Figure 1.

Mutagenesis and heteroduplex structure. (A) In a first round of oligonucleotide mutagenesis, two MfeI sites were created at positions 2401 and 2870 (position numbers of the cut sites) on the ami insert of pALT-22, leading to pALT-212. The MfeI site at 2401 was generated by an A-to-C substitution at position 2401. The MfeI site at 2870 was generated by a T-to-A substitution at position 2872. pALT-212 was then digested with MfeI and ligated in diluted conditions to close the vector. The new plasmid, pALT-215, contains a deletion of 469 bp in the center of the ami fragment. By oligonucleotide mutagenesis, ClaI and HindIII sites were created at positions 2375 and 2894 (position numbers of the undeleted original are retained). Both are single nucleotide additions, G and T, respectively. Another round of mutagenesis was done to generate an AflII site at position 2355 and an NdeI site at position 2923, both being the addition of one A. To produce large amounts of single-stranded (SS) DNA, we subcloned the mutagenized EcoRI inserts from the pALT vectors to M13 mp11. We obtained two plasmids: pCH4, which contains ClaI and HindIII sites, and pAN5, which contains AflII and NdeI sites. Each DNA was checked at each step by restriction analysis; SS pAN5 and SS pCH4 were sequenced to check sites, insert orientation, and the absence of additional mutation on the insert. (B) Replicative form (RF) of pCH4 was hybridized with SS pAN5 to generate the heteroduplex I (HetI); RF pAN5 was hybridized with SS pCH4 to generate HetII. Sequence structures of the EcoRI inserts in HetI and HetII are represented. Each added base is indicated below the site it generates. The MfeI sequence created by ligating the MfeI ends to introduce the 469-bp deletion (Δ) is indicated. Within this sequence, boxed bases are the mutagenized positions 2401 and 2872. Distances from the restriction sites to the EcoRI ends and to the boxed bases are indicated.