Establishment of the technique for the real-time fluorescence quantitative reverse transcription polymerase chain reaction by DNA melting curve analysis for detecting the CDR3 skewing of TCR alpha gene repertoire in the human peripheral blood
- VernacularTitle:采用荧光定量PCR溶解曲线法监测人TCR alpha链CDR3谱系漂移技术的初步探讨
- Author:
Xianying TANG
;
Yongping SUN
;
Rui MA
;
Hongqian ZHU
;
Zuguo TIAN
;
Wanbang SUN
;
Xinsheng YAO
- Publication Type:Journal Article
- Keywords:
T-cells receptor(TCR);
Complementarity determining region 3(TCR CDR3);
Real-time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-PCR);
Melting curve
- From:
Chinese Journal of Immunology
2000;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish the technique for real-time fluorescence quantitative reverse transcription polymerase chain reaction(FQ-PCR)by DNA melting curve analysis for detecting the CDR3 shewing of TCR alpha gene repertoire in human peripheral blood.Methods:Total RNA of peripheral blood mononuclear cell(PBMC)from 4 healthy donors and 2 patients with lymphomatous leukemia were transcripted reversely into cDNA.The cDNA of 32 TRAV gene family CDR3 was amplified by FQ-PCR.Analysis of the monoclonal/oligoclonal/polyclonal CDR3 spectratyping with DNA melting curve.Results:The FQ-PCR products of 32 TRAV family CDR3 were showedas a blur land at the predicted of products size in healthy donors and parts of TRAV family CDR3 products disappeared in patients on 1.5% agarose gel by Gold-View staining.The 32 TRAV family CDR3 were showed with different frequencies by relative fluorescence quantitative in healthy donors and the patients.The CDR3 spetratyping for 32 TRAV families was showed as polyclonal peak(Gaussian distribution)in healthy donors but showed as different monoclonal/oligoclonal/polyclonal peak in the patients with lymphomatous leukemia with DNA melting curve analysis(we called "melting curve spectratyping of CDR3")Conclusion:The study suggests that the technique of "FQ-PCR with DNA melting curve analysis be convenience and celerity for detecting the CDR3 skewing of TCR alpha gene repertoire in human peripheral blood.
