Consolandi Clarissa, Castiglioni Bianca, Bordoni Roberta, Busti Elena, Battaglia Cristina, Bernardi Luigi Rossi, De Bellis Gianluca
Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, LITA, Via Fratelli Cervi 93, 20090 Segrate Mi, Italy.
Nucleosides Nucleotides Nucleic Acids. 2002;21(8-9):561-80. doi: 10.1081/NCN-120015069.
In this report we describe two robust procedures for oligonucleotide microarray preparation based on polymeric coatings. The proposed chemical approaches include: 1) a glass functionalisation step with appropriate silanes (gamma-aminopropyltriethoxysilane-APTES or 3-glycidoxypropyltrimethoxysilane-GOPS), 2) a coating step using polymers (poly-L-Lysine or poly(acrylic acid-co-acrylamide) copolymer) covalently bound to the modified glass and 3) a surface activation step to allow for the attachment of amino-modified oligonucleotides. Results obtained using these chemistries in oligo microarray preparation show: 1) an overall high loading capacity and availability to hybridisation against targets, 2) a good uniformity, 3) resistance to consecutive probing/ stripping cycles, 4) stability to thermal cycles, 5) effectiveness in hybridisation-mediated mutation detection procedures and 6) the possibility to perform enzymatic reactions, such as ligation.
在本报告中,我们描述了两种基于聚合物涂层的用于寡核苷酸微阵列制备的稳健方法。所提出的化学方法包括:1)用合适的硅烷(γ-氨丙基三乙氧基硅烷 - APTES或3 - 缩水甘油氧基丙基三甲氧基硅烷 - GOPS)进行玻璃功能化步骤,2)使用与改性玻璃共价结合的聚合物(聚-L-赖氨酸或聚(丙烯酸 - 共 - 丙烯酰胺)共聚物)进行涂层步骤,以及3)表面活化步骤以允许氨基修饰的寡核苷酸附着。在寡核苷酸微阵列制备中使用这些化学方法获得的结果表明:1)对靶标的杂交具有总体高负载能力和可用性,2)良好的均匀性,3)对连续的探测/洗脱循环具有抗性,4)对热循环具有稳定性,5)在杂交介导的突变检测程序中有效,以及6)进行诸如连接等酶促反应的可能性。