A MODIFIED OVERLAP EXTENSION PCR METHOD TO CREATE CHIMERIC GENES IN THE ABSENCE OF RESTRICTION ENZYMES

A modified overlap extension technique for the creation of chimeric ge nes is described: the method consists in three PCR steps. The first st ep is a conventional PCR reaction, in which oligonucleotide primers ar e partially complementary at their 5' ends to the adjacent fragments t hat are fused to create the chimer. The second PCR step consists in th e fusion of the PCR fragments generated in the first step using the co mplementary extremities of the primers. The third step corresponds to the PCR amplification of the fusion product. The final PCR product is a chimeric gene built up with the different amplified PCR fragments. T he technique is illustrated by the construction of a chimeric 5-hydrox ytryptamine (5-HT, serotonin)(1B/D) receptor by combining one part of the human 5-HT1B (h5-HT1B) and two parts of the h5-HT1D receptor gene. The chimeric gene expressed in Cos-7 cells yielded similar binding pr operties as the wild type h5-HT1D receptor.