Reproducible and inexpensive probe preparation for oligonucleotide arrays.
Nucleic Acids Res
; 29(13): E66-6, 2001 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-11433042
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligonucleotídeos
/
Sondas de DNA
/
Análise de Sequência com Séries de Oligonucleotídeos
Tipo de estudo:
Guideline
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2001
Tipo de documento:
Article
País de afiliação:
Estados Unidos