Definition of recombinant DNA

Recombinant DNA  Production of a unique DNA molecule by

joining together two or more DNA fragments
Technology not normally associated with each other

 DNA fragments are usually derived from

Dr. Diah Rachmawati, M.Si. different biological sources
Fakultas Biologi UGM

History of recombinant DNA

Recombinant DNA technology technology

A series of procedures used to recombine Recombinant DNA technology was

DNA segments. Under certain conditions,
developed by two scientists named Boyer and
a recombinant DNA molecule can enter a
Cohen in 1973.
cell and replicate.

Basic principle of recombinant

Recombinant DNA Technology
DNA technology

 DNA from one organism is

transferred to a bacterial
 The DNA is inserted into another DNA plasmid for replication
molecule called ‘vector‘  Although viruses, bacterial
artificial chromosomes also
may be used for replicating
 The recombinant vector is then DNA, bacterial plasmid are
introduced into a host cell where it most commonly used in this
technology and are called
replicates itself, the gene is then vectors.
produced

1
 Applications of Recombinant Production of Human Insulin
DNA Technology
1) Obtaining the human insulin gene
Human insulin gene can be obtained by making a
 Large-scale production of human complementary DNA (cDNA) copy of the messenger RNA
(mRNA) for human insulin.
proteins by genetically engineered
bacteria.

such as : insulin, Growth hormone,

Interferons and Blood clotting factors
(VIII & IX)

3)Introducing the recombinant

2) Joining the human insulin gene
into a plasmid vector
DNA plasmids into bacteria

The bacteria E.coli is used as the host cell. If E. coli and the
 The bacterial plasmids and the cDNA are mixed together. recombinant plasmids are mixed together in a test-tube.
The human insulin gene (cDNA) is inserted into the
plasmid through complementary base pairing at sticky
ends.

4) Selecting the bacteria which have

taken up the correct piece of DNA Recombinant DNA Technology
The bacteria are spread onto nutrient agar. The agar also contains
substances such as an antibiotic which allows growth of only the
transformed bacteria.
 Source of DNA
 Restriction Enzymes
 Cloning Vector
 Transformation
 Selection of recombinant

2

Source of DNA
- Genomic DNA
- DNA copy of an mRNA atau cDNA
Figure 8-46 Molecular Biology of the Cell (© Garland Science 2008)
Genomic DNA
The genomic DNA is digested by
a restriction endonuclease, and all
fragments cloned at random into
a plasmid vector, then the majority of
genetic information will be included
in the mixture of bacteria.
Cultures of the bacteria, with each
containing only a fraction of the
genome, collectively contain all the
genes and are called a genomic
library.
Genomic Library : a collection of
DNA clones that covers the entire
genome.
Sources of DNA to Clone
 Genomic DNA: cut up whole genome and clone small pieces.
Advantage is, you get everything. Disadvantage is, a lot of it is
junk.
 Two general methods:
 1. randomly shear DNA into small pieces, then ligate linkers to the
ends: oligonucleotides that contain a useful restriction site.
 2. partially digest the DNA with a restriction enzyme that has a 4 base
recognition site. These sites will appear at random every 256 (4 4) base
pairs. Take long pieces.
 cDNA: DNA copy of mRNA, made with reverse transcriptase.
Advantage: you just get the expressed genes. Disadvantages:
you don't get control sequences or introns, and frequency
depends on level of expression.
Genomic DNA
 The coding region for a gene of interest may be
interrupted by one or more intron regions, and thus
the complete coding region could be quite long.
 To a first approximation, it does not matter which
tissue we use to isolate the genomic information,
i.e. the genomic content is the same in all tissues.
DNA copy of an mRNA atau cDNA
 Introns will be spliced out and the mRNA will contain a contiguous
coding region.
 Tissue specific expression of the protein of interest may allow us
to isolate appropriate mRNA at enhanced levels, i.e. in tissues
where the protein is expressed the mRNA levels are considerably
higher than the corresponding genomic levels (there are many
more molecules of mRNA than copies of the gene).
 "Reverse transcription" is a mechanism whereby genetic
information contained in mRNA is converted back into a double
stranded DNA form.
 mRNA is converted to cDNA by enzymatic reactions
3
DNA copy of an mRNA atau cDNA Construction of cDNA library

Construction of cDNA library

 Isolation of total cellular RNA
 mRNA molecule has at its 3’ end a run of adenin
nucleotide residues called a poly(A) tail.
 Short oligoucleotides containing 12 to18 deoxythymidines
(poly dT) acts as primers for reverse transcriptase.
 Reverse transcriptase can use RNA as a template to
synthesize a DNA strand.
 The product of reverse transcription is RNA-DNA hybrid

Construction of cDNA library Pustaka cDNA / cDNA Library

1. Populasi dari cDNA yg telah disisipkan ke dalam

vektor kloning dan ditransformasi ke E. coli.

