TEMPLATE SECONDARY STRUCTURE CAN INCREASE THE ERROR FREQUENCY OF THE DNA-POLYMERASE FROM THERMUS-AQUATICUS

Amplification of portions of the intergenic spacer between the katE ge ne and cryptic eel operon of Escherichia coli was accomplished by the polymerase chain reaction using the DNA. polymerase from Thermus aquat icus. Nine different segments were amplified and cloned without error, but one 83-bp fragment was amplified with a high error rate such that 32 of 34 selected clones had three or more nucleotide changes from th e expected sequence. The changes were all located in two 9-bp segments immediately adjacent to the 3'-ends of the two primers. Moving the en d points of the primers to increase the spacing between them resulted in the isolation of significantly fewer error-containing products. It is proposed that stem-loop structures in the template immediately down stream from the primers interfere with an early stage of elongation an d cause misincorporation. This is supported by the observation that de stabilisation of one of the stem-loop structures reduced the frequency of errors.