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Rivista di Biologia Molecolare e Biotecnologie

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Minerva Biotecnologica 2001 Dicembre;13(4):301-6

lingua: Inglese

Com­par­a­tive exam­ina­tion of ­probe label­ing meth­ods for micro­ar­ray hybrid­iza­tion

Burke D. I. 1, Woodward K. 2, Setterquist R. A. 3, Kawasaki E. S. 1

1 Pack­ard Bio­Chip Tech­nol­o­gies, Bil­ler­i­ca, MA 0182;
2 Pack­ard Bio­Chip Ven­tures, Mer­id­en, CT 06450;
3 ­Ambion, Inc., Aus­tin, TX 78744


For the detec­tion of dif­fe­ren­tial ­gene expres­sion, Pack­ard ­BioChip’s con­fo­cal ­laser ­based scan­ners are now ­capable of ana­lyz­ing micro­ar­rays ­using one to ­five wave­lengths. ­This ­allows inves­ti­ga­tors to ­choose ­among sev­er­al label­ing meth­ods. Aim of ­this ­paper is to com­pare “­direct” Cy3/Cy5 con­ju­gat­ed nucle­o­tide incor­po­ra­tions and “indi­rect” meth­ods (ami­no-­allyl and chem­i­cal medi­at­ed) for label­ing ­cDNA ­probes. We ­assessed repro­du­cible sen­si­tiv­ity of ­each ­probe prep­ar­a­tion meth­od by com­par­ing hybrid­iza­tion inten­sities for lim­it of sig­nal detec­tion. ­This was ­done ­using ­arrays of ­mixed tar­gets con­sist­ing of a seri­al­ly dilut­ed ­human spe­cif­ic ­gene frag­ment (HU1) and an undi­lut­ed E. ­Coli DNA of chlo­ram­phen­i­col ace­tyl trans­fe­rase (CAT) ­gene. We ­present pre­lim­i­nary ­results ­which sug­gest ­that ­Alexa ­dyes ­remain com­pet­i­tive giv­en ­that its dynam­ic ­range ­exceeds ­those of oth­er meth­ods.

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