Definition of recombinant DNA

Recombinant DNA
Technology
Dr. Diah Rachmawati, M.Si.
Fakultas Biologi UGM

Definition of recombinant DNA

technology
A series of procedures used to recombine
DNA segments. Under certain conditions,
a recombinant DNA molecule can enter a
cell and replicate.

Basic principle of recombinant

DNA technology

Production of a unique DNA molecule by

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

DNA fragments are usually derived from

different biological sources

History of recombinant DNA

technology

Recombinant DNA technology is one of the

recent advances in biotechnology, which was
developed by two scientists named Boyer and
Cohen in 1973.

Recombinant DNA Technology

 Recombinant DNA technology

The DNA is inserted into another DNA

molecule called vector
The
recombinant
vector
is
then
introduced into a host cell where it
replicates itself, the gene is then
produced

includes DNA cloning, gene

cloning, and molecular cloning.
 DNA from one organism is

transferred to a bacterial
plasmid for replication
 Although viruses, bacterial

artificial chromosomes also

may be used for replicating
DNA, bacterial plasmid are
most commonly used in this
technology and are called
vectors.

Applications of Recombinant
DNA Technology

Large-scale production of human

proteins by genetically engineered
bacteria.
Such as : insulin, Growth hormone,
Interferons and Blood clotting factors
(VIII & IX)

Teknologi DNA Rekombinan

Teknologi DNA rekombinan=kloning molekular
Sekumpulan teknik-teknik eksperimen yang memungkinkan peneliti untuk
mengisolasi, mengidentifikasi, memanipulasi dan melipatgandakan fragmen
DNA dalam bentuk murni.
- Teknologi kloning reproduksi
- Teknologi kloning terapi
Produk rekayasa disebut GMO (Genetically Modified Organisms) atau
organisme transgenik

Cloning involves making identical copies

Cloning
Cloning is the process of creating genetically identical
cells or organisms by using individual animals/plants.

Cloning can mean several things:

There are three main types of cloning: DNA cloning,

reproductive cloning and therapeutic cloning.

1. To make many identical copies of a DNA molecule or

particular stretch of DNA (DNA cloning and molecular
cloning).
2. To replicate an entire organism (reproductive cloning)
3. To produce undifferentiated cells (stem cells) for the
purpose of studying and treating diseases (therapeutic
cloning)

Cloned Sheep

Therapeutic cloning
Therapeutic cloning (research cloning) is when stem
cells are extracted to grow into a piece of human tissue
which is encouraged to grow into a human organ for
transplant.

 Reproductive cloning to replicate an entire organism

 Therapeutic cloning to produce undifferentiated cells (stem cells)
for the purpose of studying and treating diseases
Figure 8-6 Molecular Biology of the Cell ( Garland Science 2008)

How is it done?
DNA is extracted from a humans cell. The DNA is
inserted into a womans ovum and allowed to develop
and produce stem cells. The stem cells are removed
from the pre-embryo and are treated to grown into
whatever organ is needed. Thus, the new organ is
transplanted into the patient.

Stem cells

What are its uses?

- It is used for medical purposes, such as creating organs to transplant
into a patient in need of that organ. If replacement organs are
available to the sick and dying people, countless numbers of lives
could be saved. Therapeutic cloning is a fast and efficient way to
repair damaged organs.
- Therapeutic cloning can be used to make insulin-secreting cells to
cure for diabetes; nerve cells to cure stroke or Parkinsons disease

(Diploid )

Newly formed embryo containing DNA from somatic cell

cell division

implant

Recombinant DNA Technology

 Source of DNA

Source of DNA

 Restriction Enzymes
 Cloning Vector
 Transformation
 Selection of recombinant

- Genomic DNA
- DNA copy of an mRNA atau cDNA

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 (44) 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.

Figure 8-46 Molecular Biology of the Cell ( Garland Science 2008)

Genomic DNA

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.

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.

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.

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.

DNA copy of an mRNA atau cDNA

DNA copy of an mRNA atau cDNA

Introns will be spliced out and the mRNA will contain a contiguous
coding region.

Construction of cDNA library

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).

