PRINCIPLES OF RECOMBINANT DNA TECHNOLOGY

PRINCIPLES OF
RECOMBINANT DNA
TECHNOLOGY
Prof. Dr. Turgut ULUTİN
İ.Ü. Cerrahpaşa Tıp Fakültesi
Tıbbi Biyoloji ABD
Rekombinant-DNA teknolojisinin
gelişimindeki önemli atlama taşları:
1860-1865
Gregor Mendel
Bezelye çaprazlamaları ile
döllere “gen” geçişini ilk kez
ortaya çıkardı
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1953 Watson ve Crick : DNA' nın çift
iplikli
sarmal yapısı keşfedildi.
1957 Kornberg : DNA polimeraz izole
edildi.
1966 Nırenberg ve ark: Genetik kod
çözümlendi.
1967 Gellert
: DNA ligaz izole
edildi.
1970 Hamilton Smith
Restriksiyon enzimlerini (DNA
makasları) keşfetti
DNA yı belli noktalardan
kesebilen enzimler
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1972-73 Jackson ve ark : İlk r-DNA
molekülü
oluşturuldu.
Boyer ve ark : Gen klonlama
tekniğinde plazmidler
keşfedildi
1975 Southern : hibridizasyon
tekniği
geliştirildi. "
Southern Blot " tekniği.
Kan ve ark : ilk prenatal teşhis
1977
Walter Gilbert ve Allan
Maxam , ayrıca Fred
Sanger ayrı ayrı
laboratuvarlarda DNA
nın şifresini çözecek
DNA baz dizisini ortaya
çıkaran “DNA dizileme”
metodunu geliştirdiler
1978 Yuet Wai Kan ve Andree-Marie Dozy
“restriction-fragment-length polymorphism”
(RFLP) yöntemini geliştirdiler
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1979 Goeddel ve ark: ilk rekombinant –
insülin
1982 Palmiter ve Brinster : Transgenik
fareler
Spradling ve Rubin : Transgenik
meyve
sinekleri
üretildi.
1985-1990
Kary Mullis arkadaşları polimeraz
zincir reaksiyonu (PCR) adı verilen , hızlı
DNA klonlaması ve dizi tepitinde önemli bir
teknik geliştirdiler
1986; İlk hastalık geni pozisyonel
klonlama ile tayin edildi ;immun bir
hastalık olan kronik granulomatos
hastalığı.
1987; Rekombinant hepatit B aşısı
üretildi.
1989; ABD de İnsan Genomu Araştırma
Ulusal Merkezi kuruldu
1994; İlk transgenik domates satış izni
aldı
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1993 Stilman ve Hall : İnsan embriyosunun
klonlanması çalışmaları
.
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1997 Wilmut
: Koyun klonlanması (Erişkin
memeli hücrelerinden)
1998 İnsan Genom Projesi (İGP)
başlatıldı
2000 li yıllar: İGP tamamlandı (2003)
DNA çipleri
Farmakogenetikte atılımlar
Kök Hücre - tedavi
Rekombinant DNA teknolojisinde
kullanılan yöntemler

Özel kesici enzimler kullanarak
DNA molekülünün kesilmesi
DNA nın Klonlanması
 DNA - nukleotid dizisinin hızlı bir
şekilde saptanması
 Nukleik asit hibridizasyonu
 Genetik Mühendisliği
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It is useful consider the essential five steps in the
technology:
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Restriction enzyme cutting which is the generation
of DNA fragments in such a way that a particular
gene or DNA segment will be included.
2. Recombinant or the incorporation of these
fragments into a suitable carrier or vector.
Transformation of a host organism, e.g. the
bacterium E. coli , by the recombinant vector.
Cloning which involves growing the transformed
host organism in culture medium so producing
multiple copies of the foreign DNA fragments
incorporated in the recombinant vector.
Finally the selection of clones containing the
relevant DNA fragment.
Generation of DNA fragments:
restriction enzymes
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The most important development in recombinant
DNA when it was recognised that
certain enzymes in particular microbes, cleaved
DNA at sequence specific sites. These enzymes
are called restriction endonucleases and each
restriction endonuclease is designated according
to the organism from which it was derived.

