TaqDNA polymerase extension of internal primers blocks polymerase chain reactions allowing differential amplification of molecules with identical 5′ and 3′ ends

Polymerase chain reaction (PCR) methodology (1) has become a routine method for selectively amplifying segments of DNA from a wide variety of sources. Amplification of specific sequences is dependent upon an exact match between the template DNA and the oligonucleotide primers. Mismatches at the 3' terminus lead to greatly reduced amplification, with no detectable product when amplified under the appropriate conditions (2,3). We demonstrate here that an oligonucleotide which can be extended by Taq DNA polymerase is able to block the amplification of a PCR product when situated between two flanking PCR primers. An oligonucleotide mismatched at its 3' terminus, however, does not demonstrate this ability. This allows the development of a method for the differential amplification of molecules with identical 5' and 3' ends. Unless otherwise indicated, PCRs were performed in a programmable heating block using 20 rounds of temperature cycling (94°C for 1 min, 55°C for 2 min and 72°C for 3 min) followed by a final 10 min step at 72 °C. One microgram of each primer, 5 ng of 'blocking' oligonucleotide, 10 ng of template and 2.5 units of Taq DNA polymerase (Perkin-Elmer Cetus) were used in a final volume of 100 /il with the reaction buffer as recommended by the manufacturer. The 'blocking' oligonucleotides, designed to block PCR amplification of the DX48 template, are indicated in Table 1. The templates used were a CAMPATH-1H humanised monoclonal antibody (mAb) heavy (H) chain (4) and DX48 humanised mAb H chain (unpublished), both in cDNA context in the plasmid vector pUC18 (GffiCO/BRL). The 5' PCR primer, X (5'-GATCAAGCTTTACAGTTACTCAGCACACAG), was designed to anneal to both templates such that the 3' end was at a position 12 nucleotides (nt) upstream of the ATG initiation codon in each case, and the 3' primer, Y (5'-GATCAAGCTTTCATTTACCCGGAGACAGGGAGA), was designed to anneal with the 3' end 20 nt upstream of the termination codon. The products of PCRs carried out on both templates using these primers would be expected to be 1462 base pairs (bp) in length. Oligonucleotides A N were able to anneal to the negative strand of the CDR3 region within the DX48 PCR, and possessed combinations of mismatches and modifications at their 5' and 3' termini (Table 1). They were added to the DX48 PCR to investigate whether such modifications would allow the oligonucleotides to block the PCR amplification. Taq DNA polymerase possesses a 5'—3' polymerase-dependent exonuclease activity (5). 5 ng of 'blocking' oligonucleotide was used in each reaction, this being an approximately lO^fold molar excess of 'blocking' oligonucleotide compared to template. The products of the PCR reactions were run on an agarose gel, as shown in Figure 1. The oligonucleotides with mismatches and modifications at their 3' termini were all unable to block the amplification of the 1462 bp PCR product, suggesting that either the 5' oligonucleotide modifications were unable to prevent degradation by the 5'3' exonuclease activity of the extending Taq DNA polymerase (5), or that the polymerase possesses strand