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TATAP MUKA 8
Polymerase Chain Reaction
(PCR)
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LO 34: menjelaskan prinsip dasar PCR
LO 35: menjelaskan perancangan prmer PCRLO 36: menjelaskan reverse transcriptase PCR (RT-PCR)
LO 3: menjelaskan nested PCR
LO 3!: menjelaskan R"P#-PCR
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INSIP DASAR PCR
1. DNA temlat DNA yang sebagian urutannyaakan diamplifkasi
!. Primer • Oligonucleotide• untuk memulai amplifkasi• membatasi ragmen yang
akan diamplifkasi.". DNA olimerase
• DNA polimerase termostabil• mengamplifkasi ragmen
DNA#. $NTP
•
Monomer DNA• dATP, dTTP, dGTP, dTP
MP%N&N PCR
'% "# menelas*an rinsi $asar PCR
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D!NAT"#AT$ON %&'() Duple* DNA is +eat
denatured to giesingle strands
ANN!A-$NG %''() to oligonucleotide
primers are annealedto t+eircomplementaryse/uences on t+e
target DNA. !0T!N1$ON %23() Ta/ polymerase
%t+ermostable) is usedto synt+esise
complementary
US DAN TA+APAN PCR
'% "# menelas*an rinsi $asar PCR
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US DAN TA+APAN PCR
'% "# menelas*an rinsi $asar PCR
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RANCAN,AN PRIM&R PCR
'% "- menelas*an erancanan rmer PCR
Aspek yang +arus dipertimbangkan4
urutan primerasal inormasi urutandata sekuens asam amino
kode degenerasi genetik"rutan basa DNA yang tela+
diketa+ui
1intesis primer4Posisi 5obble6asa inosin
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RANCAN,AN PRIM&R PCR
'% "- menelas*an erancanan rmer PCR
/i. 0." Primer $esin. $n %a) t+e amino acid se/uenceand number o codons per amino acid are s+on. T+ose
amino acids it+ 7 codons %circled) are best aoided. T+ebo*ed se/uence is t+ereore selected.
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/i. 0." Primer $esin. $n %b) a mi*ed probe is synt+esisedby including t+e appropriate mi*ture o dNTPs or eac+degenerate position. Note t+at in t+is e*ample t+e fnaldegenerate position or proline is not included, giing anoligonucleotide it+ 38 bases %a 389mer). T+ere are :3;
possible permutations o se/uence in t+e mi*ture.'% "- menelas*an erancanan rmer PCR
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/i. 0." Primer $esin. $n %c) inosine %$) is used to replacet+e our9old degenerate bases, giing eig+t possiblese/uences.
'% "- menelas*an erancanan rmer PCR
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arian PCR2erse transcritase PCR (RT3PCR)
P# menggunakan m#NA sebagai templatDigunakan dalam penentuan tingkat ekspresi
gen Proses penyalinan m#NA memerlukan reerse
transcriptase Primer Oligo%dT) digunakan untuk mensintesis
untai pertama cDNA. Primer P# digunakan setela+ untai pertama
cDNA disintesis
'% "4 menelas*an reverse transcriptase PCR (RT3PCR)
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/i. 0.# RT3PCR. %a) #eerse transcriptase is used to synt+esise a cDNAcopy o t+e m#NA. $n t+is e*ample oligo%dT)9primed synt+esis is s+on.%b) T+e cDNA product is amplifed using genespecifc primers. T+e initialP# synt+esis ill copy t+e cDNA to gie a duple* molecule, +ic+ is t+enamplifed in t+e usual ay. $n many kits aailable or #T9P# t+e entire
procedure can be carried out in a single tube.'% "4 menelas*an reverse transcriptase PCR (RT3PCR)
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. Neste$ PCR
'% "0 menelas*an nested-PCR
/i. 0.4 Neste$PCR.
%a)1tandard P#amplifcation o aragment using aprimer pair.
%b)$n t+e secondP#, a set oprimers t+at lieinside t+e frstpair is used toprime synt+esis oa s+orterragment. NestedP# can be used
to increase t+e
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. RAPD3PCR
#andom amplifed polymorp+ic DNA
P# %#APD9P#) didasarkan padapenempelan primer ekurangan #APD9P#4 reproduktibilitasrenda+, yaitu kesulitan untukmemperole+ tingkat penempelan yang
sama pada percobaan berbeda.'% "8 menelas*an RAPD3PCR
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