 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 transcription" is a mechanism whereby genetic

information contained in mRNA is converted back into a double
stranded DNA form.

 Reverse transcriptase can use RNA as a template to

mRNA is converted to cDNA by enzymatic reactions

 The product of reverse transcription is RNA-DNA hybrid

synthesize a DNA strand.

Construction of cDNA library

Construction of cDNA library

Pustaka DNA / 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

 Protect bacteria from bacteriophage infection

 Restricts viral replication
 Bacterium protects its own DNA by methylating

Restriction Enzymes

Enzim restriksi endonuklease

 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

those specific sequence motifs

Restriction Enzyme Mechanisms:

Restriction Enzymes

(a)Staggered cut: leaves sticky ends

Restriction enzymes, also called restriction endonucleases,

are enzymes that cut DNA molecules in specific places

(b) Blunt End

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.

Digesti dengan enzim restriksi

Contoh enzim restriksi

Enzim

Asal mikroorganisme

EcoRI

Escherichia coli

G
A-A-T-T-C
G
C-T-T-A-A

5Phosphate
extension

BamHI

Bacillus amyloliquefaciens

G
G-A-T-C-C
C-C-T-A-G
G

5Phosphate
extension

PstI

Providencia stuarti

C-T-G-C-A
G
G
A-C-G-T-C

3Hydroxyl
extension

PvuII

Proteus vulgaris

C-A-GC-T-G
G-T-CG-A-C

Blunt end

Recognition
site

Tipe pemotongan

Primrose, 1994

Ligasi / Penyambungan DNA

1. Dalam kloning tahap ini diperlukan untuk
menyambung DNA target dg DNA plasmid vektor
2. Untuk menyambung DNA digunakan Enzim ligase,
menyambungkan ikatan fosfodiester yang terputus
3. Ujung tumpul kurang efisien penyembungannya
dibanding ujung lancip

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

Cloning 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?

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.

 Antibiotic resistance genes and/or other

selectable markers enable identification of

cells that have acquired the vector construct.

 Some vectors contain an additional eukaryotic

origin of replication allowing autonomous,

episomal replication in eukaryotic cells.
 Multiple unique cloning sites are often included

 Some vectors contain inducible or tissue-

specific promoters permitting controlled

expression of introduced genes in transfected
cells or transgenic animals.

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

Plasmid
 Adalah molekul DNA sirkular untai ganda yang banyak terdapat di dalam

sel bakteri, di luar kromosom

 Selalu membawa 1 gen, merupakan ciri penting bakteri pembawanya.

Misalnya gen tahan antibiotik

 Ukuran di alam 1 kb 250 kb

 Cosmids - an extrachromosomal circular DNA molecule that combines

 Ciri khasnya yaitu mempunyai situs untuk memulai replikasi sendiri

 Bacterial Artificial Chromosomes (BAC) - based on bacterial mini-F

 Guna: mengklon fragmen DNA besar, konstruksi pustaka DNA, subkloning,

 Yeast Artificial Chromosomes (YAC) - an artificial chromosome that

 plasmid yang sekarang digunakan untuk rekonstruksi DNA dimodifikasi

features of plasmids and phage; cloning limit - 35-50 kb

plasmids. cloning limit: 75-300 kb

contains telomeres, origin of replication, a yeast centromere, and a

selectable marker for identification in yeast cells; cloning limit: 100-1000 kb

sehingga mampu memperbanyak diri tidak tergantung kromosom.

memanipulasi DNA, mengkonstruksi DNA.

dari alam, sudah dikurangi atau ditambah dengan sifat tertentu untuk
mempermudah pekerjaan kloning

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

Plasmid vector

Vectors typically include a selectable

marker and a cloning site

 Covalently closed, circular, double stranded DNA

 selectable markers usually are a gene for a product

molecules that occur naturally and replicate

extrachromosomally in bacteria
 Many confer drug resistance to bacterial strains
 Origin of replication present (ORI)

 the cloning site on a vector is engineered with many

Plasmid pUC19

that the host cell cannot make itself, such as an

antibiotic resistance factor

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.

Producing Recombinant DNA

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

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.

