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TUGAS APLIKASI PCR

RT-PCR

( Reverse Transcription Polymerase ChainReaction)

DISUSUN OLEH :

1. IKA LAIDY S P27903114062

2. ISNI FAUZIAH ARBI P27903114063

3. MELIANA INDAH HARAHAP P27903114068

Analis Kesehatan 2B

POLTEKKES KEMENKES BANTEN

Tahun Ajaran 2014/2015

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KATA PENGANTAR

Puji syukur kami panjakan k!"a#ira Tu"an $an% &a"a Esa kar!na aas k!"!n#ak-

Nya p!nu'isan maka'a" ini #!n%an ju#u' RT-PCR) #apa !rs!'!saikan #!n%an *aik+ Da'am

k!s!mpaan ini p!nu'is m!n%"aurkan rasa ",rma #an !rima kasi" s!*!sar-*!sarnya k!pa#a

#,s!n p!n%ajar #an s!mua pi"ak yan% !'a" m!m*anu m!m*!rikan masukan saran-saran

p,sii. #a'am p!m*uaan maka'a" s!"in%%a p!nu'is #apa m!ny!'!saikan u%as maka'a" ini+

&aka'a" ini #i*ua unuk m!m!nu"i sa'a" sau u%as #a'am maa ku'ia"APLIKASI PCR

*!rujuan unuk #apa m!m*!rikan in.,rmasi k!pa#a para p!m*a/a !nan% RT-PCR+

Da'am p!nu'isan maka'a" ini p!nu'is m!rasa masi" *anyak k!kuran%an-k!kuran%an *aik

pa#a !knis p!nu'isan maupun ma!ri m!n%in%a akan k!mampuan yan% #imi'iki p!nu'is+

Unuk iu kriik #an saran #ari s!mua pi"ak san%a #i"arapkan #!mi p!ny!mpurnaan maka'a"

ini+

Tan%!ran% 01 S!p!m*!r 2034

P!nu'is

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DAFTAR ISI

KATA PENGANTAR ....................................................................................... i

DAFTAR ISI .................................................................................................... ii

BAB I PENDAHULUAN

1.1 Laar 5!'akan% +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

1.2 Rumusan &asa'a" ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

1.3 Tujuan &aka'a" +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

BAB II PEMBAHASAN

2.1 P!n%!rian P,'ym!ras! C"ain R!a/i,n (PCR6

2.2 P!n%!rian RT-PCR ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

2.3 Prinsip RT-PCR +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

2.4 Ta"apan RT-PCR #an PCR s!/ara umum ++++++++++++++++++++++++++++++++++++++++++++++++2. On! S!p #an T7, S!p +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

BAB III PENUTUP

3.1 K!simpu'an ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

3.2 Saran ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

DAFTAR PUSTAKA

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BAB I

PENDAHULUAN

1.1 LATAR BELAKANG

PCR a#a'a" suau m!,#! in 8ir, unuk m!n%"asi'kan s!jum'a" .ra%m!n DNA

sp!si.ik #!n%an panjan% #an jum'a" sku!ns yan% !'a" #i!nukan #ari jum'a" k!/i' !mp'a!

k,mp'!ks+ PCR m!rupakan suau !knik san%a kua #an san%a s!nsii. #an #apa

#iap'ikasikan #a'am *!r*a%ai *i#an% s!p!ri *i,',%i m,'!ku'!r %!n!ika p,pu'asi #an

ana'isis .,r!nsik+ T!knik DNA r!k,m*inan !'a" m!m*!rikan p!ru*a"an s!/ara si%ni.ikan

