Determination of size and orientation of DNA fragments cloned in phage M13 by S1 nuclease mapping

Cloning of DNA fragments in phage M13 is a routine procedure in DNA sequence determination. A simple procedure for the determination of DNA fragment orientation has been described earlier (1). Here we introduce a modification of this procedure which allows the simultaneous determination of DNA fragment orientation and size. Procedure: Single-stranded M13 DNAs (1, about lpg) were mixed in 5ul of 5mM Tris-HCl (pH 8.0), l OOmM NaCl, 0.5mM EDTA, heated to 80°C for 5 min and allowed to cool to room temperature for 15 min. The ionic conditions were then adjusted to 30mM sodium acetate (pH 4.6), l OOmM NaCl, lmM ZnSC>4 and the DNA digested with 1 unit Si nuclease (Amersham) for 30 min at 37°C in a total volume of IOJJI. Afterwards the digest was fractionated on a 0.8% agarose gel (2). Fig.l: Agarose gel electrophoresis Lane 1:single-stranded(ss)M13 DNA with 3kb insert,no SI treatment;lane 2:ss M13 DNA with 3kb insert,both orientations.no Sj __ treatment plane 3:same as lane l,but Si-treated; lane 4:same as lane 2,but Sj-treated ;lane 5:ss Ml 3 DNA with 3kb insert + ss ~~ ' 7 M13 DNA with 2.4kb insert of opposite orientation,Sl-treated;lane 6:same as lane 5,but both inserts with same orientation; lane 7:replicative form(RF)Ml3 DNA with 2.4kb insert,cut with restriction enzyme Pstl;lane 8:ss M13 DNA with 3kb insert + ss H13 DNA with 0.6kb insert of opposite orientat ion;lane 9:same as lane 8,but both inserts with same orientation;lane 10:RF M13 DNA with 0.6kb insert,cut with PstI. Fig.l shows that single-stranded (ss) M13 DNA (lane 1) forms a slower migrating DNA structure (partial hybrid) with ss M13 DNA containing the same insert in opposite orientation (lane 2). S^ nuclease treatment generates a double-stranded (ds) DNA fragment corresponding in length to the insert length (lane 4). Ttiis ds DNA is not formed when the inserts have the same orientation (lane 3). Lanes 5-7 and 8-10 demonstrate that the S^ nuclease-resistant ds DNA fragments (lanes 5,8) indeed correspond exactly to the insert length (lanes 7,10). Ttiese data show that ss M13 DNA hybridization followed by S, nuclease treatment allows the determination of insert length and orientation in one step.