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REKOMBİNANT DNA
TEKNOLOJİSİ III
Doç.Dr.Öztürk ÖZDEMİR
2004-2005
The Tools of Molecular
Biology
REKOMBİNASYON
Rekombinasyon: Yenibileşim - yenidenoluşum. Bir
molekülün-hücrenin, atasal “wild type” yada
ilkin(orijinal) yapısından farklılık göstermesi
durumudur.
I - In vivo rekombinasyon
II- In vitro rekombinasyon
Klon (Clone);
 Bir tek atasal diploid
hücreden mitoz bölünme yoluyla
birden fazla hücre eldesine denir.
 Rekombinant DNA
teknolojisi ile sentezlenen
identik DNA/gen kopyalarına
denir. ”Gene cloning”
Genetik Klonlamada Tarihçe
Gelişme
Araştırıcı
Yıl
 Deniz hayvanlarında döllenme
O.Hertwig
1875
 İlk kez anne rahmi dışında döllenme L.Schenk
1878
 Tüp ortamında insan yumurta hücresi döllendi MF Menkin 1944
 Dondurulmuş sperm ile inek yumurtası döllendi
1952
 Deney tüpünde döllenen bir memeli yavru doğdu
1959
 Dondurulmuş embriyodan yavru fareler elde edildi
1972
 Louise Brown isimli bebek deney tüpünde döllendi anne
rahmine yerleştirilerek sağlıklı doğum yaptırıldı
1978
 Avusturalyada donmuş embriyodan sağlıklı bir kız çocuğu
elde edildi
1984
Genetik Klonlamada Tarihçe
Gelişme
Yıl
 Kiralık anne Mary Beth bebeğini vermeyi redetti. 1986
 Embriyo hücrelerinin çoğaltılmasıyla çok sayıda kuzu
elde edildi
1987
 İnsan embriyosu klonlandı çok tepki aldı J.Hall
 Dolly klonlandı
1993
I.Wilmut 1997
 Fransada bir dananın 63 klonu elde edildi
 Totipotent stem hücrelerinden deneysel organogenezis
 Farede gen klonlama yöntemiyle insan kulağı gelişimi sağlandı
 Avusturalyada bir at klonlama ile Coada eşek doğurdu
1999
2000
2001
2002
 Amerikada ex vivo yapay rahim geliştirildi
2002
Klonlama Tipleri

DNA /Gen düzeyinde klonlama
 Hücre düzeyinde klonlama
 Organizma /çekirdek düzeyinde
klonlama
Başarılı Klonlama Yapabilmek İçin
Gen;
 Bağımsız olarak replike olabilmeli
 Konak hücreye kolaylıkla transfer
edilebilmeli
 Seleksiyona olanak tanımalı
Memeli Hücrelerine Gen
Transfer Teknikleri
 Microinjection***
 DAAE-Dextran Mediated
 Electroporation
 Lipofection
 Calcium Phosphate***
 Protoplast Fusion
 Polyprene
 Viral infection***
(Lentivirus, Retrovirus, Adenovirus)
*: Yaygın kullanılan yöntemler
Genetik Klonlamada Kullanılan
Vektörler
 Plazmid
 Bakteriyofaj
 Cosmid
 YAC
 Baculovirus
 BAC
 PAC
 Lentivirus **
5-10 kb
20 kb
50 kb
100 kb
150 kb
200 kb
250-300 kb
~ 100-200 kb
The construction of Mammalian Transfection Vector For
Expression of Cytosine- 5 Specific DNA Methyltransferase
Gene M.Msp1 In Cultured Cells
Ozturk OZDEMIR
Received 01.05.1997
STOPLAZMİK KALITIM
(YUMURTA HÜCRESİ)
• Regülatör - modülatör proteinler
• Yumurta polarity genler
• Segmentasyondan sorumlu genler (25 adet)
• Yumurta hücresindeAnterioposterior gradiyent farkı
• Remodelling faktörler ;
•
•
•
•
•
zigotik effect
integrinler
transkripsiyon faktörleri
pair-rule genler
segment polarity genler
• Homeodomeik, Hox (Homeobox) fetusa ait genler
Nükleer Transplantasyon
• Wilmut ve arkadaşları donör hücre olarak 6
yaşında sağlıklı bir koyunun meme epitel
hücresi ve resipient hücre olarak ise aynı
koyunun metafaz II evresinde bekletilmiş
enucleated yumurta hücresi kullandılar.
Klonlama sonrası elde edilen ve annesiyle
%100 aynı genotip ve fenotipte olan
sağlıklı kuzuya DOLLY adını verdiler.
