Sensitive detection of telomerase activity based on isothermal exponetial amplification reaction

doi:10.3969/j.issn.1000-1565.2016.02.008

Abstract

Abstract: Telomerase elongated product has many repeat sequences-(ggttag)_n.A specific DNA probe-(ctaacc)₂ which is matched with two repeat sequences is used to hybridize with telomerase elongated product.Excess DNA probes are attached to magnetic graphene and removed by magnetic separation.The DNA probes hybridized with telomerase elongated product is rapidly amplified by isothermal exponential amplification reaction(IEXPAR).SYBR Green I is utilized for real-time fluorescent detection of IEXPAR products.Under the optimal conditions,the telomerase activity in only 50 cancer cells can be easily detected.Sensitive detection of telomerase activity under isothermal condition is achieved.

Key words: telomerase activity, isothermal exponential amplification reaction, magnetic graphene, real-time fluorescence detection

CLC Number:

ZHOU Xiaoyu,ZHANG Jiayu,ZHOU Man,JIA Hongxia. Sensitive detection of telomerase activity based on isothermal exponetial amplification reaction[J]. Journal of Hebei University (Natural Science Edition), 2016, 36(2): 155-161.

References

[1] FENG J,FUNK W D,WANG S S,et al.The RNA component of human telomerase [J].Science,1995,269:1236-1240.
[2] COHEN S B,GRAHAM M E,LOVRECZ G O,et al,Protein composition of catalytically active human telomerase from immortal cells [J].Science,2007,315:1850-1853.DOI:10.1126/science.1138596.
[3] KIM N W,WU F.Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol(TRAP)[J].Nucleic Acids Res,1997,25:2595-2597.
[4] HOU M,XU D W,BJORKHOLM M,et al.Real-time quantitative telomeric repeat amplification protocol assay for the detection of telomerase activity [J].Clinical Chemistry,2001,47:519-524.
[5] WEGE H,CHUI M S,LE H T,et al.SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity [J].Nucleic Acids Res,2003,31:e3.DOI:10.1093/nar/gng003.
[6] SZATMARI I,ARADI J.Telomeric repeat amplification,without shortening or lengthening of the telomerase products:a method to analyze the processivity of telomerase enzyme [J].Nucleic Acids Res,2001,29:e3.
[7] SZATMARI I,TOKES S,DUNN C B,et al.Modified telomeric repeat amplification protocol:a quantitative radioactive assay for telomerase without using electrophoresis [J].Anal Biochem,2000,282:80-88.DOI:10.1006/abio.2000.4589.
[8] MAESAWA C,INABA T,SATO H,et al.A rapid biosensor chip assay for measuring of telomerase activity using surface Plasmon resonance [J].Nucleic Acids Res,2003,31:e4.DOI:10.1093/nar/gng004.
[9] SATO S,KONDO H,NOJIMA T,et al.Electrochemical telomerase assay with ferrocenylnaphthalene diimide as a tetraplex DNA-specific binder [J].Anal Chem,2005,77:7304-7309.DOI:10.1021/ac0510235.
[10] ESKIOCAK U,OZKAN A D,OZSOZ M,et al.Label-free detection of telomerase activity using guanine electrochemical oxidation signal [J].Anal Chem,2007,79:8807-8811.DOI:10.1021/ac071014r.
[11] PATOLSKY F,GILL R,WEIZMANN Y,et al.Lighting-up the dynamics of telomerization and DNA replication by CdSe-ZnS quantum dots [J].J Am Chem Soc,2003,125:13918-13919.DOI:10.1021/ja035848c.
[12] WEIZMANN Y,PATOLSKY F,POPOV I,et al.Telomerase-generated templates for the growing of metal nanowires [J].Nano Lett,2004,4:787-792.DOI:10.1021/nl049939z.
[13] XIAO Y,PAVLOV V,NIAZOV T,et al.Catalytic beacons for the detection of DNA and telomerase activity [J].J Am Chem Soc,2004,126:7430-7431.DOI:10.1021/ja031875r.
[14] VAN NESS J,VAN NESS L K,GALAS D J,Isothermal reactions for the amplification of oligonucleotides [J].Proc Natl Acad Sci USA,2003,100:4504-4509.DOI:10.1073/pnas.0730811100.
[15] LEE J,PARK II-S,JUNG E,et al.Direct,sequence-specific detection of dsDNA based on peptide nucleic acid and graphene oxide without requiring denaturation [J].Biosensors And Bioelectronics,2014,62:140-144.DOI:10.1016/j.bios.2014.06.028.