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Learning Outcome (LO)
LO 11: menjelaskan tahapan isolasi DNA danRNA
LO 12: menjelaskan jenis label dan carapelabelan asam nukleat
LO 1: menjelaskan prinsip hibridisasi asamnukleat
LO 1!: menjelaskan metode sekuensing DNA"anger#$oulson
LO 1%: menjelaskan metode sekuensingotomatis
&orking 'ith nucleic acids
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Metode dasar diperlukan dalam teknik
manipulasi gen: penanganan pengukuran analisis molekul asam nukleat.
Percobaan manipulasi gen membutuhkansumber asam nukleat, baik dalam bentukDNA atau RNA
Ada tiga persyaratan dasar untukmendapatkan/ mengisolasi asam nukleat:membuka sel untuk mengeluarkan asam
nukleat
pemisahan asam nukleat dari komponensel lainn a
1 solation o* DNA and RNA
LO 11: menjelaskan tahapan isolasi DNA dan RNA
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olation o* DNA and RNA
cel
lcell disruption• enzymatic degradation o cell !all material "i
present#• and detergent lysis o cell membranes
deproteinisation• phenol or phenol/chloroorm mi$tures
nucleic
acids
precipitation • isopropanol or ethanol
% a DNA preparation is re&uired, the enzymeribonuclease "RNase# can be used to digest theRNA in the preparation.
LO 11: menjelaskan tahapan isolasi DNA dan RNA
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% mRNA is needed orcDNA synthesis, aurther puri'cation can
be perormed by usingoligo"d(#)cellulose tobind the poly"A# tails oeukaryotic mRNAs "*ig.+.#.
(his gi-es substantialenrichment or mRNAand enables mostcontaminating DNA,
rRNA and tRNA to beremo-ed.
+ig 1 ,reparation o* mRNA b-a.nit- chromatograph- usingoligo(d/)0cellulose
LO 11: menjelaskan tahapan isolasi DNA dan RNA
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A maor problem encountered in many cloningprocedures is that o keeping track o the smallamounts o nucleic acid in-ol-ed.
(his problem is magni'ed at each stage o theprocess, because losses mean that the amount omaterial usually diminishes ater each step.
ne !ay o tracing the material is to label thenucleic acid !ith a radioacti-e molecule "usually adeo$ynucleoside triphosphate "dN(P#, labelled !ith+0 or +1P#, so that portions o each reaction may be
counted in a scintillation counter to determine theamount o nucleic acid present.
diolabelling o* nucleic acids
LO 12: menjelaskan jenis label dan cara pelabelan asam
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A second application o radiolabelling is in theproduction o highly radioacti-e nucleic acidmolecules or use in hybridisation e$periments.
2uch molecules are kno!n as radioacti-e probes,and ha-e a -ariety o uses "see 2ections +.3 and4.1#.
(he di5erence bet!een labelling or tracingpurposes and labelling or probes is largely one ospeci'c acti-ity, i.e . the measure o ho!radioacti-e the molecule is.
*or tracing purposes, a lo! speci'c acti-ity !illsu6ce but or probes, a high speci'c acti-ity is
necessary. %n probe preparation the radioacti-e label is usually
the high)energy β)emitter +1P. 2ome common methods o labelling nucleic acid
molecules are described belo!.LO 12: menjelaskan jenis label dan cara pelabelan asam
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21 nd labelling %n this techni&ue the enzyme polynucleotide kinase
is used to transer the terminal phosphate group o
A(P onto 7)hydro$yl termini o nucleic acidmolecules. % the A(P donor is radioacti-ely labelled, this
produces a labelled nucleic acid o relati-ely lo!speci'c acti-ity, as only the termini o each
molecule become radioacti-e "*ig. +.1#.
+ig 2 nd labelling DNA using pol-nucleotide kinase (,N)"a) DNA is dephosphorylated using hosphatase, to generate 7)0groups. "b) (he terminal phosphate o 8γ +1P9A(P "solid circle# is thentranserred to the 7 terminus by PN. (he reaction can also occur as ane$change reaction !ith 7)phosphate terminiLO 12: menjelaskan jenis label dan cara pelabelan asam
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22 Nick translation (his method relies on the ability o the enzyme DNA
polymerase % "see 2ection 3.1.1# to translate "mo-e
along the DNA# a nick created in the phosphodiesterbackbone o the DNA double heli$. Nicks may occur naturally, or may be caused by a
lo! concentration o the nuclease DNase % in thereaction mi$ture.
