POLYMERASE CHAIN REACTION (2).pptx

8/17/2019 POLYMERASE CHAIN REACTION (2).pptx

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POLYMERASE CHAIN

REACTION (2)

Sagung Chandra Yowani

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PCRMelting

94 oC

Melting

94 oCAnnealing

Primers

50 oC

Extension

72 oC

T e m p e r a t u r e

100

0

50

T i m e

30x

3’5’

5’3’5’

5’

5’

5’

5’

5’

2


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PCRMelting

94 oC

Melting

94 oCAnnealing

Primers

50 oC

Extension

72 oC


100

0

50

T i m e

30x

3’5’

5’3’5’

5’

5’

5’

5’

5’

Heat

Heat

3


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PCRMelting

94 oC

Melting

94 oCAnnealing

Primers

50 oC

Extension

72 oC


100

0

50

T i m e

30x

3’5’

5’3’5’

5’

5’

5’

5’

5’

5’

5’

5’

5’

4


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Fragments ofdefined length

PCR Melting

94 oC

Melting

94 oCAnnealing

Primers

50 oC

Extension

72 oC


100

0

50

T i m e

30x

3’5’

5’3’5’

5’

5’

5’

5’

5’

5’

5’

5’

5’


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Mor! C"#$!% & Mor! 'NA

Number of cycles

0 10 15 20 25 30

Size

Marker


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PCR M!hod%

1. Multiplex PCR

2. Nested PCR

3. Reverse transcriptase PCR & Real timePCR

*


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Mu$i+$!, PCR

• A-+$i.#aion o/ %o-! %!0u!n#!% inon! i-! running

• I %hou$d ! running a h! %a-!!-+!raur!

• Th! %i! o/ PCR +rodu# -u% !di!r!n#!

• A-+$i.a%i !!ra+a uruan da$a-%au wau !r5a$an

• Iu haru% !r5a$an +ada %uhu "ang%a-a 7


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Multiplex PCR• Multiplex PCR is a variant of PCR which enalin! simultaneous

amplification of man" tar!ets of interest in one reaction " usin! more than

one pair of primers.

• Multiplex PCR adalah varian dari PCR "an! memun!#in#an amplifi#asi

simultan dari an"a# tar!et "an! menari# di satu rea#si den!an

men!!una#an leih dari satu pasan! primer.

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Nested PCR

• Two pairs $instead of one pair% of PCR primers are used to amplif" a

fra!ment.

• irst pair 'amplif" a fra!ment similar to a standard PCR. (econd pair of

primers'nested primers $as the" lie ) are nested within the first fra!ment%

ind inside the first PCR product fra!ment to allow amplification of a

second PCR product which is shorter than the first one.• *ua pasan! $u#an satu pasan!% primer PCR di!una#an untu#

memper#uat fra!men.

• Pasan!an pertama 'amplif" fra!men mirip den!an PCR standar.

Pasan!an #edua primer'ersaran! primer $#arena mere#a erohon! )ersaran! di dalam fra!men pertama% men!i#at dalam fra!men produ#

pertama PCR untu# memun!#in#an amplifi#asi produ# PCR #edua "an!

leih pende# dari "an! pertama.

1:


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Nested PCR

• Advantage' +er" low proailit" of nonspecific amplification

• +er" usefull for a ver" small amount of *N,

• ,dvanta!e' proailitas san!at rendah amplifi#asi non

spesifi#

• (an!at er!una untu# -umlah "an! san!at #ecil dari *N,

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12


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Why real time PCR ?

•

QUANTITATION OF mRNA – northern blotting

– ribonuclease protection assay

– in situ hybridization

– RT-PCR

• most sensitive

• can discriminate closely related mRNAs

•

technically simple• but difficult to get truly quantitative results using

conventional !R

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Real-Time PCR

Real-time PCR monitors the fluorescence emitted during the

reaction as an indicator of amplicon production at each PCR cycle

(in real time) as opposed to the endpoint detection

Real-time PCR memonitor fluoresensi dipancarkan selama reaksisebagai indikator produksi amplikon pada setiap siklus PCR

(secara real time) yang bertentangan dengan deteksi titik akhir

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• Tradiiona$ PCR ha% ad;an#!d /ro- d!!#ion a h! !nd 1


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PCR APPLICATION

• PCR ha% !!n a++$i!d o a $arg! nu-!r o/ -!di#a$ +ro#!dur!%?

