摘要
探讨了2种不同硅烷化试剂3-氨基丙基三甲氧基硅烷、三甲氧基丙基二乙烯基三胺及其不同处理时间对寡核苷酸探针固定效率的影响.结果表明3％的3-氨基丙基三甲氧基硅烷的95％丙酮溶液处理2h,然后用5％戊二醛溶液处理1 h,可以获得很好的杂交信号.对比polyT15空间子,含有相同寡核苷酸序列的PEG6、PEG12空间子能使杂交信号增强6、8倍.在以pH值9-11的碳酸盐缓冲液为点样液时,杂交信号随着pH值的逐渐升高而略有下降.寡核苷酸探针的浓度和荧光标记样品的浓度影响杂交结果,当寡核苷酸探针的浓度为25μmol、所用长度为110bp的荧光标记的PCR产物为2μL,经杂交液6×SSEPT稀释至10μL时,可以获得很好的杂交信号.
Silanization of clean glass slides with 3% AFTES (3'-aminopropyltrimethoxysilane) or 3% DETA (trimethoxysi-lylpropyldiethylenetriamine) was performed by incubation with different time respectively, followed by disposal with 5% glutaralde-hyde in water for 1 hour. The results showed that incubation with 3% AFTES (dissolved by 95% acetone) for 2 hours was enough to get strong fluorescence signal. Compared with polyT15 spacer, probes with PEG6 and PEG12 spacer could improve signal intensity to 6-and 8-folds. When using carbonate buffer with 9-11 pH value as spotting solution, there's no significant decrease of signal intensities as pH value grows up. Best signal intensity could be produced when 25μM oligonucleotide probe hybridized with 2μL fluorescence labeled 110bp asymmetry PCR product which was diluted to 10 μL by 6 × SSEPT buffer.
出处
《山东大学学报（理学版）》
CAS
CSCD
北大核心
2004年第3期92-96,共5页
Journal of Shandong University(Natural Science)
基金
山东省科技厅重点攻关项目基金资助(003100104)
关键词
硅烷化
空间子
寡核苷酸探针
杂交
Silanization
Spacer
Oligonucleotide probe
Hybridization
