Radical generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization.

Journal Article · Tue Jan 01 04:00:00 EST 2002 · Anal. Biochem.

DOI:https://doi.org/10.1016/S0003-2697(02)00388-3· OSTI ID:961433

Kelly, J J; Chernov, B N; Mirzabekov, A D; Bavykin, S G

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.

• Research Organization: Argonne National Laboratory (ANL)
• Sponsoring Organization: DOD
• DOE Contract Number: AC02-06CH11357
• OSTI ID: 961433
• Report Number(s): ANL/BTC/JA-47765
• Journal Information: Anal. Biochem., Journal Name: Anal. Biochem. Journal Issue: 2002 Vol. 311; ISSN ANBCA2; ISSN 0003-2697
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
99 GENERAL AND MISCELLANEOUS
AUTOMATION
BACILLUS
CENTRIFUGATION
COMPLEXES
DNA
EDTA
FRAGMENTATION
HYBRIDIZATION
NUCLEASES
NUCLEIC ACID HYBRIDIZATION
NUCLEIC ACIDS
OLIGONUCLEOTIDES
PHENANTHROLINES
RADICALS
RNA
SAMPLE PREPARATION
SPECIFICITY