HYBRID PCR SEQUENCING - SEQUENCING OF PCR PRODUCTS USING A UNIVERSAL PRIMER

We describe a general method for making template DNA for sequencing of PCR products. The procedure may be particularly useful for PCR produc ts where mini mal sequence information is known or as an alternative t o primer walking when sequencing long PCR products. A cassette contain ing the hybridization site for the M13 sequencing primer is ligated to a sample PCR product. Using one phosphorylated primer specific for th e cassette together with one primer specific for the sample PCR produc t subsequent PCR amplifies one hybrid construct directionally. This al lows utilization of the universal M13 primer when sequencing of one st r and after the removal of the complementary strand using lambda-exonu clease.