Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization.
DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms. We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.
Kelly
John J
JJ
Northwestern University, Department of Civil Engineering, 2145 Sheridan Road, Evanston, IL 60208, USA.
Chernov
Boris K
BK
Tovstanovsky
Igor
I
Mirzabekov
Andrei D
AD
Bavykin
Sergei G
SG
D004247:DNA / Q000737:chemistry; D004492:Edetic Acid; D005296:Ferrous Compounds; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D005609:Free Radicals / Q000737:chemistry*; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009942:Organometallic Compounds; D010618:Phenanthrolines; D012313:RNA / Q000737:chemistry
C095824:5-(bromoacetamido)-1,10-phenanthroline copper complex; D005296:Ferrous Compounds; D005456:Fluorescent Dyes; D005609:Free Radicals; D009696:Nucleic Acids; D009942:Organometallic Compounds; D010618:Phenanthrolines; C055526:Fe(II)-EDTA; D012313:RNA; D004247:DNA; D004492:Edetic Acid