• Th! .r% a++$i#aion o/ PCR wa% /or genetic testing@ wh!r! a %a-+$! o/'NA i% ana$"!d /or h! +r!%!n#! o/ g!n!i# di%!a%! -uaion%>Pro%+!#i;! +ar!n% #an ! !%!d /or !ing g!n!i# #arri!r%@ or h!ir#hi$dr!n -igh ! !%!d /or a#ua$$" !ing a!#!d " a di%!a%!> 'NA%a-+$!% /or Pr!naa$ !%ing #an ! oain!d " a-nio#!n!%i%@ #horioni#

;i$$u% %a-+$ing@ or !;!n " h! ana$"%i% o/ rar! /!a$ #!$$% #ir#u$aing in h!-oh!r% $ood%r!a-

• PCR !$ah di!ra+an unu %!5u-$ah !%ar +ro%!dur -!di%?

• A+$ia%i +!ra-a dari PCR ada$ah unu +!ngu5ian g!n!i@ di -ana%a-+!$ 'NA diana$i%i% unu !hadiran -ua%i +!n"ai g!n!i>

orang ua #a$on da+a diu5i unu -!n5adi o+!raor g!n!i@ aauana


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PCR APPLICATION

• > PCR ana$"%i% i% a$%o !%%!nia$ oPr!i-+$anaion g!n!i# diagno%i%@wh!r! indi;idua$ #!$$% o/ a d!;!$o+ing

!-r"o ar! !%!d /or -uaion%>

• Ana$i%i% PCR 5uga +!ning unudiagno%i% g!n!i Pr!i-+$anaion@ di

-ana %!$


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PCR APPLICATION

• PCR #an a$%o ! u%!d a% +ar o/ a %!n%ii;! !% /or tissue typing@ ;ia$ oorgan ran%+$anaion> A% o/ 2::7@ h!r! i% !;!n a +ro+o%a$ o r!+$a#! h!radiiona$ aniod" B" u%ing PCR<a%!d !%% o %ud" h!%! -uaion%@ h!ra+" r!gi-!n% #an %o-!i-!% !indi;idua$$" #u%o-i!d o a +ai!n>

• Ana$i%i% PCR 5uga +!ning unu diagno%i% g!n!i Pr!i-+$anaion@ di-ana %!$ Padaahun 2::7@ ahan ada u%u$an unu -!ngganian !% !ra%i%aniodi radi%iona$ unu go$ongan darah d!ngan !% !ra%i% PCR

• Ban"a !nu an!r -!$iaan +!ruahan ! onog!n> '!ngan-!nggunaan !% !ra%i% PCR unu -!-+!$a5ari -ua%i ini@ r!5i-!n!ra+i adang

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http://en.wikipedia.org/wiki/Tissue_typinghttp://en.wikipedia.org/wiki/Organ_transplantationhttp://en.wikipedia.org/wiki/Blood_typehttp://en.wikipedia.org/wiki/Oncogenehttp://en.wikipedia.org/wiki/Oncogenehttp://en.wikipedia.org/wiki/Blood_typehttp://en.wikipedia.org/wiki/Organ_transplantationhttp://en.wikipedia.org/wiki/Organ_transplantationhttp://en.wikipedia.org/wiki/Tissue_typing


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PCR APPLICATION

• Chara#!riaion and d!!#ion o/ in/!#iou%di%!a%! organi%-% ha;! !!n r!;o$uioni!d" PCR?