2. Lebih baik dibandingkan dengan membuat pustaka

genom, sebab tdk semua DNA diekspresikan

3. Pembuatannya di mulai dari: (1) isolasi RNA, (2)

pencetakan ds cDNA/complementary DNA, (3) cDNA
diligasikan dg vektor, (4) vektor ditransformasikan ke
E. coli

Origin and function

 Bacterial origin = enzymes that cleave foreign
DNA
 Named after the organism from which they were
derived
 EcoRI from Escherichia coli
 BamHI from Bacillus amyloliquefaciens
Restriction Enzymes  Protect bacteria from bacteriophage infection
 Restricts viral replication

 Bacterium protects it’s own DNA by methylating

those specific sequence motifs

4
 Enzim restriksi endonuklease Restriction Enzymes
 Enzim Endonuklease Restriksi : memotong DNA dengan cara mengenal
urutan spesifik DNA yang akan dipotong dulu, baru melakukan
pemotongan di dalam sekuen pengenal tersebut dengan hasil potongan
sticky end (ujung lancip) atau blunt end (ujung tumpul)
 Umumnya diisolasi dari bakteri, diberi nama asal bakterinya, fungsi
asalnya; menghalangi DNA masuk ke dalam sel bakteri. DNA bakteri
terlindungi sebab mempunyai enzim yang memodifikasi enzim restriksi
hingga jadi tidak berfungsi.
 Sekuen pengenal 4,6,8 pasang basa

 Sifatnya palindromik: jika ditarik garis sumbu ditengah sekuen pengenal

akan terlihat urutan basa yang simetris.
 Memotong ikatan fosfodiester sehingga menghasilkan satu ujung
mempunyai gugus fosfat dan ujung lainnya gugus OH
Restriction enzymes, also called restriction endonucleases,
are enzymes that cut DNA molecules in specific places
Restriction enzymes can be used to isolate a specific gene.
Once a gene has been isolated, it can be transferred by
a cloning vector to an organism.

Restriction Enzyme Mechanisms:

(a)Staggered cut: leaves “sticky ends” Contoh enzim restriksi
Enzim Asal mikroorganisme Recognition Tipe pemotongan
site
EcoRI Escherichia coli GA-A-T-T-C 5‘Phosphate
C-T-T-A-AG extension
BamHI Bacillus amyloliquefaciens GG-A-T-C-C 5‘Phosphate
C-C-T-A-GG extension

PstI Providencia stuarti C-T-G-C-AG 3‘Hydroxyl

GA-C-G-T-C extension
(b) Blunt End

PvuII Proteus vulgaris C-A-GC-T-G Blunt end

G-T-CG-A-C

Digesti dengan enzim restriksi Ligasi / Penyambungan DNA

1. Dalam kloning tahap ini diperlukan untuk

menyambung DNA target dengan DNA vektor

2. Untuk menyambung DNA digunakan Enzim ligase,

menyambungkan ikatan fosfodiester yang terputus

3. Ujung tumpul kurang efisien penyembungannya

dibanding ujung lancip

Primrose, 1994

5
Enzim yang berperan dalam manipulasi DNA
Nuklease: memotong, memendekkan, mendegradasi
a. Eksonuklease: memotong basa satu persatu dari ujung DNA
b. Endonuklease: memotong ikatan fosfodiester DNA
Ligase : menyambung DNA ss dan ds yang terputus
Polimerase : membuat kopi DNA baru berdasarkan cetakan
DNA/RNA
Enzim pemodifikasi: menghilangkan atau menambah gugus
kimiawi pada DNA
Choosing the Vector
 Depends on the size of DNA to be cloned
 Is the protein encoded by the DNA going to be
expressed in a prokariotic or eukaryotic cell?
 Antibiotic resistance genes and/or other
selectable markers enable identification of
cells that have acquired the vector construct.
 Some vectors contain inducible or tissue-
specific promoters permitting controlled
expression of introduced genes in transfected
cells or transgenic animals.
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
Requirements of a vector to serve as a
carrier molecule
 Most vectors contain a prokaryotic origin of
replication allowing maintenance in bacterial
cells.
 Some vectors contain an additional eukaryotic
origin of replication allowing autonomous,
episomal replication in eukaryotic cells.
 Multiple unique cloning sites are often included
for versatility and easier library construction.
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
6
 Plasmid Plasmid vector
 Adalah molekul DNA sirkular untai ganda yang banyak terdapat di dalam
sel bakteri, di luar kromosom
 Covalently closed, circular, double stranded DNA
 Selalu membawa  1 gen, merupakan ciri penting bakteri pembawanya.
Misalnya gen tahan antibiotik molecules that occur naturally and replicate
extrachromosomally in bacteria
 Ukuran di alam 1 kb – 250 kb
 Many confer drug resistance to bacterial strains
 Ciri khasnya yaitu mempunyai situs untuk memulai replikasi sendiri
sehingga mampu memperbanyak diri tidak tergantung kromosom.  Origin of replication present (ORI)
 Guna: mengklon fragmen DNA besar, konstruksi pustaka DNA, subkloning,
memanipulasi DNA, mengkonstruksi DNA.
 plasmid yang sekarang digunakan untuk rekonstruksi DNA dimodifikasi
dari alam, sudah dikurangi atau ditambah dengan sifat tertentu untuk
mempermudah pekerjaan kloning

