Biomedical Engineering Reference
In-Depth Information
100
90
80
70
60
50
40
30
100% GC
50% GC
20
10
0
0
5
10
15
20
25
30
35
40
Length of DNA oligos
Figure 1.2
(See companion CD for color igure.) . Calculated. melting. points. for. duplex. DNA. (250.nM). in. 10.mM. MgCl 2 ,.
50.mM. NaCl,. pH. 8. buffer.. Melting. point. is. highly. dependent. on. the. GC. content. of. the. sequence. (50%. GC.
content.=.red. squares,. 100%. GC. content.=.blue. diamonds).. (Data. from. http://www.idtdna.com/analyzer/
Applications/OligoAnalyzer/,.a.free.web.calculator.for.DNA.melting.points,.IDT,.accessed.January.4,.2012.)
target..However,.just.because.a.reaction.is.thermodynamically.favored.does.not.mean.it.
can.happen.quickly..In.order.for.the.long.strand.to.displace.the.shorter.one.at.a.reasonable.
rate,.a.short.(4-6.bp).ss.sticky.end.that.is.complementary.to.the.long.strand.can.be.provided..
There.is.little.kinetic.impediment.for.binding.of.the.long.strand.to.this.toehold,.and.once.
it.is.anchored,.the.long.strand.can.compete.much.more.effectively.with.the.shorter.strand.
1.4 Branched DNA
1.4.1  Holliday Junction
Holliday's.classic.paper.proposed.a.mechanism.for.explaining.genetic.recombination.that.
involved. a. structure. now. called. the. “Holliday. junction”. (Holliday. 1964).. The. Holliday.
junction.is.a.crossover.structure.formed.when.two.duplex.DNA.strands.exchange.partners..
These.junctions.were.observed.by.electron.microscopy.in.1970.(Potter.and.Dressler.1970),.
and.a.detailed.molecular.structure.was.proposed.in.1972.(Sigal.and.Alberts.1972)..More.
detail. is. now. available. from. crystal. structure. studies. (a. review. in. Curr. Opt. Struct. Biol ..
summarizes. six. such. studies. [Ho. and. Eichman. 2001]).. The. key. observation. is. that. the.
Holliday. junction. can. adopt. either. a. splayed-out. coniguration,. in. which. four. duplexes.
originate. at. the. point. of. the. junction,. or. a. linked-duplexes. coniguration.. In. the. latter.
coniguration,.two.straight.duplex.segments.of.DNA.are.joined.at.the.point.of.the.junction.
by.two.oligonucleotide.strands.that.cross.over.from.one.duplex.to.the.other..This.crossover.
structure.(Figure.1.3).forms.the.basis.for.most.engineered.DNA.nanostructures.
In. 1993,. Fu. and. Seeman. mapped. out. basic. permutations. for. DNA. tiles. that. consist.
of. two. DNA. helices. held. together. by. pairs. of. crossover. sites. (Fu. and. Seeman. 1993)..
Using. denaturing. and. native. polyacrylamide. gel. electrophoresis. (PAGE). and. radical.
autofootprinting,. they. determined. that. antiparallel. arrangement. of. the. DNA. duplexes.
gave. stable. tiles. in. which. the. crossover. sites. were. well. protected. from. radical. cleavage,.
implying.that.they.were.buried.between.the.two.helices..Parallel.DNA.helices.lacked.this.
 
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