DOLLY
Doç.Dr.Öztürk ÖZDEMİR
Nükleer Klonlamanın Önemi
 Yumurta hücresinin embriyogeneziste spermden
farklı artı(+) öneminin olduğu,
 Ökaryotik hücrenin G0 evresinde totipotent
kromatin organizasyonu kazandıği,
 Metafaz II evresinde yumurta hücresinin
klonlama için en uygun stage olduğu,
 Memelilerde eşeysiz üremenin mümkün olduğu,
 Bir gen yerine çekirdeğin tamamının transplante
olabileceği gösterildi
Klonlama Sonrasında;
 Unipotent hücrenin totipotent hücreye dönüştürülmesi,
 Sinir hücrelerinin rejenerasyonu,
 Telomerlerde ” end replicatin problem”giderilerek,
yaşlanmanın geciktirilmesi,
 Stem hücrelerinden spesifik doku eldesi,
 Epigenetik modifikasyonu ile kanser tedavisine
yeni bir yaklaşım,
“Ex vivo gene replacement” ile genetik tedavi ve
 İnsan genom projesi önemli bir ivme kazanmıştır.
Goals of DNA Technology
1. Isolation of a particular gene or sequence
2. Production of large quantities of a gene product
1. Protein or RNA
3. Increased production efficiency for
commercially made enzymes and drugs
4. Modification/improvement of existing organisms
5. Correction of genetic defects
Amplifying DNA
Often we need large quantities of a
particular DNA molecule or fragment for
analysis. Two ways to do this:-
1. Insert DNA mol. in a plasmid and let it
replicate in host >>> many identical copies
(= ‘DNA cloning’)
2. Use PCR technique - automated multiple
rounds of replication >>> many identical
copies.
DNA Cloning
1. Purpose:- to amplify (bulk up) a small amount of
DNA by inserting it into in a fast growing cell e.g.
bacterium, so as bacterium divides we will have
many copies of our DNA
2. 1. Obtain a DNA vector which can replicate inside
a bacterial cell (plasmid or virus) which
3. 2. Insert DNA into vector - use restriction enzyme
3. 3. Transform host cells i.e. insert vector into host
cell (e.g. bacterium)
4. 4. Clone host cells (along with desired DNA)
5. 5. Identify clones carrying DNA of interest
Vectors are convenient
carriers of DNA. They are
often viruses or plasmids.
Usually are small circular
DNA molecules and must be
capable of replicating in the
host cell
The DNA of interest must
be inserted into the vector.
Restriction Enzymes
Target or recognition
sequence
Restriction enzymes
(R.E.) recognise
target sequences and
cut DNA in a specific
manner.
Cuts here
This R.E. leaves TTAA single stranded ends (‘sticky
ends’)
If you cut DNA of interest and plasmid with same
restriction enzyme then you will have fragments with
identical sticky ends.
Sticky ends will
readily rejoin - so
its possible to join
2 DNA’s from
different sources
AATT
TTAA
Plasmids are
usually chosen to
have only one
target site. DNA of
interest can then
insert into this site
Recombinant plasmid
Transformation of
host and selection of
desired clones
• Bacteria are made to take up the
recombinant plasmid & grown
(cloned) in large numbers
(TRANSFORMATION)
• Bacteria carrying desired sequence
can be selected.
• Large amounts of DNA or
proteins can be extracted
work with gene
work with protein
Making a Genomic Library
Genomic library = a complete
collection of DNA fragments
representing an organism’s
entire genome.
1. Cut up genome into
thousands of fragments with
an R.E.
2. Insert each of these into
separate plasmids and then
into separate host cells.
3. Result - a collection of
bacterial colonies (clones)
carrying all the foreign
DNA fragments i.e.
a genomic library
A question for you - how will a cDNA
library differ from a genomic library ?
• Which would have more genes ?
• What would be present in the clones in each
case?
–
–
–
–
Promoters ?
Enhancers
Introns ?
Poly-T (from poly-A tail)?
How do we identify DNA
mols. of different sizes ?
long short
DNA DNA
Gel Electrophoresis
Standards of known M.W.
Run DNA fragments through a gel
under influence of an electric
current. Each of the DNA
fragments travels through the gel
at a constant speed appropriate
for its size.
Longer molecules move more
slowly so don’t travel as far.
See Fig 20.8
Polymerase Chain Reaction (PCR)
Small amount of DNA can be amplified greatly automated process involves:• A DNA polymerase which is stable at high
temperatures
• specific primers to start off replication at
known position.
