TM 03 Asam Nukleat (Biologi Molekuler 2014).pptx

8/21/2019 TM 03 Asam Nukleat (Biologi Molekuler 2014).pptx

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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.



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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



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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. +.+#.



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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#.



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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

LO 1: menjelaskan prinsip hibridisasi asam nukleat


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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


18/23LO 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;/

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TM 03 Asam Nukleat (Biologi Molekuler 2014).pptx

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