Comparison of Different Methods for Preparing Single Stranded DNA for Oligonucleotide Microarray

Abstract

Single stranded DNAs (ssDNA) were crucial reagents for nucleic acid analysis on oligonucleotide microarrays. Methods for preparing ssDNAs have been reported, but the systematic study about them was still lacking now, especially for oligonucleotide microarrays. Using oligonucleotide microarrays, some of the most popular methods were compared in this report, including asymmetric polymerase chain reaction (PCR), magnetic beads based method, denaturation of double stranded DNA (dsDNA) by heating or by alkaline treatment. All the ssDNA targets prepared by the four methods were applied to oligonucleotide microarrays. The results indicate that the ssDNA targets generated by asymmetric PCR and magnetic beads based method have shown high sensitivity and specificity in hybridization, but those generated by denaturation of dsDNA via heating or alkaline treatment produced serious false negative signals under the same hybridization conditions. Considering the simplicity and the cost of the method, asymmetric PCR is a more favorable approach for oligonucleotide microarrays. In view of the fact that most of the present asymmetric PCR procedures were in two steps, and a purification process was necessary before the second step amplification was carried out, a one-step asymmetric PCR was attempted in this work. The results demonstrate that it was a preferred approach for ssDNA preparation for oligonucleotide microarrays use.