Research On Gene Detection Method Of Lung Cancer Based On Ion Torrent Platform

Objective Lung cancer have become the leading cause of cancer deaths worldwide,with1.5 million new cases worldwide each year which pose a serious threat to human health.Among them,non-small-cell lung cancer?NSCLC?accounts for 80%to 85%of lung cancer.However,many patients are found to be in the late stage and lost the chance of surgery.With the introduction of precision medicine,the individualized treatment of tumor,namely targeted drug,plays an important role in tumor therapy.In order to provide recommendations to clinicians in the treatment of lung cancer targeted therapy,this study proposes a method for the detection of non-small cell lung cancer drugs based on Personal Gene Machine platform.Methods The exons 18,19,20,21 of EGFR,exons 11,15 of BRAF,exons 2,3,4 of KRAS are selected as the target region for the study of gene detection in lung cancer,which is completed by combining with cosmic,NCBI,ensemble database,technical guidelines for individualized treatment of tumor and reading relevant literatures.Firstly,we used primer 5.0 designs the specific primers which are used for the Amplif ication of night items sequence.primers are verified after design and synthesized.Secondly,according to the Fusion Method of life,the Fusion primers based on Ion Torrent PGM platform were designed,namely,the primers for PGM amplification subsets,and 5 sets of Fusion primers were designed,totaling 45 pairs.The primers are then certificated.At the same time,we also design nine pairs of quantitative primers which are used to detect the homogeneity of multiple PCR amplif ication.A549 DNA is extracted by the tissue and cell DNA extraction kit of TIANGEN Company from A549 cells as experimental materials.Then,we optimize the multiple PCR reaction conditions and reaction system.The optimization is carried out on the basis of result of QPCR detection.we also construct wild plasmid and mutant plasmid as a reference which both include nine item sequences.Finally,library is constructed with optimized multiple PCR reaction system and conditions.Sequencing library by the Ion Torrent PGM and then analysis data.Results 1.Specific primers designed for the selected nine item sequence are verified by PCR,and then PCR products are detected by agarose gel electrophoresis and Agilent 2100 DNA.The results show that the primers were designed successfully.The PCR products sizes as follows,177 bp,173bp,262 bp,208bp,257 bp,218bp,124bp,296bp,264bp.Five sets of fus ion primers based on PGM platform were designed,namely,the primers for constructing amplicon library.The PCR amplified products are detected by agarose gel electrophoresis and Agilent 2100 DNA lab chip,target fragment size is corrective.The PCR product sizes as follows,240bp,231bp,316bp,263bp,273 bp,313 bp,182bp,352bp,and 321bp respectively.The sequence are then verified by Sanger sequencing which show that five sets of fusion primers based on PGM platform are successfully designed and could be used to construct PGM platform library.2.We have successfully constructed wild plasmid and mutant plasmid containing nine item sequences which are verified by Sanger sequencing.The constructed mutant plasmid comprises the following mutant sites:EGFR18?G719A?,EGFR19?E746_A750del/D761Y?,EGFR20?T790M/D770_N771ins CCA?,EGFR21?L858R?,BRAF11?G464E/G466A/G469A?,BRAF15?V600E?K2?G12D/G13D?,k3?Q61H?,k4?A146T?.At the same time,nine pairs of quantitative primers are successfully designed to detect the homogeneity of multiplex PCR amplification.3.After continuous conditional optimization,the final multiplex PCR system consisted of10?l of 2x Multiplex PCR Master Mix,1?l of template DNA,and 7.7?l of Primer Mix?0.6?l/0.5?l/1.3?l/0.55?l/1.3?l/0.9?l/0.65?l/1.1?l/0.8?l?,ddH₂0 1.3?l.The multiplex PCR reaction system is 20?l.The final multiplex PCR reaction conditions is as follow,95°C 15min;?95°C 30s,55°C 30s,72°C 45s?26 cycles.72°C 30min.Amplicons library construction is performed using 5 sets of fusion primers following the optimized 9-plex PCR conditions.After the library is sequenced by the PGM,we analysis date by plug-ins of ion torrent PGM and Nextgene,as a word,date quality is good.Conclusion This study initially established a method for the detection of genes for non-small cell lung cancer based on the PGM platform,which can provide recommendations for individualized treatment of non-small cell lung cancer patients,which is given suggestions to clinicians to rational drug use.