• Th! Human Immunodefciency i!us (orHI)@ o/ aniodi!% o h! ;iru% #ir#u$aing inh! $ood%r!a-> How!;!r@ aniodi!% dona++!ar uni$ -an" w!!% a/!r in/!#ion@

-a!rna$ aniodi!% -a% h! in/!#ion o/ an!worn@ and h!ra+!ui# ag!n% o .ghh! in/!#ion don a!# h! aniodi!%

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http://en.wikipedia.org/wiki/HIVhttp://en.wikipedia.org/wiki/HIV


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PCR APPLICATION

• PCR !%% ha;! !!n d!;!$o+!d ha#an d!!# a% $i$! a% on! ;ira$g!no-! a-ong h! 'NA o/ o;!r

:@::: ho% #!$$%> In/!#ion% #an !d!!#!d !ar$i!r@ dona!d $ood #an! %#r!!n!d dir!#$" /or h! ;iru%@

n!worn% #an ! i--!dia!$" !%!d/or in/!#ion@ and h! !!#% o/ani;ira$ r!a-!n% #an ! 0uani.!d>

2:

http://en.wikipedia.org/wiki/HIV_testhttp://en.wikipedia.org/wiki/Viral_loadhttp://en.wikipedia.org/wiki/Viral_loadhttp://en.wikipedia.org/wiki/HIV_test


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PCR APPLICATION

• So-! di%!a%! organi%-%@ %u#h a% ha /orTu"e!culosis@ ar! di#u$ o %a-+$! /ro-+ai!n% and %$ow o ! grown in h!

$aoraor"> PCR '!ai$!d g!n!i# ana$"%i% #an a$%o

! u%!d o d!!# aniioi# r!%i%an#!@a$$owing i--!dia! and !!#i;! h!ra+"> Th! !!#% o/ h!ra+" #an a$%o !i--!dia!$" !;a$ua!d>

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http://en.wikipedia.org/wiki/Tuberculosishttp://en.wikipedia.org/wiki/Tuberculosis_diagnosishttp://en.wikipedia.org/wiki/Sputumhttp://en.wikipedia.org/wiki/Sputumhttp://en.wikipedia.org/wiki/Tuberculosis_diagnosishttp://en.wikipedia.org/wiki/Tuberculosis


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PCR APPLICATION

• Th! %+r!ad o/ a disease o!ganism hrough +o+u$aion% o/do-!%i# or wi$d ani-a$% #an !

-onior!d " PCR !%ing> In -an" #a%!%@h! a++!aran#! o/ n!w ;iru$!n %u

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http://en.wikipedia.org/wiki/Influenzahttp://en.wikipedia.org/wiki/Chickenhttp://en.wikipedia.org/wiki/Goosehttp://en.wikipedia.org/wiki/H5N1http://en.wikipedia.org/wiki/Influenza_pandemichttp://en.wikipedia.org/wiki/Influenza_pandemichttp://en.wikipedia.org/wiki/H5N1http://en.wikipedia.org/wiki/Goosehttp://en.wikipedia.org/wiki/Chickenhttp://en.wikipedia.org/wiki/Influenza


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PCR APPLICATION

• Th! d!;!$o+-!n o/ PCR


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PCR APPLICATION

• L!%% di%#ri-inaing /or-% o/'NA .ng!r+rining #an h!$+ inPa!ental testing@ wh!r! an indi;idua$ i%-a#h!d wih h!ir #$o%! r!$ai;!%> 'NA /ro-unid!ni.!d hu-an r!-ain% #an ! !%!d@and #o-+ar!d wih ha /ro- +o%%i$!

+ar!n%@ %i$ing%@ or #hi$dr!n> Si-i$ar !%ing

#an ! u%!d o #on.r- h! io$ogi#a$ +ar!n%o/ an ado+!d (or idna++!d) #hi$d> Th!a#ua$ io$ogi#a$ /ah!r o/ a n!worn #an a$%o! #on.r-!d (or ru$!d ou)>

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http://en.wikipedia.org/wiki/DNA_fingerprintinghttp://en.wikipedia.org/wiki/Parental_testinghttp://en.wikipedia.org/wiki/Anna_Nicole_Smithhttp://en.wikipedia.org/wiki/Paternity_testinghttp://en.wikipedia.org/wiki/Paternity_testinghttp://en.wikipedia.org/wiki/Anna_Nicole_Smithhttp://en.wikipedia.org/wiki/Parental_testinghttp://en.wikipedia.org/wiki/DNA_fingerprinting


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PCR APPLICATION

• PCR ha% !!n a++$i!d o -an" ar!a% o/ r!%!ar#h in-o$!#u$ar g!n!i#%?

