Subtractive hybridization of biotinylated DNA in phenol emulsion.
A method of subtractive hybridization using biotinylated DNA and phenol emulsion reassociation technique (PERT) has been proposed. A possibility of combining these techniques has been shown for the first time. The effect of biotinylation degree on the formation of water-insoluble Bio-DNA aggregates was studied. The conditions when Bio-DNA aggregation is actually absent were revealed. A possible use of the above method in hybridization experiments in a wide range of DNA concentrations has been shown. The time of hybridization was 0.5-1 h. The method was approved on a model system, and its possible application for the enrichment of rare mRNA was shown. No less than 300-fold enrichment is achieved for a rare transcript (IL-2) in three cycles of subtractive hybridization.
Laman
A G
AG
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Region, Moscow, Russian Federation.
Kurjukov
S G
SG
Bulgakova
E V
EV
Anikeeva
N N
NN
Brovko
F A
FA
D019921:Biotinylation; D004247:DNA / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis; D019169:Jurkat Cells; D009693:Nucleic Acid Hybridization; D019800:Phenol / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors
D018076:DNA, Complementary; D007376:Interleukin-2; D012333:RNA, Messenger; D019800:Phenol; D004247:DNA