USe of PCR primers containing a 3' terminal ribose residuce to prevent cross-contamination of amplified sequences

Cross-contamination with previously amplified products poses a serious limitation In the use of PCR for clinical testing and In certain research applications as well. In the present study we report the use of novel primers containing a 3'-termlnal ribose residue to circumvent this problem. Extension of the primer by Taq DNA polymerase generates a cleavable ribonucleotide linkage within the amplified product. Cleavage of the primer by base or with a ribonuclease interferes with further replication of the product should carry over to another sample occur. Primers terminating In any of the 4 ribose residues function equally well as all DNA primers. Taq DNA polymerase is thus able to both efficiently extend and copy the single ribose residue. In translating from all DNA primers to ones containing a 3'-rlbose residue no modification of the PCR protocol Is required. The products formed can be used in all applications of the PCR. Since neither the original sample DNA, the primers or the extension products are modified by base or ribonuclease treatment both pre- and postamplification sterilization can be carried out. Preamplification treatment with RNase A can yield as high as 10Mold sterilization. Under these conditions the addition of β-mercaptoethanol or other sulfhydryl reducing agent is necessary to inactivate the enzyme during thermocycllng. Post-amplification treatment with NaOH readily yields at least 108fold sterilization. This alone Is sufficient for most, if not all, applications of PCR. It is especially useful for quantitative RT-PCR, since the original target RNA sequence, which may be present in high copy numbers, Is also destroyed.