3. A series of procedures used to recombine
DNA segments. Under certain conditions, a
recombinant DNA molecule can enter a cell
and replicate.
Definition of recombinant DNA
technology
4.
5. Basic principle of 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(called cloning)
6. 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)
9. Cloning Vectors
A vector is used to amplify a single molecule
of DNA into many copies. A DNA fragment
must be inserted into a cloning vector. A
cloning vector is a DNA molecule that has an
origin of replication and is capable of
replicating in a bacterial cell.
10. Cloning Vectors
Most vectors are genetically engineered
plasmids or phages. There are also cosmid
vectors, bacterial artificial chromosomes, and
yeast artificial chromosomes.
11. 2,686 bp
pUC 18
A Typical Plasmid
Lac Z
Gene
Multiple Cloning
Site
aagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaat
HindIII SphI PstI SalI XbaI BamHI XmaI KpnI SstI EcoR
AccI SmaI BanII
HincII
BspMI
Origin
of Replication
Ampr
Gene
13. Enzymes are the Tools of DNA
Technology
The tools of DNA technology are the same enzymes
used by cells to modify their own DNA:
DNA Polymerases
DNA Ligase
One special class of enzyme is pivotal to the cloning
of DNA and many other techniques used in DNA
Technology
These enzymes are the restriction
endonucleases
14. Restriction Endonucleases
There are a number of different sub classes
of restriction endonucleases
Type II restriction endonucleases are the
most useful class as they recognize specific
palindomic sequences in DNA and cut the
sugar phosphate backbone within the
palindrome
15. What is a Palindrome?
A palindrome is anything that reads the
same forwards and backwards:
English palindromes:
Dad
Able was I ere I saw Elba (supposed said by
Napoleon)
16. DNA Palindromes
Because DNA is double stranded and the
strands run antiparallel, palindromes are
defined as any double stranded DNA in
which reading 5’ to 3’ both are the same
17. DNA Palindromes
Some examples:
The EcoRI cutting site:
–
5'-GAATTC-3'
–
3'-CTTAAG-5'
The HindIII cutting site:
–
5'-AAGCTT-3'
–
3'-TTCGAA-5'
18. Uses of Restriction
Endonucleases
Because restriction endonucleases cut
specific sequences they can be used to make
“DNA fingerprints” of different samples of
DNA. As long as the cutting site changes on
the DNA or the distance between cutting
sites changes, fragments of different sizes
will be made.
19. Gel Electrophoresis
Separates DNA (or RNA or Protein)
fragments on the basis of charge and
size
Because DNA is an acid, it looses
protons in basic buffers, thus it has a
negative charge that should be uniform
per unit length
27. Uses of Restriction
Endonucleases
Because Type II restriction endonucleases
cut only at palindromes, they leave “sticky
ends” that will base pair with any other
fragment of DNA cut with the same enzyme.
This is useful in cloning.
28. G
CTTAA
AATTC
G
1 Digestion
2 Annealing of sticky ends
3 Ligation
Ligase
G
CTTAA
AATTC
G
EcoRI
EcoRI
R. E.s and DNA Ligase
Can be used to make recombinant DNA
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
AATTC
G
4 Recombinant DNA
29. Host Cell
Cloning Into pUC18
pUC18
LacZ
Ampr
R. E.
Digestion
Addition of ligase joins
nicks and makes a
single recombinant
plasmind
R. E.
Digestion
Matching sticky
ends anneal
Transformation
of cells with the
recombinant
plasmid
31. Libraries
If all the DNA from an organism is
digested with a restriction enzyme and
cloned into a plasmid, many different
recombinant plasmids will be made,
each with a different fragment of DNA
cloned into it
Once inserted into host cells , this
collection of many different recombinant
plasmids is called a “library”
33. cDNA Libraries
Because of the large size of the
genomic libraries and the tedium of
screening, anything that can be done to
limit library size is a good thing
Protein coding regions of most
eukaryotic genomes make up only a
small percentage of the total DNA (2%
in humans)
34. cDNA Libraries
Most cells only express a small subset
of an organism’s genes
By using reverse transcriptase, a cDNA
copies of the mRNA being produced in a
group of cells can be made
Cloning cDNA to make a library
produces a much smaller library
enriched with the part of an organism’s
genome that is of most interest
