title: Hybridisation of N4-methylcytosine-containing amplicons on DNA microarrays.
abstract: 5'-Cy5-labelled PCR amplicons containing the analogue base, N(4)-methylcytosine, instead of cytosines were compared in microarray hybridisation experiments with the corresponding amplicons containing the canonical set of bases, with respect to the intensity of the fluorescence signal obtained, and cross hybridisation to non-corresponding probes. In general, higher hybridisation temperatures resulted in reduced signal intensities, particularly in the case of the N(4)-methylcytosine containing amplicons. At the lower hybridisation temperatures tested (40 °C, 30 °C), these modified amplicons gave about equal or stronger fluorescence signal than the corresponding regular amplicons. With the two GC-richest amplicons tested, in one instance the N(4)-methylated target gave a dramatically higher signal intensity than the unmodified amplicon, interpreted as reflecting the reduced formation of hairpin structures in the target sequence, due to the lower thermodynamic stability of the G:N(4)-methylC base pair, making the target more accessible, while in the other case no hybridisation was observed with either version of the amplicon, probably due to interference from a G-tetrad structure. Both for the regular and the N(4)-methylated amplicons, no significant cross hybridisation was seen in these experiments.
authors.affiliation: CICATA, Instituto Politécnico Nacional, Querétaro, Querétaro 76090, Mexico.
authors.forename: Cyntia R
authors.initials: CR
authors.lastname: Flores-Juárez
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Leberre Anton
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Trévisiol
authors.affiliation: Instituto de Neurobiología, Universidad Autónoma de México, Juriquilla, Querétaro 76230, Mexico.
authors.forename: Anaid
authors.initials: A
authors.lastname: Antaramian
authors.affiliation: CICATA, Instituto Politécnico Nacional, Querétaro, Querétaro 76090, Mexico.
authors.forename: Eva
authors.initials: E
authors.lastname: González-Jasso
authors.affiliation: CICATA, Instituto Politécnico Nacional, Querétaro, Querétaro 76090, Mexico. Electronic address: rpless@ipn.mx.
authors.forename: Reynaldo C
authors.initials: RC
authors.lastname: Pless
mesh_terms: D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
chemical_list: C039052:N(4)-methylcytosine; D003596:Cytosine
keywords: GC-rich DNA; Microarrays; N(4)-methylcytosine; PCR; dCTP analogue