Simplified Method for the Telomeric Repeat Amplification Protocol (TRAP)

Since it was first described by Kim et al. in 1994 (4), the telomeric repeat amplification protocol (TRAP) has been used extensively to detect telomerase activity in a variety of tissues. It is a highly sensitive polymerase chain reaction (PCR)-based assay that has been used to detect telomerase activity in a high proportion of cancers of various origins as well as low levels of activity in certain normal progenitor cells. The function of telomerase is to extend the length of the telomeric DNA on the ends of chromosomes by adding extra telomeric repeats. This overcomes the end replication problem faced by linear chromosomal DNA and prevents chromosomal shortening that could otherwise trigger cellular senescence. The TRAP assay is essentially a two-part process. In the first reaction, a synthetic oligonucleotide (TS) containing the telomeric repeat sequence recognized by telomerase is mixed with the tissue homogenate to be tested. If telomerase is present, it will add further telomeric repeats (TTAGGG) onto the end of the TS oligonucleotide. In the second part of the reaction, another oligonucleotide (CX) is introduced that is complementary to the repeat sequence, and the extended synthetic oligonucleotides are then amplified by PCR. The PCR product lengths vary depending on the number of repeat units incorporated but always increase in size by multiples of six (the length of the repeat unit). After gel electrophoresis, a characteristic DNA ladder is produced, with the bands 6 bp apart. The original TRAP method entailed lyophilizing the CX primer in the bottom of the reaction tube and sealing it with wax. The remaining reagents required for both telomeric extension and PCR were then added above the wax barrier, and the tube was incubated for 10 min to allow the telomerase to extend from the TS oligonucleotide. The tube was then heated to 95°C, at which time the reagents sank below the wax, mixing with the second primer (CX), and PCR could then be performed. A disadvantage of this technique was the requirement for prior preparation of the reaction tubes. Lyophilization of the CX primer and sealing with wax beads is quite time-consuming, and pipetting 10 μL of molten wax into tubes is quite difficult to do without the wax setting. A slight alteration to this technique is to add an entire wax bead (AmpliWax PCR Gem; Perkin-Elmer, Norwalk, CT, USA) to each tube containing lyophilized oligonucleotide, heat the tubes briefly in a thermal cycler to melt the wax, then cool them so the wax sets and forms a barrier (2,6). Disadvantages of this are that the use of one wax bead per reaction increases the reagent cost significantly and that when the reaction vessels were transferred from the thermal cycler back to a pre-PCR setup area, the risk of contamination significantly increased. Most methods now used do not separate the PCR primer from the remaining reagents with a wax barrier but instead add the Taq DNA polymerase and/or the CX primer to the reaction only after the telomeric extension step has been carried out and the sample has been heated to 95°C (1,3). This method is less laborious than preparing the reaction tubes with lyophilized CX primer and wax beads; however, it does require an extra pipetting step and reopening of the reaction tubes before the PCR, which can increase the risk of contamination. We have modified the protocol to incorporate the use of AmpliTaq Gold