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PCR Cloning
by:
Prof. Dr. drh. Aris Haryanto, M.Si.

Department of Biochemistry
Faculty of Veterinary Medicine
Gadjah Mada University
Yogyakarta
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DNA Molecule
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Terms used in Cloning

 DNA recombination.
The DNA fragment to be cloned is inserted into a vector.
 Transformation.
The recombinant DNA enters into the host cell and
proliferates.
 Selective antibiotics.
A specific antibiotic is added to kill E. coli without any
protection. The transformed E. coli is protected by the
antibiotic-resistance gene
 Isolation of desired DNA clones
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DNA CLONING
 DNA cloning is a technique for reproducing DNA fragments.
 It can be achieved by two different approaches:
▪ cell based
▪ using polymerase chain reaction (PCR).
 A vector is required to carry the DNA fragment of interest into
the host cell.
 DNA cloning allows a copy of any specific part of a DNA (or
RNA) sequence to be selected among many others and
produced in an unlimited amount.
 This technique is the first stage of most of the genetic
engineering experiments:
▪ production of DNA libraries
▪ PCR
▪ DNA sequencing
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Cloning Vectors
 The molecular analysis of DNA has been made possible by
the cloning of DNA. The two molecules that are required for
cloning are the DNA to be cloned and a cloning vector.
 Cloning vector - a DNA molecule that carries foreign DNA
into a host cell, replicates inside a bacterial (or yeast) cell
and produces many copies of itself and the foreign DNA
 Three features of all cloning vectors
 sequences that permit the propagation of itself in bacteria
(or in yeast for YACs)
 a cloning site to insert foreign DNA; the most versatile
vectors contain a site that can be cut by many restriction
enzymes
 a method of selecting for bacteria (or yeast for YACs)
containing a vector with foreign DNA; usually
accomplished by selectable markers for drug resistance
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Types of Cloning Vectors
 Plasmid - an extrachromosomal circular DNA molecule
that autonomously replicates inside the bacterial cell;
cloning limit: 100 to 10,000 base pairs or 0.1-10
kilobases (kb)
 Phage - derivatives of bacteriophage lambda; linear
DNA molecules, whose region can be replaced with
foreign DNA without disrupting its life cycle; cloning limit:
8-20 kb
 Cosmids - an extrachromosomal circular DNA molecule
that combines features of plasmids and phage; cloning
limit - 35-50 kb
 Bacterial Artificial Chromosomes (BAC) - based on
bacterial mini-F plasmids. cloning limit: 75-300 kb
 Yeast Artificial Chromosomes (YAC) - an artificial
chromosome that contains telomeres, origin of
replication, a yeast centromere, and a selectable marker
for identification in yeast cells; cloning limit: 100-1000 kb
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General Steps of Cloning with
any Vector

 prepare the vector and DNA to be cloned by digestion with

restriction enzymes to generate complementary ends
(exception Topo cloning )
 ligate the foreign DNA into the vector with the enzyme
DNA ligase
 introduce the DNA into bacterial cells (or yeast cells for
YACs) by transformation
 select cells containing foreign DNA by screening for
selectable markers (usually drug resistance)
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Restriction and Ligation
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Insertion of Restriction Fragment
into Vector

Multi Cloning Site

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The pUC 18 or 19 Cloning Vector

Lac Z α

RE Sites in blue occur only once in the plasmid

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Transformation and Selection

White White Blue Dead

Screening
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PCR cloning strategies

 Cloning methods for PCR products are divided into three

types:
a. blunt-end cloning
b. sticky-end cloning
c.T-A cloning
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PCR Cloning Considerations

 Nature of the Insert: not all PCR fragments will clone

with the same efficiency into the same vector.
 Insert Size:The size of the fragment being cloned is a
primary contributor to the overall cloning efficiency. Large
fragments of DNA (≥ 5 kb) are amenable to cloning in
high-copy number vectors, yet at a much lower efficiency.
 Vector-to-Insert Ratio:Optimization of molar
concentration ratios of the vector to insert is critical to
ensure efficient cloning. insert ratios: 1:1, 1:3,
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T - A Cloning

 When DNA fragments are generated Taq polymerase adds

1 or 2 extra adenines onto the end of 3’ end of blunt ds DNA
 Several commercially available kits take advantage of this
ability
 Use a plasmid vector with thymidine residues linked onto
the 3’ ends of linearised plasmid DNA
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TA CLONING VECTOR
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T-A Recombinant Vector
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Why are theTopo Cloning?

