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Lect 4 Replikasi N PCR
Lect 4 Replikasi N PCR
FARMASI UTA 45
2013
Priyo Wahyudi
1
REPLIKASI & PCR
A. Replikasi pada prokariot dan eukariot
B. Prinsip PCR
C. Modifikasi PCR
2
Dogma Sentral Biologi
Molekuler
Replikasi
Duplikasi DNA
Transkripsi
Sintesis RNA
Inti Sel
3
Protein
Alir Informasi Genetik
4
Tipe Sel
Prokariotik: Struktur
sel mikroorganisme dimana
sel tidak memiliki membran
inti sel
9
DNA Replication
Replication = to copy
Process of copying
DNA in the
chromosomes
Without DNA
replication, a new cell
would only have half
of its information.
Kemungkinan Replikasi DNA
In the late 1950s, three different mechanisms
were proposed for the replication of DNA
Conservative model
Both parental strands stay together after DNA replication
Semiconservative model
The double-stranded DNA contains one parental and one
daughter strand following replication
Dispersive model
Parental and daughter DNA are interspersed in both strands
following replication
Kemungkinan Replikasi
DNA
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Kemungkinan Replikasi
DNA
13
Replikasi DNA Semikonservatif
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DNA Replication
DNA Replication
Because of the pairing- if you have
one strand, it is easy to predict the
replicated strand.
T-A-G-C-C-G-T
Remember: A goes with T, C goes
with G
A-T-C-G-G-C-A
DNA Replication
Remember: The nitrogen bases are held
together by hydrogen bonds
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Replication origins
Replikasi Bakteri
34
The four standard phases of a eucaryotic cell
DNA replication occurring at S Phase (DNA synthesis phase)
G1 and G2, gap between S and M
Multiple ORI and Replication Forks During
Eukaryotic DNA Synthesis
A replicating Drosophila
chromosome
Different regions of a chromosome are replicated at different times
Arrows point to the replicating regions at different times
Replikasi DNA
Mekanisme
semi-konservatif
Masing-masing pita DNA (yg dalam rantai
ganda) menjadi cetakan (template)
Nukleotida-nukleotida DNA akan tersusun
secara komplementer terhadap pasangan
basa Nitrogennya
Sintesis DNA terjadi pada orientasi 5 --> 3
Nukleotida disambungkan pada gugus OH
pada atom C 3
40
Replikasi DNA: datangnya nukleotida
G berpasangan dgn C
P04 berikatan pada 3 OH 41
Replikasi DNA: datangnya
nukleotida
T berpasangan dgn A
P04 berikatan pada 3 OH 42
Replikasi DNA
43
Replikasi DNA
44
Animasi Replikasi
45
Replication at a Glance
46
PCR
Penemu teknik dan mesin PCR
Reaktan :
a. Fragmen DNA templat
b. Sepasang primer (primer forward & reverse)
c. Ensim DNA polymerase termostabil
d. Deoxynucleotide triphosphates = dnTP (dATP, dTTP, dGTP,
dCTP)
PCR reaction
The cycling reactions : There are three major steps in a PCR,
which are repeated for 20 to 40 cycles. This is done on an
automated Thermo Cycler, which can heat and cool the
reaction tubes in a very short time.
Denaturation at around 94C :
During the denaturation, the double strand melts open to
single stranded DNA, all enzymatic reactions stop (for
example the extension from a previous cycle).
Annealing at around 54C :
Hydrogen bonds are constantly formed and broken between
the single stranded primer and the single stranded
template. If the primers exactly fit the template, the
hydrogen bonds are so strong that the primer stays
attached
Extension at around 72C :
The bases (complementary to the template) are coupled to
the primer on the 3' side (the polymerase adds dNTP's from
5' to 3', reading the template from 3' to 5' side, bases are
PCR
Polymerase Chain Reaction) prinsipnya adalah
meniru (artificial) proses replikasi yang terjadi pada
sel Bakteri
Suatu teknik yang dilakukan dalam suatu tabung reaksi
pada sebuah mesin yang disebut PCR
Consumables
Instrumentation
PCR Animation
Multiplex PCR
58
PCR has numerous applications :
1) Gene isolation
3) Molecular taxonomy
Suatu teknik yang dilakukan dalam suatu tabung reaksi pada sebutah mesin yang
disebut PCR
Reaktan :
a. mRNA
b. Sepasang primer (primer forward & reverse)
c. Ensim Reverse Transcriptase
d. Ensim DNA polymerase termostabil
e. Deoxynucleotide triphosphates = dnTP (dATP, dTTP, dGTP, dCTP)
Instrumentation
Consumables
Reverse Transcription-PCR
(RT-PCR) Amplified
copies of
specific
1. Extract total mRNA
RNA
2. Reverse 3.
Transcription PCR
dNTP
prim s
er
Double
Reverse stranded
Transcriptase Copy DNA
Beberapa Aplikasi RT PCR:
Isolasi DNA / Gen eukariot
Mengisolasi DNA / Gen yang tidak dapat di Klon
Mapping DNA / Gen dari suatu sifat yang komplex
Membuat sekuens DNA buatan (Assembling
artificial sequences)
Menambahkan situs ensim, start atau stop codon
Site-directed mutagenesis
Molecular taxonomy
Forensic DNA
Skrining / deteksi klon colony PCR
Deteksi mRNA Reverse transcriptase PCR
Diagnosis penyakit infeksi / degeneratif
Aplikasi RT PCR
Cloning genes expressed forms (not genomic
version)
Monitor a genes expression level in any tissue
Monitor expression changes following treatments
Sophisticated RT-PCR: The real time PCR
sequencing a whole mRNA profile
EST (Expressed Sequence Tags) database
Microarray (DNA chip)
Diagnose and easily differentiate between
different cancer types
Early detection of hidden illnesses
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