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Indirect Fluorescent Labeling of Microarray
Targets Using ImProm-II™ Reverse Transcriptase
By Herbert Kasler, Ph.D., Christopher Barker, Ph.D.,
Yanxia Hao, Ph.D. and Eric Verden, M.D.
J. David Gladstone Institutes, University of California, San Francisco
Historically, fluorescent cDNA targets used for microarray hybridization
have been produced using direct incorporation of nucleotides coupled to
bulky fluorescent dyes such as Cy®3 and Cy®5. These modified nucleotides
are not efficiently incorporated by reverse transcriptases. Moreover,
reverse transcriptases incorporate different dye-modified dNTPs at
different rates. Recently, indirect labeling of cDNA target populations,
using aminoallyl-derivatized dUTP incorporation followed by amino-coupling
of fluorescent dyes, has been gaining ground on the traditional method of
direct incorporation.
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