• Three step cycle:
1. Heat to separate DNA strands = Denaturation
2. Cool and allow primers to bind (Annealing)
3. Polymerize new DNA strands (Extension)
Repeat steps 25 – 35 times >>> millions of copies of
original DNA
Polymerase chain reaction
Denaturation (95C)
Primer
annealing (50C)
15
Polymerase chain reaction
Extension (72C)
Polymerase chain reaction
Denature
13
Polymerase chain reaction
Denature
Anneal primers
Polymerase chain reaction
Denature
Anneal primers
Extend
Polymerase chain reaction
Denature
Polymerase chain reaction
Denature
Anneal primers
Polymerase chain reaction
Denature
Anneal primers
Extend
Extend
Anneal primers
Denature
Polymerase chain reaction
Bacterial Plasmids
• Plasmids are small, circular DNA molecules in bacteria.
• By inserting genes into plasmids, scientists can combine
eukaryotic and prokaryotic DNA. (Recombinant DNA)
• Bacterial cells continually replicate the foreign gene along
with their DNA.
• Cloning using plasmids can be used to:
– Identify a particular protein a gene makes (ie: for study)
– Produce large amounts of a particular protein/gene (ie:
for use in medicine)
Restriction Enzymes
• Also used to
make
recombinant
DNA.
• Specifically
cut DNA
molecules at
precise base
locations.
(restriction)
Making Recombinant DNA (Fig 20.3)
Making Recombinant DNA (Fig 20.3)
…Still Making Recombinant DNA
…Almost Recombinant
Why Use Bacteria as vectors?
1. Plasmids are easy to use to manipulate
which genes are expressed in clones.
2. Bacteria replicate very quickly and
allow you to produce a large number of
a desired gene.
Identifying Clones
• Not all of the reproduced bacteria are
clones carrying the desired gene.
• Two ways to identify which are clones:
– Look for the gene
– Look for the protein the gene codes
for
Nucleic Acid Hybridization
• If you know the sequence of the cloned gene you are
looking for, you can make a nucleic acid probe with
a complementary sequence.
• The probe is radioactively labeled and allowed to
base pair with the denatured (separated strands)
DNA.
• The probes H-bond with their complement (cloned
gene), thus identifying the cloned cells.
• Identified cells are cultured to produce more.
Figure 20.4 Using a nucleic acid probe to identify a cloned gene
Expressing Euk. Proteins in Bacteria
• It is more difficult to get the bacteria to
translate the proteins because of differences in
promotor sequences b/t prokaryotes and
eukaryotes.
• Expression vectors are plasmids that contain
the promotor sequence just before the
restriction site.
• This allows the insertion of a eukaryotic gene
right next to the prokaryotic promotor.
Expressing Euk. Proteins in Bacteria
• Bacteria also lack the enzymes needed to
remove introns from DNA.
• Therefore, cDNA (no introns) is inserted into
plasmids to allow expression of the eukaryotic
gene.
• Reverse transcriptase is the enzyme used to
make cDNA from a fully processed mRNA
strand.
Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene
Another Solution: Use Yeast (eukaryotic)
• Why?
– They grow quickly like bacteria
– They are eukaryotes (similar enzymes,
metabolic mechanisms, protein mods)
– They have plasmids (rare for eukaryotes)
– Can replicate artificial chromosomes as well
as DNA in plasmids
Genomic Libraries
• Plasmids and phages used to store copies of specific
genes.
Polymerase Chain Reaction (PCR)
PCR
• Faster and more specific method for amplifying
short DNA sequences
• After DNA is denatured (split), primers start new
complementary strands with each strand
producing more molecules of the sequence.
• In vitro = doesn’t require living cells
– In test tube: denatured DNA, free nucleotides,
DNA primers (specific to gene desired),
“special” DNA polymerase (can withstand
high heat w/o denaturing)
Analyzing DNA
• Gel electrophoresis separates molecules based
on size, charge, density, etc.
• Linear DNA – mainly separated by fragment
length (size)
• Molecules of DNA are separated into bands of
molecules of the same length.
Gel Electrophoresis
Restriction Fragment Analysis
Southern Blotting
Southern Blotting
1. Produce restriction fragments of DNA (restriction
enzyme used)
2. Separate fragments (gel electrophoresis)
3. Blotting
 Transfer DNA to nitrocellulose paper

Hybridize with radioactive probes

Autoradiography to identify which have probes.
RFLPs
• Polymorphisms that result from differences in
noncoding regions of DNA.
• Restriction enzymes cut DNA into different
fragments in each variant.
• RFLP markers allowed scientists to more accurately
map the human genome.
• Genetic studies do not have to rely on phenotypic
(appearance/proteins) differences to guide them
anymore.
In Situ (on a slide) Hybridization
• Radioactively (or fluorescently) labeled probes
base pair with complementary denatured DNA
on a microscope slide.
• Autoradiography and staining identify the
location of the bound probe.