 PCR generates the exact gene fragment we want to clone.

 PCR products and no other DNA are ligated into the Topo
vector by the topoisomerase.
 Ligation is highly efficient (as high as 90%).
 Selection of transformants is highly efficient
 A very large number of white colonies is generated
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Features of Topo Vector

 EcoR I sites flanking the PCR product insertion site

for easy removal of inserts
 Kanamycin and ampicillin resistance genes for your
choice of selection in E. coli
 Easy blue/white screening of recombinant colonies
 Promoter/priming sites for in vitro transcription
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The Topo TA PCR Cloning
Vector
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The Topo TA Cloning Process
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Kloning dan Ekspresi

Gen Penyandi IFN
pada Kucing dengan
Champion pET SUMO
Oleh:
Dr. drh. Aris Haryanto, M.Si.
Prof. Dr. drh. Ida Tjahajati, MP
drh. Penny Humaidah, M.Biotech.
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Pendahuluan
PEMBUATAN REKOMBINAN DAN MONOKLONAL ANTIBODI
PRIMER INTERFERON-GAMMA (IFN) UNTUK PEMBUATAN
KIT DIAGNOSIS TUBERKULOSIS PADA ANJING DAN
KUCING

Hibah Kompetensi Dikti Angkatan II (2009-2010)

Kloning dan Ekspresi Gen Penyandi IFN

pada Kucing dengan Champion pET SUMO
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Latar Belakang
• Diagnosis tuberkulosis masih merupakan masalah besar baik pada
manusia maupun pd hewan kesayangan termasuk anjing & kucing.
• Diagnosis tuberkulosis didasarkan pengenalan antigen spesifik oleh
sel T, yg akan direspon dg produksi sitokin interferon-gamma (IFN-γ)
• Berdasarkan pada sifat respon limfosit T yang spesifik antigen maka
dapat diciptakan kit diagnosis yang spesifik dan akurat.
• IFN-γ dihasilkan oleh limfosit T sebagai respon imun spesifik
terhadap adanya rangsangan dari makrofag yang
mempresentasikan antigen melalui MHC-II.
• Respon imun spesifik sel T, terhadap pengenalan antigen
M.tuberculosis memungkinkan pengembangan diagnosis secara in
vitro berdasarkan IFN-γ yang dihasilkan oleh sel T setelah
terstimulasi dgn antigen yg pernah dikenalinya (Andersen, 2004).
• Hal ini menjanjikan untuk mengembangkan tool diagnostik penyakit
tuberkulosis pada hewan yang spesifik, cepat, dan akurat.
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Tujuan
Tujuan penelitian adalah untuk men-subcloning dan
mengekspresikan gen penyandi IFN melalui pembuatan
DNA rekombinan IFN sebagai dasar untuk pembuatan kit
diagnosis penyakit tuberkulosis pada kucing.
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pET Sumo Protein Expression System
Pengantar
• Cat. No. K300-01.
• Small Ubiquitin-like Modifier (Sumo)
• Digunakan untuk mengekspresi, mempurifikasi dan
memproduksi rekombinan protein pada E. coli.
• Sumo protein adalah protein Smt3 dari S. cerevisiae dengan BM
11 kDa.
• Protein interes kita merupakan protein fusi dgn protein Sumo
• Fusi protein Sumo akan meningkatkan ekspresi protein
rekombinan.
• Struktur tersier protein Sumo dikenali secara spesifik dan
dipotong oleh enzim Sumo protease.
• Apabila protein Sumo difusikan pada bagian N terminus protein
rekombinan kita maka pemotongan protease Sumo ini akan
dihasilkan protein rekombinan murni.
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Kelebihan pET Sumo

