Nucleic Acids Research, 2001, Vol. 29, No. 13 e66
© 2001 Oxford University Press
Reproducible and inexpensive probe preparation for oligonucleotide arrays
We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.
* To whom correspondence should be addressed at: Department of Radiation Therapy, Longwood Radiation Oncology Center, Brigham and Women’s Hospital, Level L2, 75 Francis Street, Boston, MA 02115, USA. Tel: +1 617 6326905; Fax: +1 617 6326900; Email: mmakrigiorgos{at}partners.org