 Plasmid yang digunakan untuk kloning umumnya berukuran antara 2-4 kb.

Vectors typically include a selectable Plasmid pUC19

marker and a cloning site

 selectable markers usually are a gene for a product

that the host cell cannot make itself, such as an
antibiotic resistance factor

 the cloning site on a vector is engineered with many

possible sites for restriction enzyme cutting, where
foreign DNA can be inserted

Plasmid
 Ori/origin of replication
Digunakan untuk memperbanyak diri tanpa tergantung
perbanyakan kromosom inang.

 marker seleksi/selectable marker

untuk proses seleksi plasmid yang membawa rekombinan,
umumnya barupa gen tahan antibiotik.

 situs kloning/cloning site
Tempat dimana DNA yang akan diperbanyak disisipkan;
panjangnya beberapa puluh-ratusan pasang basa; terdapat
beberapa situs enzim restriksi, situs restriksinya satu satunya
ditempat itu.
A cloning vector is a carrier that is used to clone a gene and transfer it
from one organism to another
the piece of foreign DNA inserted at a cloning site is said to be cloned,
and the combined foreign DNA + vector is called recombinant DNA

7
Producing Recombinant DNA

Introducing recombinant DNA into Cells

 DNA-mediated transformation
 Microinjection
 Electroforation

The combination of DNA from two or more sources is called

recombinant DNA. Inserting a donor gene, such as the human
gene for insulin, into a cloning vector, such as a bacterial
plasmid, results in a recombinant DNA molecule.

Transformation
Transformasi DNA rekombinan ke E.coli
The uptake of free foreign DNA into the cell
 a piece of DNA to be inserted into a vector.
 piece of DNA with a restriction enzyme and then ligate the DNA insert into
 Secara alami bakteri mampu mengambil molekul DNA
the vector with DNA Ligase. The insert contains a selectable marker which dari media tempat tumbuhnya
allows for identification of recombinant molecules.
An antibiotic marker is often used so a host cell without a vector dies when
exposed to a certain antibiotic, and the host with the vector will live  E. coli dalam keadaan normal hanya mampu
because it is resistant.
mengambil DNA dalam jumlah terbatas.
 The vector is inserted into a host cell (bacteria), in a process called
transformation. Selectable markers can be for antibiotic resistance, color  Harus ada perlakuan fisik dan kimiawi tertentu untuk
changes, or any other characteristic which can distinguish transformed
hosts from untransformed hosts. meningkatkan kemampuan mengambil sel yang telah
diperlakukan disebut sel kompeten.
 Different vectors have different properties to make them suitable to
different applications. Some properties can include symmetrical cloning
sites, size, and high copy number.

Transformasi DNA terkonstruksi ke E.coli Electroporation

 Untuk membuat sel kompeten biasanya dimasukkan

larutan garam 50mM kalsium klorid dingin.
 Kemungkinan CaCl2 menyebabkan perubahan struktur
dinding sel bakteri sehingga mudah menyerap DNA
 Efisiensi transformasi 0.01%

 Perlu DNA penanda/selectable marker berupa DNA

resistensi terhadap antibiotik misalnya pUC19 Electric Shock Opens Pores in Cell Wall
seleksinya dengan ampisilin.

8
 Microinjection Microparticle Bombardment
 In microinjection, the DNA is injected directly into the
nucleus of the cell being transformed.
 In biolistics, the host cells are bombarded with high
velocity microprojectiles, such as particles of gold or
tungsten that have been coated with DNA.

Shoots projectiles of gold or tungsten coated with

DNA or RNA into cells
Generally used with Eucaryotes