[Detection of IgH rearrangements using real-time quantitative PCR and its reaction parameters].
To investigate the optimal reaction conditions of clonal rearrangements of immunoglobulin heavy chain (IgH) gene using polymerase chain reaction (PCR) with consensus primers and the feasibility of detecting this gene using SYBR green I real-time quantitative PCR with consensus primers, the systemic experiments were performed, which included annealing temperature, concentration of primer, the amount of Taq enzyme, concentration of dNTP and Mg(2+), number of PCR cycles. Detection of this gene on SYBR green I real-time quantitative PCR with consensus primers was carried out under the optimal reaction parameters obtained from previous study, and the sensitivity of IgH rearrangements gene was examined by using SYBR green I real-time quantitative PCR. The results indicated that the optimal annealing temperature was 60 degrees C, the optimal concentration of primers was 0.8 micromol/L, the satisfactory Taq enzyme amount was 0.5 U, the optimal concentration of dNTP was 100 micromol/L, the optimal concentration of Mg(2+) was 3.0 mmol/L, the suitable number of cycle was 40 cycles. Amplification of IgH rearrangement gene and detection of desired gene fluorescence signal on SYBR green I real-time PCR were performed. The sensitivity of IgH gene using this quantitative PCR was 10(4)/ml. It is concluded that the optimal reaction parameters for amplification of clonal IgH rearrangements gene by using PCR technique was determined, and stable and specific amplification of desired gene with consensus primers was performed. Basically, IgH rearrangement gene was successfully detected by SYBR green I real-time PCR.
Li
Hui-Min
HM
Department of Hematology, The First Affiliated Hospital, Kunming Medical College, Kunming 650032, China.
Shi
Lan
L
Duan
Yong
Y
Liu
Hua
H
Zhang
Xue-Mei
XM
D052160:Benzothiazoles; D003959:Diamines; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D015203:Reproducibility of Results
D052160:Benzothiazoles; D003959:Diamines; D007143:Immunoglobulin Heavy Chains; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I