿法相對最弱；同一種方法內(nèi)部比較顯示隨著檢材量的減少,三種方法所提取的基因組DNA的電泳譜帶亮度均逐漸減弱。OD260／OD280比值,改良醋酸銨鹽析法在1.6～2之間(1.962±0.195),酚／氯仿法大于2(2.110±0.470),Chelex-100法在1左右(1.018±0.124)。從所得DNA的量上來看,Chelex-100法所得DNA的量明顯高于其他兩種方法；改良醋酸銨鹽析法次之,酚／氯仿法最少；且隨著檢材量的減少,這種趨勢更明顯。PCR-PAGE結(jié)果顯示改良醋酸銨鹽析法提取的DNA均可擴增出清晰的譜帶；酚／氯仿法提取的DNA擴增譜帶較弱,且隨著檢材量的減少,譜帶亮度明顯減弱,當檢材量減小為1／8片時已基本顯示不出譜帶；Chelex-100法提取的DNA在1片時未擴增出譜帶,1／2片時譜帶隱約可見,而在1／4片、1／8片時可見清晰譜帶,但亮度不及改良醋酸銨鹽析法所提取的DNA的亮度。 6、對改良醋酸銨鹽析法所提取的模板DNA進行PCR前修復,PCR-PAGE結(jié)果顯示采用Taq DNA聚合酶修復,修復組均擴增出清晰的譜帶,而未修復組無譜帶或譜帶較弱；采用T4連接酶修復,修復組和未修復組在譜帶亮度上未見顯著性差異；采用Taq DNA聚合酶和T4連接酶聯(lián)合修復,結(jié)果顯示兩個樣品的修復組均可見清晰譜帶,而未修復組均未見譜帶。 結(jié)論 1、改良醋酸銨鹽析法具有簡單、經(jīng)濟、無毒等優(yōu)點；與酚／氯仿法、Chelex-100法相比,提取基因組DNA的量更大、質(zhì)量更好,適合微量檢材DNA的提取。 2、使用Taq DNA聚合酶修復技術(shù)可以極大地提高高度降解檢材STR分型的成功率,適合高度降解檢材STR分型的應用。 3、改良醋酸銨鹽析法結(jié)合酶修復技術(shù)實現(xiàn)了微量FFPET檢(0.5cm×0.25cm×7μm)STR的成功分型,在特殊生物性檢材的個人識別中具有重要意義。
[Abstract]:Formalin-fixed and paraffin-embedded tissue (FFPET) is often used in clinical pathology, forensic pathology, and medical research because of its ability to preserve tissue or prepare tissue specimens for testing over a long period of time. Endogenous nuclease is destroyed, but formaldehyde not only leads to DNA strand breakage and degradation, DNA depurination and DNA-protein cross-linking, resulting in changes in the quality of DNA immobilized tissue, but also its residual in template DNA seriously affects PCR amplification.
Studies have shown that there are many factors affecting the extraction of FFPET DNA, such as the type of stationary solution, fixation time, slice thickness, slice storage time and different extraction methods, which can affect the quality of FFPET DNA extraction, thus affecting its genotyping. It can increase the quantity or specificity of amplified products, but does not significantly improve the detection rate; by shortening the length of amplified products, such as the development of mini-short tandem repeat (mini-short tandem repeat) system or SNP (single nucleotide polymorphism) system with smaller amplified fragments, although it improves the success rate of DNA typing, it is still popular. In addition, the length of amplified fragments in miniSTR system can not be reduced indefinitely (basically between 50 and 200 bp); SNP analysis technology needs more loci to achieve the same recognition rate as STR kits currently used, which also limits its practical detection. Application.
Based on the actual needs of forensic medical examination and previous research results, this study improved the ammonium acetate salting-out method and improved the quality of template DNA by combining the pre-PCR repair technology, in order to provide a new method for the STR typing of FFPET DNA in forensic medical examination.
Materials and methods
1. The kidney tissue (1cm *1cm *0.5cm) and heart blood (15ml) within 24 hours after death were provided by the Department of Forensic Pathology, China Medical University.
