寡核苷酸芯片探针固定化和杂交条件的优化

Immobilization of oligonucleotide probe on DNA chip and optimization of hybridization conditions

目的 探讨寡核苷酸芯片探针固定化的方法 ,优化杂交条件。方法 设计spacer为poly(dT) 10～ 2 0 的长度为 15～ 30mer的探针 ,与 12 2～ 10 6 7bp的荧光标记靶序列杂交 ,杂交温度选择 4 2℃～ 6 5℃ ,杂交时间选择 1～ 3h。扫描分析杂交结果 ,通过Quantarray定量分析软件进行定量。结果 较短的如 10 0bp左右的靶序列 ,spacer为poly(dT) 10 、探针为 2 0mer左右即可获得非常理想的杂交结果。较长的如 75 0bp左右的靶序列 ,较短的spacer不能获得理想的杂交结果 ,可以选择poly(dT) 15或poly(dT) 2 0 ,探针应为 2 0mer以上 ,2 5mer和 30mer均能获得较好的杂交结果。 1k以上的靶序列与 15～ 30mer的探针杂交信号较弱。

Objective To investigate the immobilization of oligonucleotide probes and optimizition of the hybridization conditions.Methods The probes of 15 30 mer length with poly(dT) 10～20 as spacer were designed,and then hybridized with the fluorescence labeled target sequence of 122～1067 bp at temperature of 42℃～65℃ for 1～3 h.The chip was scaned to acquire the image and the signols were quantified by Quantarray software.Results For the shorter target circa 100 bp,the probe of 20 mer with spacer of poly(dT) 10 was enough to gain perfect result,while for the longer one circa 750 bp or so the length of probe should be 25 or 30mer and the spacer should be more than 15.In the case of that the sequence of target was longer than 1k the hybridization with probes of 15～30 mer was notable to gain well image.Conclusion Elevated hybridization efficiency can be gained by optimization of hybridization conditions.