DNA polymerase % catalyses a strand replacementreaction !hich incorporates ne! dN(Ps into the DNAchain.
% one o the dN(Ps supplied is radioacti-e, the result
is a highly labelled DNA molecule "*ig. +.+#.
LO 12: menjelaskan jenis label dan cara pelabelan asam
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+ig Labelling DNA b- nick translation. "a# A single)strand nick is introduced into the phosphodiester backbone o aDNA ragment using DNase %. "b# DNA polymerase % thensynthesises a copy o the template strand, degrading thenontemplate strand !ith its 7;+ e$onuclease acti-ity. % 8α)+1P9dN(P is supplied this !ill be incorporated into the ne!lyLO 12: menjelaskan jenis label dan cara pelabelan asam
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Labelling b- primer e3tension (his term reers to a techni&ue !hich uses random
oligonucleotides "usually he$adeo$yribonucleotide
molecules < se&uences o si$ deo$ynucleotides# toprime synthesis o a DNA strand by DNApolymerase.
(he DNA to be labelled is denatured by heating, andthe oliognucleotide primers annealed to the single
stranded DNAs. (he leno! ragment o DNA polymerase "see
2ection 3.1.1# can then synthesise a copy o thetemplate, primed rom the +) hydro$yl group o the
oligonucleotide. % a labelled dN(P is incorporated, DNA o -ery high
speci'c acti-ity is produced "*ig. +.3#.
LO 12: menjelaskan jenis label dan cara pelabelan asam
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+ig ! Labelling DNA b- primer e3tension(oligolabelling). "a# DNA is denatured to gi-e single)strandedmolecules. "b# An oligonucleotide primer is then added to gi-e ashort double)stranded region !ith a ree +)0 group. "c# (heleno! ragment o DNA polymerase % can then synthesise acopy o the template strand rom the primer, incorporating 8α)+1P9dN(P "'lled circles# to produce a labelled molecule !ith a-er hi h s eci'c acti-it .LO 12: menjelaskan jenis label dan cara pelabelan asam
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cleic acid h-bridisation
LO 1: menjelaskan prinsip hibridisasi asam nukleat
+ig 214 /he principle o* nucleic acidh-bridisation
(his eature o DNA molecules is a critical part omany o the procedures in-ol-ed in genemanipulation and is also an essential eature o lieitsel. (hus, the simple =・ > and A ・ ( base pairing
has proound implications or li-ing systems and or
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Nucleic acid hybridisation can be used as ane$tremely sensiti-e detection method, capable o
picking out speci'c DNA se&uences rom comple$mi$tures.
?sually a single pure se&uence is labelled !ith +1Pand used as a probe.
(he probe is denatured beore use so that thestrands are ree to basepair !ith theircomplements.
(he DNA to be probed is also denatured, and isusually '$ed to a supporting membrane made rom
nitrocellulose or nylon. 0ybridisation is carried out in a sealed plastic bag
or tube at @7 or se-eral hours to allo! theduple$es to orm.
(he e$cess probe is then !ashed o5 and the degree
cleic acid h-bridisation
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(he ability to determine the se&uence o bases in
DNA is a central part o modern molecular biology,and pro-ides !hat might be considered as theultimate structural inormation.
Rapid methods or se&uence analysis !erede-eloped in the late CEs,and the techni&ue is
no! used in laboratories !orld!ide. (here are t!o main methods or se&uencing DNA. %n one method, de-eloped by Allan Ma$am and
Falter =ilbert, chemicals are used to clea-e the
DNA at certain positions, generating a set oragments that di5er by one nucleotide.
(he same result is achie-ed in a di5erent !ay in thesecond method, de-eloped by *red 2anger and Alan
>oulson, !hich in-ol-es enzymatic synthesis o DNAstrands that terminate in a modi'ed nucleotide.
