Figure 1.

Methods to detect rtA181T mutations and pre-S internal deletion mutations. (A) Rationale of the amplification created restriction enzyme method to assist the detection of a small percentage of the rtA181T mutant. Partial amino acid sequences of the surface and polymerase genes were shown. Arrow (left), a mismatched primer with two nucleotides (CA) mismatches; Arrow (right), a matched antisense primer. A BST XI site was created if the amplicon was wild type. (B) Digestion of the PCR products with BST XI separated the wild type amplicon from the mixture. The undigested (mutant) DNA was gel-purified and submitted for sequence verification. In a 10⁵ copies of mixture, this method could detect 1/1000 (10² copies) of mutant. (C) Detection of the pre-S internal deletion mutants. The PCR product was analyzed by Southern blot to detect large deletion (b). The pre-S sequence with or without small deletions (a) was gel-purified and sent for sequence analysis.