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Fluoride-cleavable biotinylation phosphoramidite for 5'-end-labeling and affinity purification of synthetic oligonucleotides.

Abstract：

:A fluoride-cleavable phosphoramidite for biotinylation was designed, synthesized and coupled efficiently to the 5'-end of DNA on an automatic synthesizer. The diisopropylsilyl acetal functionality was used to link the biotin moiety through a tertiary hydroxide group to the 5'-end of DNA. This linkage proved to be completely stable under certain post-synthetic DNA cleavage/deprotection conditions [0.05 M K(2)CO(3) in MeOH, room temperature, 24 h and MeNH(2) (approximately 40%)/NH(4)OH (approximately 29%), 1:1 v/v, 65 degrees C, 30 min] while it can be readily broken by fluoride ion, releasing unmodified DNA. To demonstrate the use of this DNA biotinylation method, we applied this method in affinity purification of synthetic DNA. As revealed by HPLC analysis, biotinylated full-length DNA can be efficiently attached to NeutrAvidin coated microspheres, and failure sequences can be readily removed. Subsequent treatment of the microspheres with pyridine/HF released high quality full-length unmodified DNA in good yield.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Fang S,Bergstrom DE

doi

10.1093/nar/gkg130

keywords:

subject

Has Abstract

pub_date

2003-01-15 00:00:00

pages

708-15

issue

2

eissn

0305-1048

issn

1362-4962

journal_volume

31

pub_type

杂志文章

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