#a'am i'mu %!n!ika kar!na m!mun%kinkan !rja#inya is,'asi #an karak!risik %!n-%!n

m!mp!'ajari s!/ara rin/i .un%si #an !kspr!si s!'ama pr,s!s p!rk!m*an%an !rja#i s!*a%ai

r!sp,n !r"a#ap 'in%kun%an+

Reverse Trancription Polymerase Chain Reaction (RT-PCR)m!rupakan 8ariasi #ari

Polymerase Chain Reaction (PCR) s!*ua" !knik 'a*,ra,rium yan% *iasanya #i%unakan

#a'am 5i,',%i &,'!ku'!r unuk m!n%"asi'kan s!jum'a" %an#aan *a%ian !r!nu unai DNA

aau yan% #ik!na' #!n%an amp'i.ikasi+ Da'am RT-PCR s!*a'iknya unai RNA p!rama-ama

#i ranskrip *a'ik m!nja#i DNA k,mp'!m!n (/DNA6 m!n%%unakan !n9im r!8!rs!

rans/ripas! #an /DNA yan% #i"asi'kan akan #i%an#akan s!p!ri "a'nya PCR pa#a

umumnya+

1.2 R!"!#$% M$#$&$'

3+ Apa yan% #imaksu# #!n%an P,'ym!ras! C"ain R!a/i,n (PCR6

2+ Apa yan% #imaksu# #!n%an RT-PCR

;+ Apaka" prinsip #ari RT-PCR

1+ Apa saja a"apan-a"apan #ari RT-PCR #an PCR s!/ara umum

4+ Apa yan% #imaksu# On! S!p #an T7, S!p #ari RT-PCR

1.3 T!(!$%

1. Unuk m!n%!a"ui apa yan% #imaksu# #!n%an P,'ym!ras! C"ain R!a/i,n (PCR6

2. Unuk m!n%!a"ui apa yan% #imaksu# #!n%an RT-PCR

3. Unuk m!n%!a"ui apaka" prinsip #ari RT-PCR

4. Unuk m!n%!a"ui apa saja a"apan-a"apan #ari RT-PCR #an PCR s!/ara umum

. Unuk m!n%!a"ui apa yan% #imaksu# On! S!p #an T7, S!p #ari RT-PCR

1.4 M$%)$$*

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&an.aa yan% #ip!r,'!" #ari p!nu'isan maka'a" ini a#a'a" #apa m!mp!r,'!" p!n%!a"uan

!nan% P,'ym!ras! C"ain R!a/i,n (PCR6 k"ususnya m!n%!nai RT-PCR) !nan%

p!n%!rian a"apan #an prinsip #ari RT-PCR+

BAB II

PEMBAHASAN

2.1 D+)i%i#i P,&-"+$#+ /'$i% R+$*i,% P/R

R!aksi *!ranai p,'im!ras! aau '!*i" umum #ik!na' s!*a%ai PCR (P,'ym!ras! C"ain

R!a/i,n6 m!rupakan suau !knik aau m!,#! p!r*anyakan (r!p'ikasi6 DNA s!/ara

!n9imaik anpa m!n%%unakan ,r%anism!+ D!n%an !knik ini DNA #apa #i"asi'kan #a'am

jum'a" *!sar #!n%an 7aku r!'ai. sin%ka s!"in%%a m!mu#a"kan *!r*a%ai !knik 'ain yan%

m!n%%unakan DNA+ T!knik ini #irinis ,'!" Kary &u''is pa#a a"un 3

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Reverse Trancription Polymerase Chain Reaction (RT-PCR)m!rupakan 8ariasi #ari

Polymerase Chain Reaction (PCR) s!*ua" !knik 'a*,ra,rium yan% *iasanya #i%unakan

#a'am 5i,',%i &,'!ku'!r unuk m!n%"asi'kan s!jum'a" %an#aan *a%ian !r!nu unai DNA

aau yan% #ik!na' #!n%an amp'i.ikasi+ Da'am RT-PCR s!*a'iknya unai RNA p!rama-ama

#i ranskrip *a'ik m!nja#i DNA k,mp'!m!n (/DNA6 m!n%%unakan !n9im r!8!rs!

