三种荧光标记法对寡核苷酸芯片杂交信号的影响

The impact of three fluorescence marker methods to oligonucleotide microarray hybridize signal

目的 观察标记引物法、随机渗入标记法、末端转移标记法三种荧光标记法对寡核苷酸芯片杂交信号的影响。方法 将淋球菌 gyrA基因 91位密码子突变型探针倍比稀释成终浓度为 10 0 0 μmol/L～ 0 2 5 μmol/L的梯度浓度点样并制作寡核苷酸芯片 ,PCR扩增荧光标记包含gyrA基因突变的目的DNA片段 ,与芯片杂交 ,分别记录三种标记方法的杂交信号强度。结果 标记引物法的荧光信号最强 ,随机渗入标记法的荧光信号强度次之 ,其Cy5 dUTP最佳浓度为0 1mmol/L(dTTP :Cy5 dUTP =10∶1) ,末端转移标记法反应时间在 6 0min和 12 0min时荧光信号值相近。点样的探针浓度达到 31μmol/L以上 ,荧光信号为平台期。 结论 明确了三种荧光标记法对寡核苷酸芯片杂交信号的影响 。

Objective To observe the impact of three fluorescence marker methods of primer marker, random inleakage marker and terminal transfer marker to Oligonucleotide microarray hybridize signal. Methods The Ser91 code mutation probes of Neisseria gonorrhoeae gyrA gene were doubly serial dilutions made twelve times, the end concentration were 1 000 , 500, 250, 125, 62 5, 31, 15, 7 5, 3 75, 1 9, 1 0, 0 5 and 0 25 μmol/L, respectively. The oligonucleotide microarray was fabricated by dotting specimen instrument. Three sorts Cy5 labeled PCR objective segment of gyrA gene were hybridized with the probes, respectively, and their fluorescence signal intensities were noted. Results The fluorescence signal of primer marker method was the strongest, terminal transfer marker method was the lowest. In random inleakage marker method, when dTTP/Cy5 dUTP was equal to 10∶1, the fluorescence signal was the best. In terminal transfer marker method, the fluorescence signals were closer when reaction times were 60 min and 120 min. When probe concentration was more than 31 μmol/L, the fluorescence signal reached flat roof location. Conclusion We definitude the impact of three fluorescence marker methods to oligonucleotide microarray hybridize signal that help us to choose better method.