Detection Of IDH Mutation In Gliomas Using The Combination Of PCR With MALDI-TOF MS Technique

Recently, the research of cancer and oncology has become hot topic in field of biology and medicine. It is shown that the reason for occurrence and development of tumor is change in genetic level, and these changes are predictable and recognizable. Early detection, timely diagnosis and classification can help patients to prevent and treat tumor positively and effectively. Currently, the "gold standard" used to detect the gene mutation was direct sequencing. However, this method is not fit for high throughput clinical samples. In order to solve this difficult problem, an analysis and detection technique which combing PCR-based method with MALDI-TOF MS was proposed in this paper. And this technique could be used for rapid identification of gene mutations in gliomas histopathology. The main efforts in this paper were listed as below:1) The genomic DNA of U87 MG was collected through culture in vitro. The sample pretreatment included amplification of gene sequence, restriction enzyme digestion etc. The MALDI-TOF mass spectrometry was used to analyze the data, and a method of detection IDH mutation in glioma cells or tissues was built. The result showed that molecular weight of nucleic acid fragment between 2,700 and 6,600 Da could be detected by mass spectrometry. The precision of signal to noise ratio and resolution was enough to the detection. No mutations were found in three gene loci of the U87 MG cell lines, including IDH1 R132, IDH2 R140 and IDH2 R172, this result was consistent with the data reported and it proved the reliability of the technique.2) The above method was confirmed by ESI-Orbitrap mass spectrometry and Gene sequencing. Nucleic acids with multiple charges were detected in ESI source, and their molecular weight through calculation was consistent with those obtained from MALDI-TOF MS. The gene mutations could be found directly through the gene sequencing result. Besides, an indirect comparison could be made base on the results of MALDI-TOF MS, and it is consistent with the data in the sequence information. The two methods all could be used to verify the feasibility of MALDI-TOF MS.3) 102 tissues of patients with brain tumor were collected and analyzed through the above method. It is shown that 33 of 74 patients’ glioma samples had gene mutations in IDH1 and the mutation probability was 45%; the mutation probability of IDH1R132 H accounted for 97% of the total; there was no mutation in IDH2 among these samples. No IDH mutation was found in other brain tumors samples.The gene mutation detection technique was verified by sequencing and analysis of clinical samples. It can be used to detect the mutations of single nucleotide in prescient nucleic acid sequence. This technique can be used to detect the mutation of IDH gene in samples. If the PCR primers are designed according to the target gene sequence information, it also can be used for detection mutation of other genes. Meanwhile, this technology can be used in genetic research in both cellular and tissular level, especially suitable for high-throughput tissue samples.