Department of Chemistry, University of Toronto, Toronto, Canada.
Anal Chim Acta. 2010 Feb 19;661(1):103-10. doi: 10.1016/j.aca.2009.12.001. Epub 2009 Dec 6.
Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (DeltaT(m)) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1-0.6 M). Increases of DeltaT(m) when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1 bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.
最近的初步研究表明，由寡核苷酸和聚（2-羟乙基甲基丙烯酸酯）（PHEMA）组成的混合膜可以提高对 3 碱基错配（3 bpm）寡核苷酸靶标的检测选择性。现在已经完成了使用这种混合膜检测单核苷酸多态性（SNP）的选择性评估。通过考虑完全互补靶标和 SNP 的熔融曲线的锐度和熔融温度差异（DeltaT(m)）来定量确定选择性。研究了严格条件，并确定当使用中等离子强度（0.1-0.6 M）时，选择性最大化。与仅含有固定化寡核苷酸探针的表面相比，使用混合膜时 DeltaT(m) 的增加高达 3 倍。同时，对于混合膜，1 bpm 靶标的熔融曲线锐度的增加高达 2 倍。PHEMA 的共固定化导致寡核苷酸探针在表面上的更均匀分布。用 Cy3 染料标记的固定化寡核苷酸探针的荧光发射寿命测量表明，在 PHEMA 存在下，固定化寡核苷酸的微环境存在差异。
