Murat Sayan 45 Clonal analysis Cloning of PCR product Can overcome this problem Single nucleotide mismatch may escape detection Detects known and potential new mutations Requires large number of clones (works consistently only for viral loads of over 1E+3 IU/ml) Expensive Time – consuming, requires highly- skilled personnel and expensive equipment unsuitable RFLP PCR and restriction fragment length polymorphism analysis 5% populations of mutants Must be designed specifically for each mutant of interest Only known mutations Separate sets of endonuclease reactions must be designed (works consistently only for viral loads of over 1E+3 IU/ml) Expensive Time – consuming, requires highly- skilled personnel unsuitable Table 2. Comparison of the genotyping methods for diagnosis of drug resistance mutation in hepatitis B virus (6, 32, 40). Direct sequencing Line probe assay Technique Population based sequencing Differential hybridization on the membrane-bound oligonucleotide probe Minor subpopulations may escape detection Affects sequence context Minor subpopulations may escape detection Mutation detectability Detects known and potential new mutations Sensitivity Analytic sensitivity Specificity Usability Cost Difficulty Routines Affects sequence context and secondary structures in the target, minor subpopulations may escape detection 10% populations of mutants 5% populations of mutants Single nucleotide mismatch may escape detection New set of specific probes are required Requires sufficient PCR yield and perfect purification (works consistently only for viral loads of over 1E+3 IU/ml) Expensive but acceptable Expensive Time - consuming, requires highly- skilled personnel and expensive equipment Acceptable Only presents mutations Requires sufficient PCR yield (works consistently for viral loads over 990 copies/ml (95% CI) a Requires highly- skilled personnel Acceptable Obtaining In - house Commercial kit b In - house In – house a 990 copies/ml =1.7E + 2 IU/ml in AmpliPrep/Cobas TaqMan 48 HBV test (Roche Diagnostics GmbH, Mannheim, Germany) b INNO-LiPA DR v2 (Innogenetics,Ghent, Belgium) the HBV population, with a sensitivity of 990 copies/ ml at a 95% confidence interval (CI). However, one disadvantage of the assay (INNO - LiPA DR v2) is the limited scope of the mutations represented in the assay, and it only detects known mutations for LAM and ADV currently with wild type variants (20, 22, 30). However, in this technique, recombination of genotypes or new mutations cannot be detected (32). Periodic updating with new probes specific to novel mutations is required, because HBV strains resistant to newly developed antiviral drugs have been isolated and characterized (Table 2) (33). On the other hand, an INNO-LiPA DR v3 that confers resistance to ETV therapy has not yet been released as a commercial kit. This prototype line-probe assay allows the detection of variants rtA194T and rtI233V associated with resistance to TDF and ADV, respectively (22). Clonal Analysis of HBV Variants Minor HBV populations can be identified by large - scale cloning and sequencing protocols; however, this is laborious and beyond the capacity of clinical laboratories. This method, however, is sufficiently sensitive for the detection of emerging, resistant mutants that are present in low concentrations (34). For the cloning of the HBV pol region, a 700 bp PCR amplification covering the whole HBV pol gene can be us ed with the following primers; CLC188 5’-TCCCCAACCTCCAATCAC-3’ and CLC887 5’- AAACCCAAAAGACCCACAA-3’ (designed in our laboratory). The amplified 700 bp HBV pol region can be cloned into a TA vector by using the TOPO- XL-PCR cloning kit (Invitrogen, CA, USA) and the constructs can then be sequenced using the Big Dye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, Foster City, CA, USA) in an ABI Prism 3130 XL Genetic Analyser (Perkin Elmer, Foster City, CA, USA) according to the manufacturer’s instructions (35). Conclusions Other powerful technologies are in development, including high-throughput systems capable of detecting polymorphisms in the entire HBV genome using gene chip technology (36). Emerging technologies that have not yet penetrated significantly Hepatitis Monthly, Winter 2010; 10(1): 42-47