Because of the complementary pairing of bases in the
DNA molecule, the restriction endonucleases always
create double-stranded breaks. Depending on the
particular base sequence which is cleaved either a
staggered end results, when DNA has been cleaved by
a restriction enzyme which produces staggered termini
with complementary nucleotide sequences, these
termini are referred to as being “sticky” since they will
unite with complementary sequences produced by the
same enzyme on DNA if a suitable vector. The
cohesive termini are held together by hydrogen
bonding but are then sealed and stabilised with an
enzyme called DNA ligase. The union of the foreign
DNA fragment with that of the vector produces what
is variously referred to as a hybrid, or recombinant
molecule.
Rekombinant DNA teknolojisinde
kullanılan yöntemler - 1

Özel kesici enzimler kullanarak DNA
molekülünün kesilmesi
HpA I
5' G-T-T * A-A-C 3'
3' C-A-A * T-T-G 5'
Eco RI
5' G *A-A-T-T-C 3'
3' C-T-T-A-A * G 5'
Hind III
5' A *A-G-C-C-T 3'
3' T-T-C-G-G * A 5'
Pst I
5' C-T-G-C-A * G 3'
3' G * A-C -G-T-C 5'
makasları)
Restriksiyon enzimleri (DNA
Restriksiyon enzimleri
Enzim
Elde edildiği kaynak
Tanıma bölgesi
BamHI
Bacillus amyloliquefaciens H
GGATCC
EcoRI
Escherichia coli RY13
GAATTC
HaeIII
Haemophilus aegyptius
GGCC
HindIII
Haemophilus influenzae Rd
AAGCTT
HpaI
Haemophilus parainfluenzae
GTTAAC
HpaII
Haemophilus parainfluenzae
CCGG
MboI
Moraxella bovis
GATC
NotI
Nocardia otitidis-caviarum
GCGGCCGC
SfiI
Streptomyces fimbriatus
GGCCNNNNNGGCC
TaqI
Thermus aquaticus
TCGA
Restriksiyon enzimi (DNA makası) olan EcoRI çembersel
DNA molekülünü keserek yapışkan uçlara sahip doğrusal
şekle getirir
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Different sources of DNA fragments can be used
to make these recombinant molecules.
DNA from nucleated cells is termed total or
genomic DNA made by the action of the
enzyme reverse transcriptase on mRNA is called
complementary or cDNA for short.
Vectors
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A vector is the term used for the carrier of the
foreign DNA fragment used in the cloning process.
Essentially there are three types of vectors used in
this work: plasmids, phages and cosmids. All
replicate within the host bacterial cell and are
therefore sometimes referred to as replicons.
Plasmids occur naturally in bacteria to which they
confer resistance to various antibiotics, heavy
metals, etc. They are stably inherited in an
extrachromosomal state, and consist of a circular
duplex of DNA.
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Bacteriophages are viruses which infect bacteria and
the DNA is usually in the form of a linear duplex.
The may also be used to introduce foreign DNA into
the host cell. The most extensively studied and
utilized is the so-called lambda () phage.
Finally, a cosmid may be used as a vector. A cosmid
is essentially a plasmid which has had all but the
minimum of the vector DNA removed necessary for
propagation to allow or the largest possible foreign
DNA fragment.
Transformation of host organism
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After the foreign DNA has been incorporated into the
plasmid the plasmid is introduced into the host bacterial
cell by exposing the latter to calcium salts which make
the cell membrane permeable to the plasmid. This is
referred to as transformation.
The next step is to grow the host-vector in culture
medium to produce clones . If the restriction enzyme
used to insert the DNA fragment cuts within the drug
resistance gene of the vector then this can be used as a
screening procedure. Thus in pBR322 if the enzyme Pst
I were used to generate DNA fragments and to open up
the plasmid, then any recombinant plasmids produced
would make the bacterial host cell it transforms sensitive
ampicilin but remain resistant to tetracycline. This would
allow one to differentiate the recombinant clones from
those without inserts in which the vector has merely
been relegated to itself since any host cells transformed
by the latter will still be resistant to both antibiotics.
Rekombinant dna teknolojisinde
kullanılan yöntemler - 4
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DNA Klonlaması
Vektör kullanarak canlı hücrede
DNA çoğaltımı ;
*Plazmid
*Virus genetik materyeli
Polimeraz Zincir Reaksiyonu (PCR) ile
çoğaltım işlemi (tüpte)
genetik haritası.
İki tane antibiyotik dirençlilik geni taşır
Plasmid PBR322 nin
Kimerik DNA (veya Rekombinant- DNA) oluşturma:
Plazmid DNA sının yabancı DNA molekülü ile birleştirilmesi
Selection of clones with specific
DNA sequences
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A number of ingenious techniques have been developed
for detecting the insertion of specific DNA sequences. For
example, if the inserted sequence alters the antigenic
properties of the host cell then the recombinant can be
screened for using immunochemical methods. However,
the most widely used method for identifying clones with
specific DNA fragments is by nucleic acid hybridisation .
From the plated colonies of bacteria, a replica is plated on
to a nitrocellulose filter to which nucleic acids. The replica
colonies are then allowed to hybridise with a radioactively
labeled gene-specific DNA or RNA probe and monitored by
autoradiography, exposure to an X-ray .
Once a particular DNA sequence of interest has been
cloned, it is possible using other techniques to “sequence”
it, i.e. determine the order of the individual nucleotides of
that DNA fragment.
Restrıctıon Mappıng

