Byproduct with altered fluorescent properties is formed during standard deprotection step of hexachlorofluorescein labeled oligonucleotides.
HEX-labeled oligonucleotides obtained via typical synthetic protocols may contain more than 15% of material with altered spectral characteristics. We discovered hexachlorofluorescein residue transformation unknown earlier for standard DNA ammonolysis step. HEX residue reacts with ammonium hydroxide yielding acridine derivative, which has differed UV-VIS and fluorescent properties compared to HEX. Therefore, for critical bioassays where sensitivity and/or fluorescent signal differentiation (e.g., in quantitative or multiplexed assays) are essential, the careful RP-HPLC purification step is required.
Chuvilin
A N
AN
Research Institute of Physical-Chemical Medicine, Moscow, Russian Federation. achuvilin@yandex.ru
Serebryakova
M V
MV
Smirnov
I P
IP
Pozmogova
G E
GE
D000166:Acridines / Q000138:chemical synthesis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005452:Fluoresceins / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*
D000166:Acridines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009841:Oligonucleotides