rans/ripas! #an /DNA yan% #i"asi'kan akan #i%an#akan s!p!ri "a'nya PCR pa#a

umumnya+ RT-PCR s!rin%ka'i #iarikan #!n%an real-time polymerase chain reaction

(qPCR/QqRT-PCR)yan% *iasanya !rja#i k!sa'a"an #a'am p!n%%unaan sin%kaan+ Pa#a"a'

RT-PCR m!rupakan sin%kaan #ari Reverse Trancription Polymerase Chain Reaction #an

real-time polymerase chain reaction #isin%ka m!nja#i BPCR aau uaniai8! PCR+

Da'am RT-PCR p,'a RNA p!rama #ik,n8!rsi k! #a'am DNA k,mp'!m!n!r (/DNA6

m!n%%unakan suau r!8!rs! rans/ripi,n+ /DNA k!mu#ian #i%unanakan s!*a%ai p,'a unuk

amp'i.ikasi s!/ara !ksp,n!nsia' m!n%%unakan PCR *iasa+ RT-PCR saa ini m!rupakan

m!,#! aau !knik yan% pa'in% s!nsii. #a'am #!!ksi RNA yan% !rs!#ia+ P!n%%unaan RT-

PCR unuk #!!ksi RNA ranskrip !'a" m!r!8,'usi su#i m!n%!nai !kspr!si %!n #a'am

*!*!rapa p!mikiran s!*a%ai *!riku :

S!/ara !,riis m!mun%kinkan unuk m!n#!!ksi ranskrip s!iap %!n s!/ara ranskrip

&!mun%kinkan amp'i.ikasi #an !'iminasi samp!' yan% #iin%inkan unuk k!*uu"an

akan k!'impa"an ma!ri

&!ny!#iakan ,'!ransi unuk #!%ra#asi RNA s!'ama RNA m!n/akup prim!r uu"

Pa#a a7a'nya s!*!'um a#anya RT-PCR m!,#! yan% #i%unakan #a'am m!n#!!ksi

kuani.ikasi RNA a#a'a" Northern Blot.N,r"!rn *', m!rupakan !knik m!nrans.!r DNA

k! k!ras D5& #an *iasanya m!n#!!ksi RNA+ N,r"!rn *', #ik!na' s!jak a"un 3

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!rara"kan #a'am p!n%!m*an%an RT-PCR s!*a%ai m!,#! pi'i"an unuk #!!ksi RNA #an

kuani.ikasi yan% m!n%%anikan p,sisi n,r"!rn *', s!*!'umnya+ 5!*!rapa a'asan yan%

#i*!rikan para p!n!'ii yan% m!nja#ikan RT-PCR s!*a%ai pa,kan !kn,',%i unuk

m!n#!!ksi aau p!r*an#in%an in%ka RNA anara 'ain :

Ti#ak m!m!r'ukan PCR a7a'

Dapa m!n%ukur *!r*a%ai r!nan% k!'impa"an RNA (F30 .'ip6

&!ny!#iakan #aa kua'iai. #an kuaniai.

2.3 Pi%#i5 RTP/R

RT-PCR m!n%%unakan s!pasan% prim!r yan% *!rk,mp'!m!n #!n%an s!Bu!ns yan%

j!'as #ari masin%-masin% #ua unai /DNA+ Prim!r !rs!*u k!mu#ian #ip!rpanjan% #!n%an

*anuan !n9im DNA p,'ym!ras! #an akan m!n%"asi'kan s!*ua" unai %an#aan pa#a s!iap

sik'usnya #an s!!rusnya m!n%ikui amp'i.ikasi ',%arimik+

2.4 T$'$5$% RTP/R $% P/R #+$$ !"!"

RT-PCR m!'ipui i%a a"ap uama yaiu :

