1. Telomerase is a ribonucleoprotein complex that consists of telomerase RNA (which contains several tandem hexanucleotide repeats complementary to the telomere repeats in the substrate) and a reverse transcriptase. This complex is able to synthesize telomeric repetitive DNA sequences at the end of linear DNA molecules. Telomerase RNA binds to the DNA strand, and complementary dNTPs are incorporated into newly synthesized, extended DNA strands by reverse transcriptase. Thus, dNTPs must also have been present during step 1 of the TRAP assay (step 1).
2. The PCR reaction mixture also contained a heat-resistant DNA polymerase (Taq polymerase).
3. Sample 3 contained cell extract isolated from immortal cells. In the presence of the telomerase, products of different lengths were synthesized during step 1 and amplified during PCR. The different fragment lengths are the result of the RNA hexanucleotide sequence binding to a different telomeric repeat on the substrate. The ladder-like appearance of the bands thus corresponds to a set of products that differ from each other by 6 base pairs. In sample 9, purified telomerase was used (positive control) resulting in the synthesis of similar DNA fragments. These two samples prove that the immortal cell line expressed active telomerase.
4. In the absence of either the telomerase substrate (sample 1) or the cell extract (sample 2), telomeric DNA synthesis and amplification do not occur. Synthesis and amplification of telomeric repeats occurs only in the presence of both the immortalized cell extract and the telomerase substrate (samples 1 and 2 are negative controls). Without added telomeric repeats, the primer complementary to three telomere repeats cannot bind to the telomerase substrate, so amplification cannot happen. (Under the reaction conditions used, at least three repeats must be added to the telomerase substrate for efficient PCR amplification.)
5. Immortalized cells contain catalytically active telomerase; fibroblasts (mature, differentiated cells) do not.
6. RNase treated samples show no evidence of telomeric DNA fragment synthesis. Telomerase RNA serves as the template in the reverse transcription reaction.
7. Heating and phenol extraction denature proteins; protease degrades proteins. In those samples, no bands appear in the gel, demonstrating the PCR did not amplify a template, presumably because there was no synthesis of telomeric repeats in them in step 1. We may conclude that a protein, presumably reverse transcriptase, is necessary for the synthesis of telomeric repeats in that step.