REPLIKASI & 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

Membran inti sel

Sitoplasma
Translasi
Sintesis Protein
Ribosom

3
Protein
Alir Informasi Genetik

4
 Tipe Sel

Prokariotik: Struktur
sel mikroorganisme dimana
sel tidak memiliki membran
inti sel

Eukariotik: Struktur sel

yang lebih kompleks,
memiliki membran inti sel
(Fungi, animal, Plant)
Sel Prokariot
Sel Eukariot
 Prokariot Eukariot

Sifat Prokariot Eukariot

Membran inti sel Tidak mempunyai Mempunyai

Jumlah kromosom 1 >1

Bentuk kromosom Bulat (sirkuler) Linier (lurus)

Mitokondria Tidak ada Ada

Mitosis & Meiosis Tidak ada Ada

Replikasi

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

12
Kemungkinan Replikasi
DNA

13
Replikasi DNA Semikonservatif

Meselson & Stahl (1958)

concluded that the mechanism
of DNA replication is the
semiconservative model.

14
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

An enzyme goes to those hydrogen bonds

and breaks them = Double Helix Unzips
 DNA Replication

Step 1: Separate the two DNA strands

 DNA Replication

Step 2: Original DNA strands act as templates &

complementary nucleotides fill in.
 DNA Replication

Step 3: Nucleotides connected and a new sugar-

phosphate backbone is formed.
 DNA Replication

Each new DNA molecule consists of one original

strand and one new strand
Replikasi Prokariot

22
Replication origins
 Replikasi Bakteri

DNA synthesis begins at a site termed the origin

of replication
Each bacterial chromosome has only one
Synthesis of DNA proceeds bidirectionally
around the bacterial chromosome
eventually meeting at the opposite side of the
bacterial chromosome
Where replication ends
Overview of bacterial DNA replication
DNA Replication in Chromosome
DNA replication in Bacterial Genome
DNA replication with two forks
Bidirectionel DNA-
Replication
of a Circular Genome
Photos of DNA
Replication
DNA Replication in Bacteria and
Eukaryotes
 Prokaryotic Replication
Bacteria have a single loop of DNA that must
replicate before the cell divides.
Replication in prokaryotes may be bidirectional
from one point of origin or in only one direction.
Replication only proceeds in one direction, from
5' to 3'.
Bacterial cells are able to replicate their DNA at
a rate of about 106 base pairs per minute.
Bacterial cells can complete DNA replication in
40 minutes; eukaryotes take hours.
 Eukaryotic Replication
Replication in eukaryotes starts at many points
of origin and spreads with many replication
bubblesplaces where the DNA strands are
separating and replication is occurring.
Replication forks are the V-shape ends of the
replication bubbles; the sites of DNA replication.
Eukaryotes replicate their DNA at a slower rate
500 to 5,000 base pairs per minute.
Eukaryotes take hours to complete DNA
replication
Replikasi Eukariot

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

Unusual Origin of PCR, Mullis KB,

Scientific American 1990,56
1993 Nobel Prize in Chemistry
 PCR
Polymerase Chain Reaction

Tujuan : memproduksi copy DNA dalam jumlah banyak

dalam waktu singkat

Suatu teknik yang dilakukan dalam suatu tabung reaksi pada

sebuah mesin yang disebut 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

2) Mapping of complex traits

3) Molecular taxonomy

4) Forensic science ( DNA fingerprinting)

5) Isolating ancient DNA sequences

6) Diagnosis of inherited disease.

59
RT PCR & Aplikasinya
 RT PCR Reverse Transcription P
olymerase Chain Reaction
Tujuan : memproduksi complementary DNA (cDNA) dalam jumlah banyak
dalam waktu singkat dari mRNA

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
Selamat Belajar ....

67