Abstract: Objectives Explore the best experiment condition for touchdown PCR and clone-based sequencing to detect the methylation status of hsa-mir-34b gene promoter.Methods After the bisulfite treatment of the DNA extracted from lung cancer cell line A549,the primer was designed to amplify the promoter region of mir-34b gene.General PCR,nest PCR and touchdown PCR were employed to amplify the target fragment with different DNA polymerase.Then direct sequencing and cloning-based sequencing methods were performed to detect the methylation status.Results Compared to general PCR,touchdown PCR and nest PCR was used alone,touchdown PCR together with nest PCR was much easier to amplification;HotStart Taq was the suitable DNA polymerase in the PCR reaction system.Compared to the direct sequencing,cloning-based sequencing could find the methylation status more clear.Conclusions HotStart Taq DNA polymerase,touchdown PCR together with nest PCR and cloning-based sequencing methods were recommend to be used to detect the methylation status after the DNA underwent the bisulfite treatment,by which we could obtain the methylation patterns clearly with high sensitivity.