Tahap pertama adalah reverse transcription (RT) aau ranskripsi *a'ik #imana RNA

#iranskrip *a'ik m!nja#i /DNA m!n%%unakan !n9im reverse transcriptase #an prim!r+ Ta"ap

ini san%a p!nin% #a'am kaiannya #!n%an p!r.,rma PCR unuk amp'i.ikasi /DNA #!n%an

*anuan DNA p,'ym!ras! s!*a* DNA p,'ym!ras! "anya #apa *!k!rja pa#a !mp'a! yan%

*!rupa DNA+ Ta"apan RT (R!8!rs! Trans/ripi,n6 #apa #i'akukan #a'am a*un% yan% sama

#!n%an PCR (,n!-s!p PCR6 aau pa#a a*un% yan% !rpisa" (7,-s!p PCR6 m!n%%unakan

su"u *!rkisar 10C sampai 40C !r%anun% pa#a karak!risik r!8!rs! rans/ripas! yan%

#i%unakan+

Tahap beriktnya adalah denatrasi ds!"A at #$%C pa#a a"ap ini #ua unai DNA

akan !rpisa" #an prim!r #apa m!n%ika pa#a unai !rs!*u jika !mp!raurnya #iurunkan

k!mu#ian yan% s!'anjunya akan #imu'ai ranai r!aksi *aru+ K!mu#ian su"u #iurunkan

"in%%a m!n/apai su"u an!a'in% yan% *!r8ariasi !r%anun% prim!r yan% #i%unakan

k,ns!nrasi pr,*! #an k,ns!nrasinya jika #i%unakan #an ju%a k,ns!nrasi kai,n+

P!r"aian uama saa m!mi'i" !mp!raur an!a'in% ,pima' a#a'a" m!'in% !mp!raur #ari

prim!r #an pr,*! (jika #i%unakan6+ T!mp!raur ann!a'in% #ipi'i" unuk PCR !r%anun%

'an%sun% pa#a panjan% #an k,mp,sisi #ari prim!r !rs!*u+ Ha' ini m!rupakan "asi' #ari

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p!r*!#aan ikaan "i#r,kar*,n anara A-T (2 ikaan6 #an G-C (; ikaan6+ T!mp!raur ann!a'in%

*iasanya*!rkisar 4 #!raja #i *a7a" Tm !r!n#a" #ari pasan%an prim!r yan% #i%unakan+

Tahap akhir adalah ampli&ikasi PCRyan% m!rupakan pr,s!s #imana #i'akukannya

p!rpanjan%an DNA m!n%%unakan Prim!r yan% m!m!r'ukan TaB DNA p,'ym!ras! yan%

!rm,sa*i' *iasanya pa#a su"u 2C yan% m!rupakan su"u ,pima' unuk aki8ias !n9im

p,'ym!ras!+ Lamanya masa inku*asi iap !mp!raur p!ru*a"an su"u #an jum'a" sik'us

#ik,nr,' s!/ara !rpr,%ram m!n%%unakanprogrammable thermal cycler+ Ana'aisa pr,#uk

PCR !r%anun% pa#a k!*uu"ann PCR+ ika m!n%%unakan PCR k,n8!nsi,na' maka pr,#uk

PCR #apa #i#!!ksi #!n%an a%ar,s! %!' !'!/r,p",r!sis #an !"i#ium *r,mi#! (aau #y!

