ligation step will give only 5 u2032 b1 3 u2032 n1 p p5 u2032 n2 3 u2032 b2 molecules that are ready to be cloned this procedure may further increase the efficiency of the bp cloning the amplification of b1 and b2 fragment can be performed using b1 and b2 primers containing an attl1 and an attl2 site respectively instead of the attb sites the procedure is identical to the one illustrated in fig1 except that the b1 b2 unit is cloned using the lr reaction into a gateway destination vector containing the attr cassette this allows the direct cloning of the chimerical dna molecule into a final vector i e an expression vector without the necessity of a first step of cloning into an entry vector and a subsequent step of cloning into the destination vector more generally it is possible to use primers with attb attp attl attr sites for the amplification providing that the cloning vector bares the corresponding cassette respectively with attp attb attr attl sites after the ligation step the population of dna fragments can be submitted to pcr using the 5 u2032 b1 and 3 u2032 b2 primers fragments with the structure 5 u2032 b1 n1 n1 5 u2032 b1 3 u2032 b2 n2 n2 3 u2032 b2 and 5 u2032 b1 n1 n2 3 u2032 b2 are amplified preferentially the pcr mixture is then used in a bp reaction only fragments.