Nanofluidics - The Nanobiotechnology - page 431

can.only.handle.relatively.short.DNA.fragments..To.cut.down.the.cost.and.improve.the.
throughput,.simpler.and.quicker.DNA.mapping.techniques.are.eagerly.pursued.
If.a.DNA.is.irst.uniformly.stretched,.then.we.can.determine.the.number.of.base.pairs.
by.optically.measuring.the.length.of.each.fragment.cut.by.the.restriction.enzyme..This.is.
called.optical.mapping.and.does.not.require.prior.sequence.knowledge..It.was.invented.
by.Schwartz.and.coworkers..The.original.idea.was.to.immobilize.elongated.and.labeled.
DNAs.in.gelling.agarose.(Schwartz.et.al..1993)..The.improved.second-generation.of.optical.
mapping.is.to.stretch.and.immobilize.DNA.molecules.on.a.positively.charged.surface.(Cai.
et.al..1995;.Samad.et.al..1995;.Aston.et.al..1999)..Optical.restriction.mapping.can.also.be.car-
ried.out.after.stretching.DNA.molecules.on.a.hydrophobic.surface.with.a.receding.menis-
cus.(Yokota.et.al..1997)..However,.stretched.DNA.molecules.are.randomly.distributed.on.
the. surface. and. their. stretching. amounts. are. not. uniform;. thus,. this. improved. optical.
mapping.still.faces.the.accuracy.problem..A.possible.solution.might.use.DNA.combing.on.
patterned.surfaces.as.mentioned.in.Section.18.2.3..This.method.can.uniformly.stretch.and.
align.DNA.molecules,.and.thus,.it.is.more.accurate.with.optical.mapping.
The.stretching.of.single.DNA.molecules.in.free.solutions.has.been.extensively.reported.
in. microluidics. (Perkins. et. al.. 1997;. Smith. and. Chu. 1998;. Smith. et. al.. 1999;. Juang. et. al..
2004),.and.fundamental.studies.have.been.carried.out.to.better.understand.their.dynam-
ics. under. spatially. nonuniform. ield. forces. such. as. luid. low. and. electric. ield. (Larson.
et.al..1999;.Larson.2005;.Hu.et.al..2009,.2011)..This.provides.an.alternative.to.map.DNAs.in.
free.solutions.instead.of.immobilizing.them.in.a.gel.matrix.or.on.the.surface..Nanoluidic.
devices.such.as.nanoslits.or.nanotubes.can.be.used.to.trap.and.conine.DNA.molecules.
since. the. strong. effect. of. coninement. dominates. once. the. device. size. is. much. less. than.
the.persistence.length.of.a.DNA.molecule.(Reisner.et.al..2005;.Hsieh.and.Doyle.2008)..This.
unique. property. has. been. utilized. to. map. DNA. molecules. with. restriction. enzymes. by.
Austin.and.coworkers.(Riehn.et.al..2005).
However,. restriction. mapping. is. a. slow. and. complicated. procedure.. Recently,. a. con-
tinuous. and. automated. DNA. mapping. technology. named. direct. linear. analysis. (DLA).
was.reported.without.using.restriction.enzymes.(Chan.et.al..2004)..This.method.stretches.
single.DNA.molecules.bound.with.site-speciic.luorescent.tags.in.tapered.microchannels.
for.single-molecule.mapping..Figure.18.4.shows.the.schematics.of.DLA.technology..First,.
DNA.molecules.bound.with.luorescent.peptide.nucleic.acids.(PNAs).tags.are.driven.by.
luid.low.and.prestretched.in.micropillar.arrays;.then,.they.go.through.a.tapered.micro-
channel.to.achieve.nearly.full.stretching..Finally,.these.stretched.DNAs.travel.through.the.
spots.where.the.excitation.and.detection.of.PNA.tags.are.achieved.by.focused.laser.light..
The.further.miniaturization.of.this.device.to.nanoscale.is.fairly.straightforward,.although.
there.might.be.some.technical.barriers..However,.the.optimal.design.of.such.a.micro-.or.
nanoluidic.device.is.dependent.on.the.size.of.mapped.DNA.molecules,.which.need.to.be.
fully.stretched.(of.course,.there.are.always.under-.or.overstretched.DNAs.due.to.the.poly-
mer.individualism,.but.those.DNAs.are.not.used.in.mapping)..This.is.probably.the.biggest.
limitation.of.the.DLA.technique.and.this.means.that.a.luidic.device.might.be.only.used.
for.DNAs.with.size.falling.in.a.certain.range.
At.the.end.of.this.section,.one.DNA.mapping.method.deserves.mentioning,.and.this.is.
nanopore.technology,.which.has.used.natural.α-hemolysin.biopores.and.artiicial.solid-
state. or. polymeric. nanopores. to. demonstrate. the. ability. to. detect. and. characterize. indi-
vidual.nucleotide.acids.(Kasianowicz.et.al..1996;.Dreamer.and.Akeson.2000;.Dreamer.and.
Branton. 2002;. Li. et. al.. 2003;. Mara. et. al.. 2004).. This. label-free. and. high-throughput. tech-
nique. is. promising. for. low-cost. and. ultrafast. DNA. sequencing,. although. there. are. still.
some.key.technological.barriers.(Branton.et.al..2008).