Immobilization of Oligonucleotides with Homo-oligomer Tails onto Amine-Functionalized Solid Substrates and the Effects on Hybridization

摘要

Microarrays have become important tools for the detectionnand analysis of nucleic acid sequences. Photochemicaln(254 nm UV) DNA immobilization onto amine-functionalizednsubstrates is often used in microarray fabricationnand Southern blots, although details of this process andntheir effects on DNA functionality are not well understood.nBy using Cy5-labeled model oligonucleotides for UVnimmobilization and Cy3-labeled complementary sequencesnfor hybridization, we measured independently the numbernof immobilized and hybridized oligonucleotides on thenmicroarray surface. By using a two-color fluorescencenLED setup and a novel method to compile the data, a fullnanalysis has been made of the effects of oligonucleotidencomposition (length and sequence) on both immobilizationnand hybridization. Short homo-oligomer sequencesn(tails) of uracils, thymines, and, to a limited extent,nguanines attached to a hybridization sequence improvenimmobilization. We propose a possible mechanism explainingnthe grafting of these nucleotides to amine-functionalizednsubstrates, and we found evidence that the DNAnbackbone is possibly involved in the immobilizationnprocess. Hybridization, on the other hand, greatly improvesnas a function of tail length regardless of tailncomposition. On the basis of statistical arguments, thenprobes increasingly bind via their tail, with the hybridizationnsequence becoming more accessible to its complement.nWe conclude that all tails, sequence independent,nimprove hybridization signals, which is caused by eithernimproved immobilization (especially thymine and uracil)nor improved hybridization (most pronounced with guaninentails).