Another tecnique with wide application from our point of view is
that of hybridization with a radioactive probe, using the socalled Southern blot method. Essentially, it consists of
subjecting the fragments of DNA, generated by a restriction
enzyme, to electrophoresis on an agarose gel which separates
the DNA fragments by size. The DNA fragments in the gel are
then denaturated with alkali which makes them single-stranded
rendering them capable of hybridizing with any complementary
DNA sequences. Permanent copy of these single-stranded
fragments is made by transferring them on to a nitrocellulose
filter. Now, in order to localize and visualize a particular
fragment on the filter, a32 P radioactively labeled DNA probe
which has been made single stranded is allowed to hybridise
with DNA fragments in the “Southern blot” . The probe can be
either a cDNA sequence to a specific gene or one produced
synthetically, the nucleotide composition being inferred from
the amino acid sequence of a particular protein, so-called
oligonucleotide probe.

The probe is made radioactive by a process
known as “nick translation” in which 32P labeled
nucleotides are introduced into the DNA
molecule. Wherever the radioactively labeled
probe hybridizes on the nitrocellulose filter this
can be localized by autoradiography.
Rekombinant DNA teknolojisinde
kullanılan yöntemler - 2

DNA - nukleotid dizisinin hızlı bir şekilde
saptanması
Enzimatik Yöntem (Sanger Yöntemi)
Kimyasal yöntem (Maxam-Gilbert Yöntemi)
DNA dizileme
yöntemi
dideoksinukleotid,
2 , 3 dideoksi CTP.
DNA dizileme yöntemi
Rekombinant DNA teknolojisinde
kullanılan yöntemler- 3

Nukleik asit hibridizasyonu:
Southern transferi : DNA hibridizasyonu
Nouthern transferi : RNA hibridizasyonu
Polimeraz Zincir Reaksiyonu (PZR veya PCR)
 Kalıp DNA
 DNA polimeraz (Yüksek ısıya dayanıklı)
 Primerler
 dNTP ler ( CTP-ATP-GTP-TTP)
Rekombinant DNA teknolojisinde
kullanılan yöntemler - 5
Genetik Mühendisliği
Transgenik hayvan ve bitkiler üretmek

Transgen: Yabancı gen (veya DNA)
Transgenik: Yabancı geni taşıyan organizma
Transgenik model oluşturmak:
 Hücrelere yeni gen nakli ( bir gen üzerinde
değişiklik yaptıktan sonra -mutant gennormal genin yerine sokulması) ile farklı
özellikler taşıyan organizmaların
üretilmesi.

