Biomedical Engineering Reference
In-Depth Information
DNA/RNA
library
+
Target molecule
+
Figure 2.1
(See companion CD for color igure.)
.General.procedure.of.SELEX..The.initial.library.contains.10
12
-10
14
.single-
stranded.DNA.or.RNA.molecules..Each.selection.cycle.involves.incubation,.partition,.and.ampliication.
library. with. target. molecules,. removing. nonbinding. sequences,. recovering. the. binding.
sequences,.and.amplifying.the.binding.sequences..The.process.begins.with.the.generation.
of.a.single-stranded.DNA.or.RNA.library,.which.typically.consists.of.10
14
-10
15
.randomized.
DNA. or. RNA. oligonucleotides.. These. oligonucleotides. are. comprised. of. three. parts:.
two.primer.binding.regions.at.both.3′.and.5′.ends.for.ampliication.and.one.randomized.
nucleotides.region.for.selection..During.the.SELEX.process,.target.molecules.are.mixed.with.
an.oligonucleotide.library.in.a.speciic.binding.solution..The.mixture.is.incubated.under.
certain.conditions.(e.g.,.on.ice,.at.room.temperature,.at.37°C).for.a.short.period.of.time.to.
allow.for.the.molecular.recognition.between.the.oligonucleotides.and.the.target.molecules..
After.this.step,.free.and.weakly.binding.oligonucleotides.can.be.removed.through.a.partition.
method.. The. binding. sequences. are. eluted. from. the. target-oligonucleotide. complex. and.
are.ampliied.by.polymerase.chain.reactions.(PCR).to.generate.an.enriched.pool.as.the.new.
library..In.order.to.avoid.false.positive.results,.it.is.also.important.to.perform.counterselection.
and.negative.selection.to.remove.the.sequences.that.bind.to.similar.molecules.or.bind.to.the.
ligand.support..The.whole.selection.process.usually.has.8-12.cycles.
In. the. SELEX. process,. the. most. critical. step. is. the. separation. of. tightly. binding.
oligonucleotides. from. the. free. or. weakly. binding. oligonucleotides.. When. SELEX. was.
initially. developed,. the. target. molecules. were. introduced. into. the. system. without.
immobilization,.and.nitrocellulose.iltration.was.used.to.separate.target-oligonucleotide.
complexes. (Ellington. and. Szostak. 1990,. Tuerk. and. Gold. 1990).. Gel. mobility. shift. (Smith.
et. al.. 1995). and. density. gradient. centrifugation. (Zhang. and. Anderson. 1998). are. also.
employed. to. separate. unimmobilized. target-oligonucleotide. complexes.. To. facilitate.
separation,.target.molecules.are.also.immobilized.to.magnetic.beads.(Lupold.et.al..2002),.
antibody-functionalized. afinity. beads. (Tsai. et. al.. 1991),. titer. plates. (Rhodes. et. al.. 2000),.
or. chromatography. columns. (Nieuwlandt. et. al.. 1995,. Muller. et. al.. 2008).. Except. for. the.
development.of.different.separation.techniques.to.accelerate.SELEX.process,.other.SELEX.
