| In order to improve the detection rate of DNA in paraffin-embedded specimens. In this paper, I research from two aspects that to improve the efficiency of PCR reaction and to improve the quality of DNA extracted from paraffin-embedded specimens.Firstly, I studied12kinds of PCR additives. The results show that trehalose, DMSO, DTT, spermidine, glutathione and betaine can improve the efficiency and stability of the PCR reaction. On this basis, I add these six kinds of additives according to a certain proportion to the conventional fluorescent PCR reaction system to establish a new type of fluorescent PCR reaction system.Secondly, I use alkaline lysis method, the TE solution heated dewaxing and proteinase K co-digestion with Chelex to extract DNA from formalin-fixed and paraffin-embedded tissue. The results showed that the quality of DNA that extracted with alkaline lysis method is poor. Therefore, this method does not apply to extract DNA from common paraffin-embedded specimens. However, the DNA obtained from Paraffin-embedded specimens in the method that use proteinase K co-digestion with Chelex after TE solution heating dewaxing can better meet the demand for PCR detection, and the DNA test results are basically the same with the Qiagen kit. Thereby establishing a new a method for extracting DNA from paraffin samples.Thirdly, I use the new DNA-extracted method with the new fluorescent PCR reaction system to detect500cases of paraffin-embedded specimens. The results showed that the detection rate was94%(471/500), significantly increased the detection rate of the DNA in paraffin-embedded specimens, and reliability.In summary, through the research, we have come to the following conclusions that the new established fluorescent PCR reaction system and the new DNA-extraction method with paraffin-embedded specimens can meet the testing requirements of the universal paraffin specimens. Thus provide a more reliable detection platform of paraffin-embedded specimens for future studies. |
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