nuk'!,i#a 'ainnya6+

2. %+ #*+5 $% T, #*+5

%+ #*+5

On! s!p RT-PCR m!n%%a*un%kan r!aksi sin!sis /DNA unai p!rama (reverse

transcription6 #an #an r!aksi PCR #a'am sau a*un% yan% sama unuk

m!ny!#!r"anakan p!n%auran r!aksi #an k!mun%kinan !rk,naminasi+ On! s!p RT-

PCR m!mun%kinkan p!mr,s!san samp!' '!*i" mu#a" #a'am jum'a" yan% *!sar #an

m!minima'kan k,naminasi /arry,8!r kar!na a*un% i#ak #i*uka anara /DNA #an

amp'i.ikasi+ D!n%an m!mp!rkua s!'uru" samp!' /DNA ,n! s!p #apa m!m*!rikan

s!#ikinya 003 p% #ari ,a' RNA+ R!aksi ,n! s!p m!m*,'!"kan p!n%%unaan

ran%kaian prim!r yan% sp!si.ik+

T, #*+5

T7, s!p PCR #imu'ai #ariranskripsi k!*a'ikan *aik #ari RNA ,a' aau p,'i

(A6 J RNA m!nja#i /DNA m!n%%unakan reverse transcriptase.T7, s!p RT-PCR

*!r%una unuk m!n#!!ksi *!*!rapa p!san #ari samp!' RNA un%%a'+ Akan

m!n#apakan .'!ksi*i'ias '!*i" *!sar apa*i'a m!mi'i" prim!r #an p,'ym!ras!#aripa#a ,n! s!p RT-PCR+ K!ika m!mi'i" 7, s!p RT-PCR kia #apa m!mi'iki

pi'i"an unuk m!n%%unakan ,'i%, (#T6 ran#,m "!@am!r aau prim!r %!n sp!si.ik

#an PCR #i'akukan ,'!" P'ainum TaB DNA P,'ym!ras! P'ainum TaB DNA

P,'ym!ras! Hi%" >i#!'iy aau *isa m!mi'i" !n9im PCR+

P+$%i%$% P,#+! %+ #*+5 $% T, #*+5 RTP/R

P,#+! *, #*+5 P,#+! ,%+ #*+5

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R$%*$i 5i"+ !*$"$

DNA +%$% :

&i, T 5i"+

H+;#$"+ $$;

Pi"+ +% #5+#i)i;

Pi"+ +% #5+#i)i;

M+%-+i$;$% : F&+;#ii&i*$#

Pi&i'$% 5i"+Pi&i'$% #i#*+"

$"5&i)i;$#i

M$"5! !%*!;

"+%-i"5$% ++$5$

#$"5+& RNA !%*!;

i!%$;$% ;+"!i$%

D$5$*

"+%,5*i"$&;$%

;+#!&i*$% !%*!; RT

P/R

K+%-$"$%$%

E%

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3.1 K+#i"5!&$%

R!8!rs! Trans/ripi,n PCR (RT-PCR6 a#a'a" sa'a" sau #ari *anyak 8arian !knik

PCR+ T!knik ini *iasanya #i%unakan #a'am *i#an% *i,',%i m,'!ku'!r unuk m!n#!!ksi

in%ka !kspr!si RNA+ S!rin% !rja#i k!sa'a" pa"aman #i anara p!'ajar #an p!n!'ii #!n%an

#!.inisi RT-PCR ini yan% m!n%an%%ap *a"7a RT-PCR a#a'a" R!a'-Tim! PCR (BPCR6+ RT-

PCR pun *!r*!#a #!n%an !knik PCR *iasa 7a'aupun k!#uanya sama-sama m!n%"asi'kan

juaan sa'inan DNA m!'a'ui amp'i.ikasi+ RT-PCR #i%unakan unuk m!n%k',nin% %!n yan%

#i!kspr!sikan ,'!" ranskripsi RNA ar%! m!nuju DNA k,mp'!m!n (/DNA6 m!n%%unakan

reverse transcriptase+ K!mu#ian /DNA *aru #iamp'i.ikasi m!n%%unakan PCR *iasa+

RT-PCR #apa #i'akukan #a'am #ua 'an%ka" p!rama #!n%an ranskripsi !r*a'ik #an

k!mu#ian PCR+ T7, s!p *iasanya '!*i" s!nsii. #aripa#a m!,#! ,n! s!p "asi' #ari ar%!yan% 'an%ka #apa #ip!r*aiki #!n%an m!n%%unakan pr,s!#ur 7, s!p+

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DAFTAR PUSTAKA

&,'!/u'ar Dia%n,si/ PCR Han#*,,k+ 1-4

N!7,n C+R+ an# A+ Gra"am+ 3