醋酸铵盐析法和DNA修复技术在福尔马林固定石蜡包埋组织DNA多态性分析中应用的研究
发布时间：2018-09-14 18:23
【摘要】： 福尔马林固定石蜡包埋组织(formalin-fixed and paraffin-embedded tissue,FFPET)因其能够较长时间地保存组织或制备检验所需的组织标本,因此在临床病理检验、法医病理学鉴定和医学科学研究过程中常被用到。这一处理过程虽然能够使DNA免受内源性核酸酶的破坏,但是甲醛不仅会导致DNA链的断裂和降解、DNA发生脱嘌呤以及DNA-蛋白质的交联,造成被固定组织DNA质量的改变,而且其在模板DNA中的残存也严重影响着PCR扩增。 研究表明,影响FFPET DNA提取的因素有很多。如固定液的种类、固定时间、切片厚度、切片存储时间以及不同的提取处理方法等均可影响FFPET提取DNA的质量,从而影响其基因分型。通过优化蛋白酶消化条件、增加循环次数或采用巢式PCR等,虽然可以提高扩增产物量或扩增特异性,但并未明显改善其检出率；通过缩短扩增产物片段长度,如开发扩增片段较小的miniSTR (mini-short tandem repeat)系统或SNP (single nucleotide polymorphism)系统,虽然提高了对降解DNA检材的分型成功率,但仍然普遍存在诸如等位基因或位点丢失、不平衡峰、伪峰等问题。此外miniSTR系统扩增片段长度不可能无限的减小(基本在50-200bp之间)；SNP分析技术要达到与目前所用STR试剂盒一样的识别率,所需的位点数较多等缺点,也限制了其在实际检案中的应用。 本研究基于法医学检案的实际需要,结合前人的研究成果,通过对醋酸铵盐析法进行改进,并结合PCR前修复技术提高模板DNA质量,以期为法医学实际检案中FFPET DNA的STR分型提供一种新的方法。 材料与方法 1、死后24h内尸体肾脏组织(1cm×1cm×0.5cm)及心血(15ml),均由中国医科大学法医病理学教研室提供。 2、经10%中性福尔马林溶液固定1d、3d、5d和7d后,进行石蜡包埋,常规切片备检。 3、血液DNA提取,新鲜组织DNA提取,FFPET切片DNA提取,对醋酸铵盐析法影响因素进行探讨,并将其与酚／氯仿法、Chelex-100法进行比较,同时探讨法医学可以实现成功分型的最小FFPET检材量。 4、对改良醋酸铵盐析法所提基因组DNA采用修复酶进行模板DNA的修复。 结果 1、不同脱蜡方法提取基因组DNA的PCR-PAGE结果显示未脱蜡和脱蜡均可得到清晰谱带；但是从谱带亮度上看95℃水浴脱蜡谱带最亮,65℃水浴脱蜡次之,后依次为二甲苯脱蜡、微波脱蜡、不脱蜡。 2、不同消化缓冲液消化提取DNA的PCR-PAGE结果显示0.5%Tween 20消化谱带最亮,1%Triton X-100消化次之,后依次为0.5%SDS消化,2%CTAB消化。 3、不同浓度醋酸铵溶液沉淀所得基因组DNA的琼脂糖凝胶电泳结果显示三种浓度醋酸铵溶液提取的DNA量无显著性差异。PCR-PAGE结果显示三种浓度醋酸铵溶液提取的基因组DNA扩增产物均可得到清晰谱带,谱带亮度上三者之间无显著性差异。 4、不同固定时间FFPET所提基因组DNA进行琼脂糖凝胶电泳结果显示不同固定时间切片组织所提取的基因组DNA的电泳谱带均成弥散状,且随着固定时间的延长,谱带弥散越明显,谱带最亮区域逐渐下移。PCR-PAGE结果显示不同固定时间切片组织均可得到清晰谱带,但随着固定时间的延长,谱带亮度逐渐减弱。 5、不同提取方法所得基因组DNA的琼脂糖凝胶电泳结果显示Chelex-100法所得DNA的电泳谱带最亮,改良醋酸铵盐析法次之,酚／氯仿法相对最弱；同一种方法内部比较显示随着检材量的减少,三种方法所提取的基因组DNA的电泳谱带亮度均逐渐减弱。OD260／OD280比值,改良醋酸铵盐析法在1.6～2之间(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)。从所得DNA的量上来看,Chelex-100法所得DNA的量明显高于其他两种方法；改良醋酸铵盐析法次之,酚／氯仿法最少；且随着检材量的减少,这种趋势更明显。PCR-PAGE结果显示改良醋酸铵盐析法提取的DNA均可扩增出清晰的谱带；酚／氯仿法提取的DNA扩增谱带较弱,且随着检材量的减少,谱带亮度明显减弱,当检材量减小为1／8片时已基本显示不出谱带；Chelex-100法提取的DNA在1片时未扩增出谱带,1／2片时谱带隐约可见,而在1／4片、1／8片时可见清晰谱带,但亮度不及改良醋酸铵盐析法所提取的DNA的亮度。 6、对改良醋酸铵盐析法所提取的模板DNA进行PCR前修复,PCR-PAGE结果显示采用Taq DNA聚合酶修复,修复组均扩增出清晰的谱带,而未修复组无谱带或谱带较弱；采用T4连接酶修复,修复组和未修复组在谱带亮度上未见显著性差异；采用Taq DNA聚合酶和T4连接酶联合修复,结果显示两个样品的修复组均可见清晰谱带,而未修复组均未见谱带。 结论 1、改良醋酸铵盐析法具有简单、经济、无毒等优点；与酚／氯仿法、Chelex-100法相比,提取基因组DNA的量更大、质量更好,适合微量检材DNA的提取。 2、使用Taq DNA聚合酶修复技术可以极大地提高高度降解检材STR分型的成功率,适合高度降解检材STR分型的应用。 3、改良醋酸铵盐析法结合酶修复技术实现了微量FFPET检(0.5cm×0.25cm×7μm)STR的成功分型,在特殊生物性检材的个人识别中具有重要意义。
[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.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919
本文编号：2243508
[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.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：D919
【参考文献】
相关期刊论文 前5条
1 杨周岐;张虎勤;张金;赵文明;;古人类骨骼DNA降解影响因素分析[J];第四军医大学学报;2006年01期
2 袁丽;鲁涤;毋丽娜;;石蜡包埋人体组织STR检验的探讨[J];法律与医学杂志;2006年01期
3 罗光华;陈玉川;成建定;王江峰;高翠莲;;DNA降解与腐败尸体死亡时间的相关性[J];法医学杂志;2006年01期
4 王会品;刘超;孙宏钰;;MiniSTR技术的研究进展[J];法医学杂志;2006年02期
5 周毅,严红,李万水,张大安,李俊涛;石蜡包埋人体组织DNA分型的研究[J];刑事技术;2003年04期
，本文编号：2243508
本文链接：https://www.wllwen.com/falvlunwen/fanzuizhian/2243508.html
