USE OF PCR PRIMERS CONTAINING A 3'-TERMINAL RIBOSE RESIDUE TO PREVENTCROSS-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 rese arch applications as well. In the present study we report the use of n ovel primers containing a 3'-terminal ribose residue to circumvent thi s problem. Extension of the primer by Taq DNA polymerase generates a c leavable 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 w ell as all DNA primers. Taq DNA polymerase is thus able to both effici ently extend and copy the single ribose residue. In translating from a ll DNA primers to ones containing a 3'-ribose residue no modification of the PCR protocol is required. The products formed can be used in al l 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 post-amplification sterilization can be carri ed out. Pre-amplification treatment with RNase A can yield as high as 10(4)-fold sterilization. Under these conditions the addition of beta- mercaptoethanol or other sulfhydryl reducing agent is necessary to ina ctivate the enzyme during thermocycling. Post-amplification treatment with NaOH readily yields at least 10(6)-fold sterilization. This alone is sufficient for most, if not all, applications of PCR. It is especi ally useful for quantitative RT-PCR, since the original target RNA seq uence, which may be present in high copy numbers, is also destroyed.