Search

A new and efficient strategy for labeling of nucleic acids prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the terminal phosphates of cleaved DNA and RNA fragments with a reporter molecule bearing aryldiazomethane group.

Pyrenyldiazomethane was shown to react quantitatively and selectively at phosphate with 2'-, 3'-, and 5'-nucleotide phosphates incorporating the different nucleic bases.

We show here a new and efficient aqueous chemistry for labeling of any class of nucleic acids for their detection on DNA chip. The labels contain a diazo function as reactive moiety and biotin as detectable unit. The highly selective reaction of diazo group on the phosphate does not disrupt base pairing recognition and hybridization specificity.

An efficient and direct labeling method based on direct alkylation of nucleic acids at phosphates by aryldiazomethane derivatives is described.

DNA and RNA labeling and detection are key steps in nucleic acid testing, particularly for molecular diagnostics applications. Here, we report a new class of aryldiazomethyl labels that include in their structure a biotin moiety as a detectable unit and a nitro substituted phenyl diazomethyl as a reactive group. The greatest reactivity towards phosphates of nucleic acids, the water solubility and the stability of these new molecules were demonstrated. These very important properties, which are the main requirements for nucleic acid labeling in aqueous conditions using automated protocols within integrated diagnostic devices, make them the perfect labeling tools for hybridization-based analysis, especially for high-density DNA chips.

8-Hydroxyl-2'-deoxyguanosine (also referred to as 8-hydroxyguanine [8-OH-dG] or 7,8-dihydro-8-oxoguanine), a common DNA adduct resulting from injury to DNA via reactive oxygen species, affects the in vitro methylation of nearby cytosine moieties by the human DNA methyltransferase. The exact position of 8-OH-deoxyguanosine relative to a CpG dinucleotide appears important to this effect. Our data indicate that 8-OH-deoxyguanosine diminishes the ability of the methyltransferase to methylate a target cytosine when the 8-OH-deoxyguanosine is one or two nucleotides 3' from the cytosine, on the same strand. On the other hand 8-OH-deoxyguanosine does not diminish the ability of the enzyme to respond to a methyl director (5-methylcytosine) when the 8-OH-deoxyguanosine is on the same strand but one or two nucleotides 3' from the methyl director. Differences in methylation rates as great as 13-fold have been detected using various 8-OH-deoxyguanosine-containing oligonucleotides as substrates in methylation assays. Our findings suggest that oxidative damage of parental strand guanines would permit normal copying of methylation patterns through maintenance methylation, while oxidative damage of guanines in the nascent strand DNA would inhibit such methylation.

Synthetic oligodeoxyribonucleotide duplexes have been used to study the methylation specificity of M · Hpa II, a bacterial DNA methyltransferase. Substrates of four types were compared. A 30-mer containing a Watson-Crick paired CCGG recognition sequence was rapidly methylated at the central cytosine on each strand in the recognition sequence. A 30-mer containing an asymmetrically methylated recognition sequence, of the type transiently produced by DNA replication, was rapidly methylated at the central cytosine on the unmethylated strand. A heteroduplex containing an A-C mispair in the recognition sequence (CCGG/CCAG) was rapidly methylated at the cytosine in the mispair. A heteroduplex containing an A-C and an adjacent C-C mispair in the recognition sequence (CCGG/CCCA) was not methylated at a significant rate. The results show that M · Hpa II can tolerate a single mispair at its recognition site in a heteroduplex without loss of activity pr specificity.