Transgenik hayvan oluşturma
yöntemleri;
1- Döllenmiş yumurtaya gen transferi
ile transgenik hayvan (fare) üretimi
2- Embriyonik kök hücrelerine (EKH)
gen transferi ile transgenik hayvan
(fare) üretimi
Transgenik fare: Döllenmiş yumurta hücresine büyüme hormonu
geni eklenen transgenik fare dev boyutlarda.
Rec- DNA Teknolojisinin
Kullanıldığı Alanlar
Table 3.2 Applications of
recombinant DNA technology
123-
4567-
Gene structure/mapping/function
e.g.globin gene
Population genetics
Relation to disease and population structure
Control of genetic disease
prenatal diagnosis
preclinical diagnosis
carrier detection
Diagnosis and pathogenesis of disease
Biosynthesis
e.g. insulin, growth hormone, interferon
Treatment of genetic disease
Insertion of cloned normal gene
Agriculture
e.g. nitrogen fixation
Table 3.3: Single gene disease in man with
their molecular basis
Disorder
Defect
Genetics
Nature of molecular
Dwafism due to growth hormone
deficiency
(rare familial form, associated with
anti- GH antibodies)
AR
Deletion of GH gene
Osteogenesis imperfecta (type
lethal in newborn period)
AR
Partial deletion in collogen genes*
Alpha-1-antitrypsin deficiency
AR
Point mutation
Congenital adrenal hyperplasia due
to 21- Hydroxylase deficiency
AR
Deletion*
Antithrombin III deficiency
AD
Deletion*
Haemophilia A
(bleeding due to Factor VIII
deficiency)
XR
Point mutations and deletion
Haemophilia B
(bleeding due to Factor IX
deficiency)
XR
Point mutation and deletion
Lesch-Nyhan syndrome (HGPRT
deficiency)
XR
Deletion*
Duchenne muscular dystrophy
XR
Deletion*
* indicates deletions have been detected in only a proportion of patients.
Table 3.4 : Proteins produced by
biosynthesis using recombinant DNA
Technology
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Protein
Disease
----------------------------------------------------------------------------
İnsulin
Growth hormone
Factor VIII
FactorIX
İnterferon
Alpha-1-antitrypsin
Somatostatin
Vaccines
İnsulin-depedent diabetes mellitus
Dwarfism due to growth hormone
deficiency
Haemophilia A
Haemophilia B
Infections, cancer (?)
Emphysema due to alpha-1-antitrypsin
deficiency (?)
Excess growth
Hepatitis B, malaria and orher tropical
disease (?)
Rekombinant DNA teknolojisinin
biyomedikal önemi
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Birçok hastalığın moleküler temelini anlamak
(ailesel hiperkolesterolemi, orak hücre
anemisi, talessemi, kistik fibroz, müsküler
distrofi)
Rec-DNA teknolojisi kullanılarak tedavi
etmek (örn: insülin, büyüme hormonu,
plazminojen aktivatörü)
Aşılar üretmek (hepatit B)
Tanı (AIDS testi)
Tedavi (gen tedavisi)
Prenatal tanıda
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Beta
thalessemia,
Duchenne
müsküler
distrofi,
Frajil-X
sendromu
Kanser teşhisinde
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retroviruslar
Onkogenler
Mutant genler
Aşı üretiminde
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Yenebilir aşıların
üretimi
Muz patates gibi
ürünlere hepatit B,
Kolera aşılarının
klonlanması
“Çocuklar ölmesin
Şekerde
yiyebilsinler”
Biyoteknolojik çalışmalarda

Çevre mühendisliğinde biyolojik
savaş
süper bakteriler
oktan, ksilen, naftalin ve karışık
hidrokarbonlar gibi petrol artıklarını
kullanabilen bakteri türlerinden ,
tüm bu özellikleri bir arada taşıyan
mikroorganizma.
Hastalıkların moleküler seviyedeki
patolojik tanıları
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Diyabet
ateroskleroz ve koroner arter
hastalıkları
Alzheimer , şizofreni, parkinson
DNA çipleri
Biyoteknolojik çalışmalarda
İlaç sanayisinde;
“Antitripsin
Kistik fibrozda”

Adli Tıpta
şüpheli suçlunun tayini
veya
babalık tayini
SUMMARY
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1- Recombinant DNA technology involves the
generation of DNA fragments using
sequence specific restriction endonucleases, the
incorporation of these fragments into a suitable
vector (plasmid, phage or cosmid), the introduction
of the vector into a host organism (usually E. coli),
and the subsequent selection of clones containing a
specific DNA sequence.
2- A DNA fragment may be made radioactive (by
labeling with 32P by the process of nick translation)
and can then be used as probe to detect
homologous DNA sequences on an electrophoretic
gel (Southern blot)


3- The technology has been used for analyzing gene
structure, the diagnosis of genetic disease (either
directly or by linkage with restriction fragment
length polymorphisms), research into the molecular
pathology of disease, biosynthesis of medically
important peptides (insulin, growth hormone,
interferon, etc. ) and perhaps one day gene therapy.
4- Concern about the possible biological hazards
and potential abuse of recombinant DNA technology
has lead to guidelines being drawn up for physical
and biological containment.
RFLP (Parça uzunluk polimorfizmi)