Minigel Procedure for Telomeric Repeat Ampli - fication Protocol

Telomeres, the ends of chromosomes, are composed of many kilobases of TTAGGG repeats in vertebrates. These repeats are added by the enzyme telomerase, a ribonucleoprotein DNA polymerase that synthesizes the repeats based on its RNA template. A sensitive assay combining telomerase activity and the polymerase chain reaction (PCR) known as the telomeric repeat amplification protocol (TRAP) has been developed (6,10). In this assay, telomerase adds TTAGGG repeats to a telomerase substrate (TS) oligonucleotide that has no telomeric sequence; the enzyme pauses after adding each repeat, presumably to permit repositioning of its RNA template prior to synthesizing the next repeat. This generates a DNA ladder of 6-bp addition products that is then sensitively detected by polyacrylamide gel electrophoresis (PAGE) after PCR amplification (using a second CX oligonucleotide as reverse primer). Telomerase activity has been detected in tumor, germ and stem cells but not in adult somatic cells. Thus, the enzyme may play a significant role in aging and in the pathogenesis of cancer (2,6). Because of this widespread interest, improvements to TRAP are continually being developed, e.g., internal standard, fluorescent TS primer and improved CX-ext primer (7,9,12). We report fluorometric detection of TRAP DNA ladders by high-resolution agarose gel electrophoresis. The human embryonic kidney (HEK) 293T cell line and the human monocytic macrophage Mono Mac 6 cell line were cultured as described (3, 5). Telomerase extracts were obtained from cells by the procedure of Kim et al. (6) with minor modifications (1,10, 11). Cells were counted, harvested and treated with ice-cold TRAP lysis buffer at 106 cells/100 μL buffer. The TRAP lysis buffer contained 0.5% 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1propanesulfonate (CHAPS), 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 5 mM EGTA, 25 mM 2-mercaptoethanol, 1 ng/mL leupeptin and 50% glycerol in diethyl pyrocarbonate (DEPC)-treated water. The lysate was kept on ice for 30 min and then centrifuged at 16 000× g for 20 min at 4°C in a Model 5415 Eppendorf® Microcentrifuge (Brinkmann Instruments, Westbury, NY, USA). Afterwards, 80 μL of supernatant were collected, flash-frozen in liquid nitrogen and stored at -75°C until used. Detection of telomerase activity from cell extracts was performed in a two-step process: (i) telomerase-mediated extension of the TS nucleotide and (ii) PCR amplification of the resultant product with both TS and the reverse CX primers (1,10,12). The oligonucleotides TS (5′-AATCCGTCGAGCAGAGTT-3′) and CX (5′-CCCTTACCCTTACCCTTACCCTAA-3′) (both from BioSynthesis, Lewisville, TX, USA) were each dissolved in DEPC-treated water at 0.1 μg/μL. Each thin-wall PCR tube contained 20 μL of a reaction mixture that included 16.5 μL of TRAP reaction buffer (20 mM Tris-HCl, pH 8.2, 63 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 0.1 mg/mL 1× bovine serum albumin [BSA] and 0.05% Tween 20), 1 μL of TS (0.1 μg), 0.5 μL of dNTPs (10 mM stock) and 2 μL cell extract (from 1000 cells). The telomerase extension reaction was completed in a Model DB 66925 Temp.Tronic Τhermal Cycler (Barnstead/Thermolyne,