In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element Specific for Bromacil

Table 1

SELEX scheme for selection of a bromacil-specific MRE.

Round	Positive selection	Negative selection
1	Immobilized target (IT) 48 hrs	Immobilization substrate (IS) 24 hrs
2	IT 18 hrs	IS 23 hrs
3	IT 12 hrs	IS 12 hrs
4	IT 6 hrs	IS 6 hrs
5	IT 1 hrs	6-Amino-3-ethyl methyl uracil (1st immobilized negative target INT-1) 19 hrs
6	IT 5 min	INT-1 19 hrs
7	IT 5 sec	INT-1 18 hrs
8	IT 5 sec, 1 μM bromacil competitive elution (CE)	1-Methyl uracil (2nd immobilized negative target INT-2)
9	IT 5 sec, 0.5 μM bromacil CE	IT 5 sec, CE with NT-1 5 sec
10	IT 5 sec, 0.1 μM bromacil CE	IT 5 sec, CE with NT-2 5 sec
11	IT 5 sec, 0.05 μM bromacil CE	IT 5 sec, CE with NT-1 5 sec, CE with NT-2 5 sec
12	IT 5 sec, 0.01 μM bromacil CE	IS 24 hrs
13	IT 5 sec, 0.05 μM bromacil CE

In vitro selection process for obtaining a bromacil-specific MRE. Immobilization target (IT) is bromacil bound to magnet beads. Immobilization substrate (IS) is streptavidin-coated magnetic beads plus blocked biotin reagent. Competitive elution (CE) is removal of bound ssDNA from target-coated magnetic beads by free pesticide in solution. INT-1 or NT-1 is the 1st immobilized or free negative target 6-amino-3-ethyl methyl uracil. INT-2 or NT-2 is the 2nd immobilized or free negative target 1-methyl uracil. Times listed are incubation times in hours (hrs), minutes (min), or seconds (sec).