Study On DNA Extraction From Formalin Fixation And Paraffin-embedded Tissues

The problem that a large amount of fresh tissue specimens cannot be available is often met in carrying out the retrospective molecular biological experiment. Although the problem can be resolved by distilling DNA from the formalin fixed-paraffin buried tissue, it easy to cause the DNA cross linking and degradation and limit the research work after the tissue is fixed by formalin and buried by paraffin. It is a problem urgent to be solved how to distill the high-quality DNA from the paraffin buried tissue. In this paper, a group of optimal conditions of distilling DNA are found out by changing some experimental parameters such as thickness of paraffin slice, tissue deparaffinating time, K density of digest protease and tissue digesting time, etc. The emphasis goes to observation of the impacts of the change of some experimental parameters such as thickness of paraffin slice, tissue deparaffinating time, K density of digest protease and tissue digesting time, etc. on the quantity and quality of distilled DNA. The OD260/OD280 of the distilled DNA in the experiment is in the range of 1.6-1.8, showing that there is no pollution of protein, RNA or phenol in the DNA.The results show the following. (1) The impacts of thickness of paraffin slice on the quantity and quality of the distilled DNA. In the study, the quality differences of distilling DNA in the three cases of 2.5μm, 5.0μm and 10.0μm-thick paraffin slices. The results show that a certain quantity of DNA can be distilled in all the above cases and there is no obvious difference between the three groups. However, the effect when the distilled DNA in the 2.5μm group is used to enlarge PCR is far less than that of the other two groups. Even after the PCR is enlarged, the visible target genic belt of PCR product electrophoresis obscure and the target gene sequencing diagram is disorderly and difficult to read the basic group sequence. In the paper, the effect when the distilled DNA from the 10.0μm group is used for PCR and sequencing observed by the author is better than that of the 5.0μm group, the available sequencing diagram has the clear curve and the basic group sequence is shown clearly. The reasons are analyzed as follows. The slice in the 2.5μm group is thin, the slicer edge seriously and mechanically damages the DNA. Although a certain quantity of DNA can be distilled, the DNA short segments are increased, which is unsuitable for PCR and DNA sequencing. It is not like that the better it is the thicker the slice. We make an attempt to increase the slice thickness to 20μm and 40μm, the quality of the distilled DNA is reduced with the increase of thickness. In the study, it is thought that the best-quality DNA can be distilled when the paraffin slice is 10.0μm thick. (2)The impacts of tissue slice deparaffinating time on the quantity and quality of the distilled DNA. The study shows that paraffin gives a big impact on the PCR reaction. Therefore, the tissue slice deparaffinating must be thorough. The author compares the two cases in which the deparaffinating time influences the quality and the distilled DNA. The results show that the deparaffinating effect with the deparaffinating time twice of 2 and 2 hours is less than that of the deparaffinating time twice of 2 and 12 hours. The thorough paraffin ting can better meet the needs of the next experiment, but the further delayed deparaffinating time will have no obvious impact on the quality of the distilled DNA. Therefore, it is unnecessary to waste time. (3) The impacts of K density of digest protease and tissue digesting time on the quantity and quality of the distilled DNA. Kosel compared the quantities of DNA distilled in the different periods of time and digested by the protease K of different densities, which shows that the higher the protease K density in the digest, the longer the tissue digesting time and the higher the digesting temperature, the larger the quantity of DNA available. The results of the study is similar to the above, but the study finds that the quantity of distilled DNA is increased, but the quality is decreased, with the protease K density increasing, that is, the content of DNA in the PCR products is reduced. The reason for this is unclear. The study results of Isola show that 20-30 5μm-thick paraffin slices digested by the protease K (0.3mg/ml) for three days can increase the output of a big segment of DNA, and its effect is better than that of the short-time digestion of the high-density protease K (3mg/ml). They think this may be related to reversion of some protease K into the cross linking phenomenon caused by the formalin fixing. We observe and find that when the paraffin slice is 10.0μm thick, the slice deparaffinating time twice is 2 and 12, respectively, the quantity of the distilled DNA is increased but not obviously, with the increase of the protease K density, and the groups with the protease K densities of 500μg/ml and 1000μg/ml in the final-row DNA sequencing can obtain the satisfactory DNA sequencing diagrams, so we think that the tissue can be completely digested when the protease K density is 500μg/ml and it is meaningful to continue increasing the protease K density. The DNA with the tissue digested for 8 hours, 12 hours or 24 hours on the optimal conditions can be applicable to the sequencing, but the products of the DNA distilled in the 8-hour group are less than those in the 12-hour group and 24-hour group after the PCR enlarging and there is no obvious difference of time between the 12-hour group and the 24-hour group. Therefore, we think the 12-hour digesting of the tissue can meet the requirements of the further experiment. (4) Other cases in the process of distilling.①The necrotic tissue can release various enzymes, causing the degradation of DNA and protein and influencing the experimental results of PCR and DNA sequencing, etc. Therefore, the necrotic tissue in the paraffin slice should be fully removed. We dye the slice HE and observe the area where the necrotic tissue of the slice and obtain good effects.②In the process of distilling DNA, DNA is liable to degradation by nuclease, so the author puts the slice in the 75℃water for 20min before the protease K digesting and after the tissue slice deparaffinating, which can fully inactivate the nuclease and reduce its digesting roles of DNA in the future steps, thus increasing the output of a bigger segment of DNA.③After the tissue is digested by the protease K, the protease K should be inactivated to prevent the protease K from influencing the next experiment. 95℃water bathing for 10min can fully inactivate the protease K.④There exist some substances repressing the PCR reaction in the formalin fixed-paraffin buried tissue and these substances are increased with the increase of the quantity of the used distilled tissue, so the reduction of quantity of the used distilled tissue is favorable to reduce the quantity of repressing substances and success of PCR reaction.⑤The reaction recycling times should be generally more than 40 when the purpose of PCR reacting model enlarging of the DNA distilled from the paraffin buried tissue is made; otherwise, it is not easy to succeed the PCR enlarging.⑥The reduction of model quantity in the PCR reaction is favorable to the success of PCR reaction. On certain conditions, the high-quality DNA can be distilled from the neutral formalin fixed-paraffin buried tissue to resolve the difficulties often met in the molecular biological study.