Wang Yijin, Wang Hong, Gao Lu, Liu Heping, Lu Zuhong, He Nongyue
Key Laboratory for Molecular and Biomolecular Electronics of Ministry of Education, Southeast University, Nanjing 210096, China.
J Nanosci Nanotechnol. 2005 Apr;5(4):653-8. doi: 10.1166/jnn.2005.086.
We reported polyacrylamide gel immobilized molecular beacon array for single nucleotide mismatch detection in this paper. Molecular beacons are oligonucleotide probes fluorescing upon hybridization to their complementary DNA/RNA targets with excellent sensitivity and high selectivity. The specially designed molecular beacon for immobilization contains a 15 base loop sequence with a 5 base pair stem, a polyT (20 bases) spacer, a 5'-end amino group for immobilization, a fluorescein in the middle of the sequence as the fluorophore, and a 3'-end DABCYL as the quencher. Between the 5'-end amino group and the stem, the polyT is used to minimize disability caused by 5'-end immobilization. The molecular beacon microarray was fabricated by a pin-based spotting robot and the hybridization was investigated by confocal microscope. A real-time hybridization process at room temperature was registered every minute for 20 min after the target solution was pumped into the hybridization cell. The result indicates that a polyacrylamide film coated glass slide provides an ideal solution-like environment for molecular beacon probes. The potential applications of this kind of molecular beacon array are mutation detection, disease mechanisms, disease diagnostics, etc. in a parallel, cost saving, and label-free detection way.
我们在本文中报道了用于单核苷酸错配检测的聚丙烯酰胺凝胶固定化分子信标阵列。分子信标是寡核苷酸探针，与它们的互补DNA/RNA靶标杂交时会发出荧光，具有出色的灵敏度和高选择性。专门设计用于固定的分子信标包含一个带有5个碱基对茎的15个碱基的环序列、一个聚T（20个碱基）间隔区、一个用于固定的5'-末端氨基、序列中间作为荧光团的荧光素以及一个3'-末端二甲基氨基偶氮苯甲酰（DABCYL）作为猝灭剂。在5'-末端氨基和茎之间，聚T用于最小化由5'-末端固定引起的失活。分子信标微阵列由基于针的点样机器人制备，并通过共聚焦显微镜研究杂交情况。在将靶溶液泵入杂交池后，在室温下每分钟记录一次实时杂交过程，持续20分钟。结果表明，涂有聚丙烯酰胺膜的载玻片为分子信标探针提供了理想的类似溶液的环境。这种分子信标阵列的潜在应用包括以平行、节省成本和无标记检测的方式进行突变检测、疾病机制研究、疾病诊断等。
