Direct primer walking on P1 plasmid DNA.

The development of vectors such as P1, P1-derived artificial chromosome (PAC) or bacterial artificial chromosome (BAC) (2,6,8) has permitted the cloning of large DNA fragments. These vectors have become common in largescale sequencing and mapping projects. Therefore, a reliable primer walking protocol for P1 plasmids is desirable. It has been demonstrated previously that sequencing of P1 plasmid DNA is generally feasible (4,11). However, the protocols included several laborious steps such as a host strain change from NS3145 to DH10B or tedious purification procedures and were limited to the use of primers located in the vector arms. Another development for sequencing P1 plasmid DNA uses a polymerase chain reaction (PCR)-based technique termed “thermal asymmetric interlaced PCR” (5); however, this method also relies on numerous experimental steps. We present an optimized sequencing protocol for direct primer walking on P1 plasmid DNA based on cycle sequencing (7). The effect of the following parameters on the success rate of P1 sequencing was evaluated: host cell type, purification procedure, template amount, primer design, primer amount and cycle sequencing conditions. Sequencing reactions were carried out with either fluorescein isothiocyanate (FITC)or Cy5-labeled primers and analyzed on the corresponding sequencing apparatus (ALF DNA Sequencer and ALFexpress; Pharmacia Biotech, Piscataway, NJ, USA). The read length assigned to sequencing results was determined by the number of nucleotides called after automatic processing by the ALF software. P1 plasmid DNA was isolated from its E. coli host strain NS3145 (library strain) and transformed into the new host strain DH10B by electroporation (3). When performing parallel P1 plasmid DNA preparations under identical conditions (2× YT medium, 0.5-L culture volume, 50 μg/mL kanamycin, single-colony inoculate, two QIAGEN 100 columns [Qiagen, Hilden, Germany]), the E. coli host strain NS3145 consistently yielded between 5 and 8 times more P1 plasmid DNA than the DH10B host strain. On average, a 0.5-L culture (NS3145 strain) yielded 80 μg (±10%) plasmid DNA. P1 plasmid DNA purification was performed with a conventional alkaline lysis/phenol:chloroform protocol (1) (Nucleobond; Macheray and Nagel, Düren, Germany) or an alkaline lysis protocol followed by column purification (Qiagen). The Nucleobond and the Qiagen protocols provided by the suppliers were both modified as follows. Lysis time was shortened from 5 to 1 min, and volumes of the resuspension, lysis and neutralization buffers were