A common problem during long term administration of chronic hepatitis B with Neocleot（s）ide analogues（NUCs）is drug resistance. NUCs resistance has been attributed to mutations at specific sites in the reverse transcriptase domain of HBV polymerase.Currently used extensive mutagenesis assay based on plasmid transfection method has some disadvantages. Here

we developed an alternative method for extensive mutagenesis phenotype assay of HBV polymerase. This method combined a randomized mutation method and golden-gate or fragment substitution reaction（FSR） strategy developed previously. Using this way

we successfully constructed multiple mutations at rt181 and rt236

the well-known mutation sites related to ADV resistance.