PCR-SSCP检测白血病患者C/EBPζ基因突变方法学建立

Establishment of PCR-SSCP Method for Detection of C/EBPζ Mutation in Patients with Leukemia

目的建立一种快速、批量筛查白血病患者骨髓C/EBPζ基因外显子突变的多聚酶链反应-单链构象多态性(PCR-SSCP)分析方法。方法用Bio-Rad iCycler梯度PCR仪对25μL PCR反应体系中的Mg++、Taq酶等浓度及反应条件进行优化,并用2%琼脂糖电泳检验产物质量。在此基础上,再对中性聚丙烯酰胺凝胶浓度、上样缓冲液种类、上样量、上样次数及相隔时间、PCR产物稀释倍数、电压、电泳温度等因素进行探讨。同时比较80个健康体检者外周血与20个胸外科良恶性肿瘤患者肋骨骨髓DNA的PCR-SSCP图谱。结果 25μL PCR反应体系中的Mg++、Taq酶浓度分别为1.5～2.0mM和1.0～1.5U,退火温度为54℃～57℃时PCR扩增效率高;应用8%～10%非变性聚丙烯酰胺凝胶,低离子强度(LIS)上样缓冲液,PCR产物稀释4～5倍,上样量为5～10μL,同一块凝胶相隔0.5～1.0小时可上样2次产物,电压为320V挂冰,室温或4℃电泳5～7小时,SSCP分析可得到较为满意的图谱。80例健康体检者外周血与20例正常肋骨骨髓DNA的C/EBPζ基因图谱相同。结论可以用正常体检者外周血DNA代替正常肋骨骨髓DNA行SSCP分析,作为C/EBPζ基因突变检测的对照图谱,两次上样的PCR-SSCP方法节约了试剂、时间、人力,可用于快速大量筛查白血病患者C/EBPζ基因突变。

Objective To establish a rapid detecting method of polymerase chain reaction-single strand conformation polymorphism （PCR-SSCP） for the screening of C/EBPζ gene mutation in bone marrow cells from leukemic patients. Methods PCR program was performed on Bio-Rad iCycler gradient PCR machine. 25μL of reaction system was optimized, including magnesium chloride and Taq polymerase concentrations. The quality of amplified products obtained by PCR was evaluated by 2% agarose electropheresis. On the basis, SSCP parameters were optimized, including neutral polyacrylamide gel concentration, the types of sample adding buffer, sample amount, sample-adding times and interval time, the dilution concentration of PCR product, voltage, electropheresis temperature. 100 DNA samples were measured by PCR-SSCP, including that the peripheral blood samples from 80 healthy individuals and from rib marrows of 20 with benign or malignant carcinomas and without abnormal peripheral blood counts after thorax surgery. Results In the 25μL PCR reaction system, magnesium chloride and Taq polymerase concentrations were 1.5 - 2.0 rnM and 1.0 - 1.5 U respectively; more PCR products were obtained at the annealing temperature of 54 - 57 ℃. Satisfactory electropheresis results were obtained using the conditions as follows： 8% - 10% non-denaturing polyacrylamide gel, sample buffer of low ion strength, PCR products diluted 4 - 5 folds, 5 - 10μL of sample amount. Two batches of PCR products were added into the same gel in turn at 0.5 - 1 hour interval, Voltage for electropheresis was 320 V and electrophoresis chambers were hanged with ice. SSCP was performed for 5 - 7 hours at room temperature or 4 %. The same electropheresis bands were obtained in the peripheral blood samples from 80 healthy individuals and the rib marrow samples from 20 patients with tumors. Conclusions The marrow samples can be substituted with peripheral blood of normal healthy individuals as the normal control for the detection of C/EBPζ gene mutation. Two sample-adding methods can save the agents, time, and labor and so can be used in the screening of C/EBPζ mutation in the patients with leukemia.