• Meningkatkan ekspresi protein fusi rekombinan

• Meningkatkan solubilitas protein fusi rekombinan
• Dapat diisolasi protein rekombinan murni dengan
pemotongan menggunakan protease Sumo
• Mudah memisahkan protein rekombinan murni dengan
protein Sumo setelah pemotongan dengan protease Sumo
melalui kromatografi afinitas dengan nickel-chelating resin
(Ni-NTA)
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Gambaran Mengenai pET Sumo
• Vektor pET Sumo didesain untuk memfasilitasi cloning
produk PCR untuk diekspresikan pada bakteri E. coli
• Memiliki promoter lac T7 untuk ekspresi protein yang tinggi,
dengan diinduksi menggunakan IPTG
• Mempunyai N terminal polihistidin (6x his) untuk deteksi,
dan purifikasi protein fusi rekombinan
• Terdapat protein fusi Sumo pada ujung N-terminal untuk
meningkatkan ekspresi dan solubilitas protein fusi
rekombinan dan untuk menghasilkan protein murni setelah
melalui prmotongan dengan protease Sumo
• Mempunyai TA Cloning Site untuk efisiensi kloning produk
PCR secara langsung
• Memiliki resistensi terhadap Kanamisin
• Berasal dari pBR322
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Prinsip TA Cloning

• Vektor pET sumo memberikan satu langkah strategi cloning

yang cepat dengan menginsersikan secara langsung produk
PCR ke dalam vektor.
• Taq Pol memiliki aktifitas menambahi satu A pada 3’ produk
PCR.
• Sedangkan vektor pET Sumo linier didesain mempunyai satu
T pada 3’
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TA Cloning Site
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Materi

• cDNA IFN kucing

• Champion pET Sumo Protein Expression System
• (Cat. No. K300-01)
• FastStart PCR Master (Cat. No. 04 710 436 001)
• LB Medium
• Bacto Agar
• Kanamisin
• dll
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Isolasi mRNA dari limfosit
Materi dan Metode
Pembuatan cDNA

PCR

Purifikasi PCR Produk

Ligasi Sekuensing

Transformasi E. Coli (Mach1) dibiakan

Check by PCR Klone (+) Ekspresi SDS PAGE

E. Coli (BL21)
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Hasil PCR gen IFN  pada Kucing

316 bp
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Hasil PCR Sebelum di Purifikasi

M 1 2 3 4

400 bp
300 bp
200 bp 316 bp
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Elektroforesis Produk Ligasi

Plasmid + insert = produk ligasi

750 bp + 316 bp = 1.066 bp

M = marker
1 = plasmid
2 = insert
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Hasil Transformasi

Kontrol (pUC 19) pET Sumo

Hasil transformasi IFN kucing pada E. Coli Mach1.

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Penanaman Klone Rekombinan
Pada LB Cair + Kanamicin
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Kultur pETfeIFN pada E. Coli
Mach1

Sebelum dikultur Setelah dikultur

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Isolasi Plasmid pET SUMO dari
E. coli Mach1
M 1 2 3 4
M = Marker DNA
1 = Kontrol negatif
2 = klone 1 (6.200 bp)
3 = klone 2 (6.200 bp)
4 = klone 3 (6.200 bp)
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Ekspresi pada E. coli BL21

Rekultur
pET-Fe IFN 120110

1. LB medium tanpa Kanamicin

2. LB medium dengan Kanamicin 5 mg/ml
3. LB medium dengan Kanamicin 5 mg/ml + glukosa 5%
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Transformasi pET FeIFN pada
E. coli BL21
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Hasil Isolasi pET FeIFN
dari E. Coli BL21

M 1 2 3 4 5 6 7
M = Marker DNA
1-7 = pET FeIFN
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Hasil PCR pET Fe IFN G
transforman E. Coli BL 21

321 bp

M = marker DNA 100 bp

1-7 = sampel kucing
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SDS PAGE Protein IFN pada Kucing

1. Marker
2. Kontrol (BL21)
3. pET FeIFN induksi IPTG jam ke 0 BM protein fusi rekombinant
4. pET FeIFN induksi IPTG jam ke 1 315 bp /3= 105 as. amino
5. pET FeIFN induksi IPTG jam ke 2 105 aa x 110 Da = 11.550 Da
6. pET FeIFN induksi IPTG jam ke 3 11, 55 kDa + 11 kDa = 22,55 kDa
7. pET FeIFN induksi IPTG jam ke 4
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