PCR-INTRODUCED LOOP STRUCTURE AS PRIMER IN DNA-SEQUENCING

The need for a primer hybridization step before sequencing has been el iminated using a stem-loop structure generated by PCR. The loop struct ure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capt ure of the PCR product, the loop is formed by elution of the non-bound strand. Here, Mit shaw that both the immobilized ann the eluted stran d call be analyzed using conventional Sanger DNA sequencing and the no vel pyrosequencing method as described previously. By using a stem-loo p structure as a primer for DNA sequencing, the risk fbr mispriming is minimized.