• PCR a$$ow% ra+id +rodu#ion o/ %hor +i!#!% o/ 'NA@!;!n wh!n nohing -or! han h! %!0u!n#! o/ h!

wo +ri-!r% i% nown> Thi% ai$i" o/ PCR aug-!n%-an" -!hod%@ %u#h a% g!n!raing %y"!idi&ation p!o"es /or Souh!rn or norh!rn $o h"ridiaion> PCR %u++$i!% h!%! !#hni0u!% wih

$arg! a-oun% o/ +ur! 'NA@ %o-!i-!% a% a %ing$!%rand@ !na$ing ana$"%i% !;!n /ro- ;!r" %-a$$a-oun% o/ %aring -a!ria$>

2

http://en.wikipedia.org/wiki/Nucleic_acid_hybridizationhttp://en.wikipedia.org/wiki/Hybridization_probehttp://en.wikipedia.org/wiki/Southern_blothttp://en.wikipedia.org/wiki/Northern_blothttp://en.wikipedia.org/wiki/Northern_blothttp://en.wikipedia.org/wiki/Southern_blothttp://en.wikipedia.org/wiki/Hybridization_probehttp://en.wikipedia.org/wiki/Nucleic_acid_hybridization


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PCR APPLICATION

• Th! a% o/ 'N seuencing #ana$%o ! a%%i%!d " PCR> Dnown%!g-!n% o/ 'NA #an !a%i$" !

+rodu#!d /ro- a +ai!n wih ag!n!i# di%!a%! -uaion>Modi.#aion% o h! a-+$i.#aion

!#hni0u! #an !,ra# %!g-!n% /ro-a #o-+$!!$" unnown g!no-!@ or#an g!n!ra! 5u% a %ing$! %rand o/

an ar!a o/ in!r!%> 2

http://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencing


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PCR APPLICATION

• PCR ha% nu-!rou% a++$i#aion% o h! -or!radiiona$ +ro#!%% o/ 'N cloning> I #an!,ra# %!g-!n% /or in%!rion ino a ;!#or/ro- a $arg!r g!no-!@ whi#h -a" ! on$"a;ai$a$! in %-a$$ 0uanii!%> 6%ing a %ing$!%! o/ ;!#or +ri-!r%@ i #an a$%o ana$"! or!,ra# /rag-!n% ha ha;! a$r!ad" !!nin%!r!d ino ;!#or%> So-! a$!raion% o h!PCR +roo#o$ #an gene!atemutations (g!n!ra$ or %i!

2*

http://en.wikipedia.org/wiki/DNA_cloninghttp://en.wikipedia.org/wiki/DNA_cloning


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PCR APPLICATION

• An !,#iing a++$i#aion o/ PCR i% h!+h"$og!ni# ana$"%i% o/ 'NA /ro-ancient sou!ces> In %o-! #a%!% h!

high$" d!grad!d 'NA /ro- h!%!%our#!% -igh ! r!a%%!-$!d duringh! !ar$" %ag!% o/ a-+$i.#aion>

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http://en.wikipedia.org/wiki/Phylogenyhttp://en.wikipedia.org/wiki/Ancient_DNAhttp://en.wikipedia.org/wiki/Ancient_DNAhttp://en.wikipedia.org/wiki/Phylogeny


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PCR APPLICATION

A #o--on a++$i#aion o/ PCR i% h!%ud" o/ +a!rn% o/gene exp!ession> Ti%%u!% (or !;!n

indi;idua$ #!$$%) #an ! ana$"!d adi!r!n %ag!% o %!! whi#h g!n!%ha;! !#o-! a#i;!@ or whi#h ha;!

!!n %wi#h!d o> Thi% a++$i#aion#an a$%o u%! 0uaniai;! PCR o0uania! h! a#ua$ $!;!$% o/

!,+r!%%ion 29

http://en.wikipedia.org/wiki/Gene_expressionhttp://en.wikipedia.org/wiki/Quantitative_PCRhttp://en.wikipedia.org/wiki/Quantitative_PCRhttp://en.wikipedia.org/wiki/Gene_expression


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Than "ou

THN* +,

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POLYMERASE CHAIN REACTION (2).pptx

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