A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR InhibitorsReportar como inadecuado




A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

1

Advanced Technology Research Laboratories, Panasonic Corporation, 3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan

2

Frontier Institute for Biomolecular Engineering Research FIBER, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan

3

Faculty of Frontiers of Innovative Research in Science and Technology FIRST, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan





*

Authors to whom correspondence should be addressed.



Abstract An assay for telomerase activity based on asymmetric polymerase chain reaction A-PCR on magnetic beads MBs and subsequent application of cycling probe technology CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences 5-TTAGGGn-3 of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5 end and a quencher at the 3 end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5 end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity. View Full-Text

Keywords: telomerase; PCR inhibitor; magnetic beads; asymmetric PCR; cycling probe technology telomerase; PCR inhibitor; magnetic beads; asymmetric PCR; cycling probe technology





Autor: Hidenobu Yaku 1,2,3, Takashi Murashima 2,3, Daisuke Miyoshi 2,3,* and Naoki Sugimoto 2,3,*

Fuente: http://mdpi.com/



DESCARGAR PDF




Documentos relacionados