Introducing recombinant DNA into Cells

Transformation
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

 DNA-mediated transformation
 Microinjection
 Electroforation
 Transfection

the vector with DNA Ligase. The insert contains a selectable marker which
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
because it is resistant.

 The vector is inserted into a host cell (bacteria), in a process called

transformation. Selectable markers can be for antibiotic resistance, color

changes, or any other characteristic which can distinguish transformed
hosts from untransformed hosts.

 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 rekombinan ke E.coli

 Secara alami bakteri mampu mengambil molekul DNA

dari media tempat tumbuhnya

 E. coli dalam keadaan normal hanya mampu

mengambil DNA dalam jumlah terbatas.

 Harus ada perlakuan fisik dan kimiawi tertentu untuk

meningkatkan kemampuan mengambil sel yang telah

diperlakukan disebut sel kompeten.

Transformasi DNA terkonstruksi ke E.coli

 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

seleksinya dengan ampisilin.

Electroporation

Microinjection


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.

Electric Shock Opens Pores in Cell Wall

Basic method of immunological screening of

recombinants

Microparticle Bombardment

Shoots projectiles of gold or tungsten coated with

DNA or RNA into cells
Generally used with Eucaryotes
Primrose, 1994

DNA status

Analyzing the status and expression of

transferred DNA

 DNA status : autonomous or integrated

 Southern Blotting -- DNA cut with restriction enzymes - probed with

radioactive DNA.

 Total DNA digest with restriction enzymes electrophoresis

 DNA status

transfer DNA into nitrocellulose membrane hybridize with probe.

 RNA analysis

 Probe is single-stranded DNA which is homolog to the DNA of interest.

 Protein: level and biological activity

Technique of Southern Blotting

Southern Blotting
 Digest DNA with restriction endonuclease
 Perform agarose gel electrophoresis of the DNA fragment from

different digests
 DNA fragments fractionated by size visible under UV light if gel

soaked in ethidium bromide

 Transfer (blot) gel to nitrocellulose filter using southern blot

technique
 DNA fragment are bounds to the filter
 Hybridize filter with radioactive labeled probe
 Expose filter to X-ray film resulting autoradiograph from

hybridized DNA fragment

Primrose, 1994

10

Protein: level and biological activity

RNA analysis

 Western blotting Protein - probed with radioactive

 How much is produced and whether it is

or enzymatically - tagged antibodies.

authentic?
 Level of RNA are quantified by Northern blotting.
 Northern Blot RNA - probed with radioactive

DNA or RNA.

 Western blotting: transfer of electrophoresed protein

bands from polyacrilamide gel on to a membrane.

 Total protein extract is separated by polyacrylamid

gel electrophoresis and the protein are stained with

Coomasie blue a new protein encoded by the
transferred DNA

Application of recombinant DNA

technology

Protein Expression
SDS PAGE
kDa M hLF NT 1

kDa M hLF NT 1

100

100
75

DNA technology can be used to cure diseases, to treat genetic

disorders, to improve food crops, and to do many other things that
may improve the lives of humans.

Western Blot
3

rhLF

75

50

50

37

37

rhLF

Production of Human Insulin

1) Obtaining the human insulin gene
Human insulin gene can be obtained by making a
complementary DNA (cDNA) copy of the messenger RNA
(mRNA) for human insulin.








Protein expression
DNA sequencing (gene mapping)
Restriction fragment length polymorphism (RFLP) analysis
Diagnosis of genetic diseases, mutation.
Studies of gene regulation, protein function, etc
Transgenic organism - improved food sources (golden
rice, insect resistance, herbicide resistance etc.)

2) Joining the human insulin gene

into a plasmid vector
 The bacterial plasmids and the cDNA are mixed together.

The human insulin gene (cDNA) is inserted into the

plasmid through complementary base pairing at sticky
ends.

11

3)Introducing the recombinant

DNA plasmids into bacteria
The bacteria E.coli is used as the host cell. If E. coli and the
recombinant plasmids are mixed together in a test-tube.

4)Selecting the bacteria which have

taken up the correct piece of DNA
The bacteria are spread onto nutrient agar. The agar also contains
substances such as an antibiotic which allows growth of only the
transformed bacteria.

12