NA se5uencing
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Although the end result is similar to that attained bythe chemical method, the 2angeroulson procedure
is totally di5erent rom that o Ma$am and =ilbert. %n this case a copy o the DNA to be se&uenced is
made by the leno! ragment o DNA polymerase"see 2ection 3.1.1#.
(he template or this reaction is single)stranded DNA,and a primer must be used to pro-ide the + terminusor DNA polymerase to begin synthesising the copy"*ig. +.4#.
"anger#$oulson (dideo3- or en6-matic)se5uencing
LO 1!: menjelaskan metode sekuensing DNA "anger#$oulson
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+ig 7 DNA se5uencingusing the dideo3- chaintermination ("anger#
$oulson) method "a# Aprimer is annealed to asingle)stranded templateand "b# the leno! ragmento DNA polymerase % used to
synthesise a copy o theDNA. A radiolabelled dN(P"oten 8α)+729dN(P, 'lledcircles# is incorporated intothe DNA. "c) >hain
termination occurs !hen adideo$y nucleosidetriphosphate "ddN(P# isincorporated. "d) A series oour reactions, eachcontaining one ddN(P in
addition to the our dN(PsLO 1!: menjelaskan metode sekuensing DNA "anger#$oulson
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(he production o nested ragments is achie-ed bythe incorporation o a modi'ed dN(P in each reaction.
(hese dN(Ps lack a hydro$yl group at the + position o
deo$yribose, !hich is necessary or chain elongationto proceed. 2uch modi'ed dN(Ps are kno!n as dideo$ynucleoside
triphosphates "ddN(Ps#. (he our ddN(Ps "A,=,( and > orms# are included in a
series o our reactions, each o !hich contains theour normal dN(Ps.
(he concentration o the dideo$y orm is such that it!ill be incorporated into the gro!ing DNA chain
inre&uently. Gach reaction thereore produces a series o
ragments terminating at a speci'c nucleotide, andthe our reactions together pro-ide a set o nestedragments.
LO 1!: menjelaskan metode sekuensing DNA "anger#$oulson
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%n C4@, Heroy 0ood and Hloyd 2mith automated2angerIs method.
%n this ne! se&uencing technology, radioacti-e markersare replaced !ith Juorescent ones.
Gach ddN(P terminator is tagged !ith a di5erent color oJuorophore: red, green, blue, or yello!.
(hus, instead o ha-ing to run our separate se&uencing
reactions, the reactions can be combined into one tube. (he 'rst automated se&uencer made use o a
polyacrylamide gel to resol-e the samples, a laser toe$cite the dye molecules as they reached a detectornear the end o the gel, and a computer to read theresults as a DNA se&uence.
%n this system each automated se&uencer !as able toproduce 34EE bases o se&uence per day.
(he current automated systems replace the old)style gel
!ith arrays o tiny capilliaries, each o !hich acts as aKlane.L
Automated DNA se5uencing
LO 1%: menjelaskan metode sekuensing otomatis
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A pump loads special capillaries !ith a polymer thatser-es as the separation matri$.
DNA samples in a C@)!ell plate are loaded into the
capillary array by a short burst o electrophoresis,called Kelectrokinetic inection.L (he capillary array is immersed in running bu5er
and the DNA ragments then migrate through thecapillary matri$ by size, smallest to largest.
As the DNA ragments reach the detection !indo!,a laser beam e$cites the dye molecules causingthem to Juoresce.
Gmitted light rom C@ capillaries is collected at
once, spectrally separated into the our colors andocused onto a >>D camera.
>omputer sot!are interprets the pattern o peaksto produce a graph o Juorescence intensity -ersustime "electropherogram#, !hich is then con-erted tothe DNA se&uence "*ig. 4.@#.LO 1%: menjelaskan metode sekuensing otomatis
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+igure 718 Automated DNA se5uencingLO 1%: menjelaskan metode sekuensing otomatis
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LO 1%: menjelaskan metode sekuensing otomatis
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/A/A, 9A !
H @: menelaskan enzim restriksiH : menelaskan nukleaseH 4: menelaskan polimerase
H C: menelaskan enzim yang digunakanuntuk memodi'kasi uung DNA
H 1E: menelaskan DNA ligase
uis n6im
N;/