2, after 10% neutral formalin solution was fixed with 1D, 3D, 5D and 7d, paraffin embedded and routine sections were used for preparation.
3. Blood DNA extraction, fresh tissue DNA extraction, FFPET slice DNA extraction, ammonium acetate salting-out method of influencing factors were discussed, and compared with phenol/chloroform method, Chelex-100 method, at the same time to explore the forensic medicine can achieve a successful classification of the minimum FFPET sample size.
4, the genomic DNA of the modified ammonium acetate salting out method was used to repair the template DNA.
Result
1. PCR-PAGE results of genomic DNA extracted by different dewaxing methods showed that clear bands could be obtained without dewaxing and dewaxing, but the brightest bands were obtained at 95 C, followed by 65 C, followed by xylene dewaxing, microwave dewaxing and non-dewaxing.
2. The results of PCR-PAGE showed that the digestive band of 0.5% Tween 20 was the brightest, followed by 1% Triton X-100, followed by 0.5% SDS and 2% CTAB.
3, agarose gel electrophoresis of genomic DNA precipitated from different concentrations of ammonium acetate solution showed that there was no significant difference in the amount of DNA extracted from three concentrations of ammonium acetate solution..PCR-PAGE results showed that the DNA amplification products extracted from three concentrations of ammonium acetate solution could get clear bands, and there was no significant difference between the three bands. Different.
4, the genomic DNA of genomic FFPET at different fixed time was agarose gel electrophoresis. The results showed that the electrophoresis bands of genomic DNA extracted from different slices of fixed time were dispersive, and with the extension of fixed time, the band dispersion became more obvious, and the brightest region of the spectrum gradually shifted downward..PCR-PAGE results showed different fixed time slice groups. Weaving can get clear bands, but with the extension of fixed time, the brightness of the band gradually decreases.
5, the agarose gel electrophoresis results of genomic DNA obtained from different extraction methods showed that the electrophoretic bands of DNA obtained by Chelex-100 method were the brightest, the ammonium acetate salting out method was the second, and the phenol / chloroform method was the weakest. The internal comparison of the same method showed that the electrophoresis band brightness of genomic DNA extracted by the three methods decreased with the decrease of the amount of samples. The ratio of OD260 to OD280 decreased gradually. The modified ammonium acetate salting-out method was between 1.6 and 2 (1.962+0.195), the phenol/chloroform method was more than 2 (2.110+0.470), and the Chelex-100 method was about 1 (1.018+0.124). The results of PCR-PAGE showed that the DNA extracted by modified ammonium acetate salting out method could amplify clear bands; the DNA extracted by phenol/chloroform method had weak bands, and the band brightness was obviously weakened with the decrease of the amount of samples. When the amount of samples was reduced to 1/8, the band brightness was basically not displayed. The DNA extracted by Chelex-100 method did not amplify the band in one piece, but could be seen faintly in one quarter and one eighth pieces, but the brightness of DNA extracted by modified ammonium acetate salting-out method was not as good as that of DNA extracted by modified ammonium acetate salting-out method.
6. The template DNA extracted by modified ammonium acetate salting-out method was repaired before PCR. The results of PCR-PAGE showed that clear bands were amplified by Taq DNA polymerase in repair group, while no bands or weak bands were found in non-repair group. A polymerase and T4 ligase were used to repair the defect. The results showed that clear bands could be seen in the repaired group, but no bands could be seen in the unrepaired group.
conclusion
1. Modified ammonium acetate salting-out method has the advantages of simplicity, economy and non-toxicity. Compared with phenol/chloroform method and Chelex-100 method, the genomic DNA extracted by modified ammonium acetate salting-out method has more quantity and better quality, which is suitable for the extraction of DNA from micro-samples.
2. Taq DNA polymerase repair technique can greatly improve the success rate of STR typing for highly degraded samples, which is suitable for the application of STR typing for highly degraded samples.
3. The improved ammonium acetate salting-out method combined with enzyme remediation technique has successfully classified the micro-FFPET (0.5cm *0.25cm *7um) STR, which is of great significance in the personal identification of special biological materials.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：D919

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