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.
Zhang
Y
Y
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
Price
B D
BD
Tetradis
S
S
Chakrabarti
S
S
Maulik
G
G
Makrigiorgos
G M
GM
D001205:Ascorbic Acid / Q000378:metabolism; D019921:Biotinylation; D017046:Cost Savings; D015342:DNA Probes / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004492:Edetic Acid / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D005363:Ficusin / Q000378:metabolism; D005434:Flow Cytometry; D011564:Furocoumarins; D017353:Gene Deletion; D020869:Gene Expression Profiling / Q000191:economics / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D008863:Microspheres; D009369:Neoplasms / Q000235:genetics; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics* / Q000378:metabolism; D010777:Photochemistry; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D015342:DNA Probes; D018076:DNA, Complementary; D005296:Ferrous Compounds; D011564:Furocoumarins; D009841:Oligonucleotides; C055526:Fe(II)-EDTA; D017665:Hydroxyl Radical; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D005363:Ficusin; D001205:Ascorbic Acid