Human Genome Project
• Attempt to map the genes on every human chromosome as
well as noncoding information.
• Three stages
– Genetic Mapping (linkage)
– Physical Mapping
– Gene (DNA) Sequencing
• Genomes of species that give insight to human codes are
also being done (fruit fly, E coli, yeast)
Genetic Mapping
• Linkage maps based on recombination
frequencies created.
• Linkage maps portray gene sequences as you
physically move along a chromosome.
• Genetic markers along the chromosome allow
researchers to use them as reference points
while studying other genes.
Physical Mapping
• Determines the actual distance between the
markers along a chromosome (# of bases)
• Utilizes chromosome walking to identify the
distance between.
– Use a series of probes to identify the DNA
sequence of various restriction fragments,
and ultimately the entire length of DNA
sample.
Chromosome Walking
DNA Sequencing
• As of 1998, 3% of the human genome had been
sequenced using automation. (Sanger Method)
• Once the sequences of all the genes are known,
scientists can begin to study all of their functions,
and manipulate their products in many ways.
Applied Genetics
• Diagnosis of Genetic Disorders
– Sequence individuals before birth to know if
their DNA contains abnormalities
• Human Gene Therapy
– Replace missing or fix damaged genes in
affected individuals
Gene Therapy
Pharmaceuticals
• Hormone production (ie: Human Growth)
• Protein supplements
– HIV treatment: “decoy” receptor protein used
to inhibit HIV virus’ ability to enter cell
• Vaccines
– Proteins that stimulate immune response can
be used instead of traditional vaccines
• Antisense Nucleic Acids
– Block translation of certain proteins
Other Uses of DNA Tech
• DNA Fingerprinting for forensic cases
• Environmental cleanup
• Agriculture
– Animal Husbandry
– Genetic Engineering of Plants
The Future of Genetics
• The future of science lies in
genetics???
Microarrays
Microarrays
• See Fig. 20.14
• All known genes are spotted on a small solid
support (chip). Many uses e.g.
• A specific cDNA is tagged with a fluorescent
marker and hybridized to the array
Microarrays
• The cDNAs would bind only to those genomic
clones that have complementary DNA sequences
• These clones would “light up”
Have been used for example to look at cancer cells - which
genes are turned ON or OFF compared to normal cells ?
DNA Sequencing
Uses dideoxy nucleotides to terminate
replication of a chain at a known base.
Automatic sequencing of DNA
Chain termination by dideoxynucleotides
Normal
nucleotides
Dideoxy
nucleotide
New nucleotide can
NOT be added on to
this 3’ end
New nucleotide
can be added on
to this 3’ end
Dideoxy sequencing
All essential components of
DNA synthesis are required, namely...
DNA polymerase
…plus ddNTPs
5
Dideoxy sequencing
Can’t
extend
any
more
Dideoxy sequencing
Result - a series of DNA fragments each 1 base longer than the next
and terminating in a specific ddbase e.g ddT
or ddG
This ddbase is the complementary one to the one on the strand being
sequenced e.g ddT
would be opposite A
Dideoxy sequencing
Heat the mixture to
separate the dd-terminated
strands from the templates
Dideoxy sequencing
• ddRibo-terminated,
-
fluorescent DNAs are
separated by size using
gel electrophoresis
• Bases color coded - easy to
read sequence.
• Sequence here is (from bottom)
CCTAGGAATCC
+
1
DNA Sequencer
machines read the
fluorescence of
each band - store
the sequence in
computers
Vast amounts of data
sequences now on
computer accessible
by online data banks.
Already many
complete genomes
sequenced.
Genome Sizes and Numbers of Genes
Organism
Genome Size
Estimated
Number of Genes
H. influenzae
(bacterium)
1.8 Mb
1,700
S. cerevisiae
(yeast)
12 Mb
6,000
C. elegans
(nematode)
97 Mb
19,000
A. thaliana
(plant)
100 Mb
25,000
D. melanogaster
(fruit fly)
180 Mb
13,000
H. sapiens
(human)
3,200 Mb
30,000 – 40,000
Southern Blotting
• Used to check for the presence
of a specific DNA sequence in a
mixture of DNA fragments.
1. Separate the mix of DNA fragments
by electrophoresis
2. Add a labeled DNA probe. It will attach
to a complementary sequence (if present)
DNA probe
3. The label will make this band ‘light up’
Northern and Western blotting
• Southern blots identify a specific DNA
sequence in a mix of DNAs
In a similar way:• Northern blots identify a specific RNA
sequence in a mix of RNAs
• Western blots identify a specific protein
sequence in a mix of proteins
In-situ hybridization
In situ hybridization probes can bind to
specific sequences on
a chromosome in a cell
prep. - show where it is
located