TY - JOUR
T1 - Immobilization of oligonucleotides with homo-oligomer tails onto amine-functionalized solid substrates and the effects on hybridization
AU - Pierik, Anke
AU - Dijksman, J. Frits
AU - Lub, Johan
AU - Stapert, Henk R.
AU - Broer, Dirk J.
PY - 2010/2/15
Y1 - 2010/2/15
N2 - Microarrays have become important tools for the detection and analysis of nucleic acid sequences. Photochemical (254 nm UV) DNA immobilization onto amine-functionalized substrates is often used in microarray fabrication and Southern blots, although details of this process and their effects on DNA functionality are not well understood. By using Cy5-labeled model oligonucleotides for UV immobilization and Cy3-labeled complementary sequences for hybridization, we measured independently the number of immobilized and hybridized oligonucleotides on the microarray surface. By using a two-color fluorescence LED setup and a novel method to compile the data, a full analysis has been made of the effects of oligonucleotide composition (length and sequence) on both immobilization and hybridization. Short homo-oligomer sequences (tails) of uracils, thymines, and, to a limited extent, guanines attached to a hybridization sequence improve immobilization. We propose a possible mechanism explaining the grafting of these nucleotides to amine-functionalized substrates, and we found evidence that the DNA backbone is possibly involved in the immobilization process. Hybridization, on the other hand, greatly improves as a function of tail length regardless of tail composition. On the basis of statistical arguments, the probes increasingly bind via their tail, with the hybridization sequence becoming more accessible to its complement. We conclude that all tails, sequence independent, improve hybridization signals, which is caused by either improved immobilization (especially thymine and uracil) or improved hybridization (most pronounced with guanine tails).
AB - Microarrays have become important tools for the detection and analysis of nucleic acid sequences. Photochemical (254 nm UV) DNA immobilization onto amine-functionalized substrates is often used in microarray fabrication and Southern blots, although details of this process and their effects on DNA functionality are not well understood. By using Cy5-labeled model oligonucleotides for UV immobilization and Cy3-labeled complementary sequences for hybridization, we measured independently the number of immobilized and hybridized oligonucleotides on the microarray surface. By using a two-color fluorescence LED setup and a novel method to compile the data, a full analysis has been made of the effects of oligonucleotide composition (length and sequence) on both immobilization and hybridization. Short homo-oligomer sequences (tails) of uracils, thymines, and, to a limited extent, guanines attached to a hybridization sequence improve immobilization. We propose a possible mechanism explaining the grafting of these nucleotides to amine-functionalized substrates, and we found evidence that the DNA backbone is possibly involved in the immobilization process. Hybridization, on the other hand, greatly improves as a function of tail length regardless of tail composition. On the basis of statistical arguments, the probes increasingly bind via their tail, with the hybridization sequence becoming more accessible to its complement. We conclude that all tails, sequence independent, improve hybridization signals, which is caused by either improved immobilization (especially thymine and uracil) or improved hybridization (most pronounced with guanine tails).
UR - http://www.scopus.com/inward/record.url?scp=76849117115&partnerID=8YFLogxK
U2 - 10.1021/ac902561w
DO - 10.1021/ac902561w
M3 - Article
C2 - 20095584
AN - SCOPUS:76849117115
SN - 0003-2700
VL - 82
SP - 1191
EP - 1199
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 4
ER -