Specific primer design and exonuclease III treatment for the reduction of nonspecific staining in direct in situ PCR.

Although direct in situ PCR is more rapid than indirect method for the in situ identification of low copy number genes, several reports indicate serious nonspecific signals with this method. Some procedures have been reported in an effort to eliminate the nonspecific signals, but the results have not been satisfactory. Exonuclease III can progressively digest blunt or recessed 3' termini of double-stranded DNA whereas DNA with 3' overhanging end is resistant to digestion. DNA fragments amplified by PCR with primers incorporating the recognition site for Sph I generate 3' four bases extensions at both end after digestion. These fragments are expected to be resistant to exonuclease III digestion. It is also expected that nonspecifically incorporated digoxigenin would be released by treatment with exonuclease III thereby reducing background. We succeeded in digesting selectively with the samples after standard PCR by Sph I and exonuclease III treatment. However, we failed to eliminate the nonspecific signals of direct in situ PCR. Southern blotting revealed that the amount of nonspecific incorporation was so huge that exonuclease III was unable to release all of nonspecifically incorporated digoxigenin and maintain specific incorporated digoxigenin simultaneously. Direct in situ PCR is a sensitive method. However, its specificity has a significant problem.