[Dye with low specificity to nucleotide sequences of DNA: use for assessing the quantity of oligonucleotides, immobilized in cells of biological microchips].
To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50 degrees C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.
Mikheĭkin
A L
AL
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia.
Chudinov
A V
AV
Iaroshchuk
A I
AI
Rubina
A Iu
AIu
Pan'kov
S V
SV
Krylov
A S
AS
Zasedatelev
A S
AS
Mirzabekov
A D
AD
D001483:Base Sequence; D004396:Coloring Agents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis; D013050:Spectrometry, Fluorescence; D013696:Temperature
D004396:Coloring Agents; D017931:DNA Primers; D004247:DNA