1. Molecular cloning involves cutting DNA from one organism and inserting it into a vector that can replicate in a host organism, allowing the DNA fragment to be amplified. Recombinant DNA technology uses restriction enzymes to cut DNA into fragments that are then ligated into cloning vectors like plasmids or bacteriophages. 2. After transforming host bacteria with the recombinant vector, clones containing the inserted DNA fragment can be selected for and amplified. Colonies containing the insert are identified through antibiotic resistance or colorimetric markers present on the vector. 3. cDNA libraries provide a way to clone and study eukaryotic genes. mRNA is isolated and reverse transcribed into cDNA, which is then ligated into vectors and

1. MOLECULAR
CLONING

2. DEFINING
CLONING
 Cutting a piece of DNA from one organism
and inserting it into a vector where it can be
replicated by a host organism. (Sometimes
called subcloning, because only part of the
organism’s DNA is being cloned.)
 Using nuclear DNA from one organism to
create a second organism with the same
nuclear DNA

3.  Recombinant DNA technology depends on the ability to
produce large numbers of identical DNA molecules (clones).
 Clones are typically generated by placing a DNA
fragment of interest into a vector DNA molecule, which
can replicate in a host cell.
 When a single vector containing a single DNA fragment is
introduced into a host cell, large numbers of this fragment
are reproduced along with the vector.
 Two commonly used vectors for cloning are E. coli
plasmid vectors and bacteriophage  vectors
DNA restriction cutting, plasmid, phage, DNA ligation, antibiotic
selection, cDNA, cDNA Library, expression vector
Molecular
Cloning

4. First recombinant DNA produced:
Stanley Cohen and Herbert Boyer cut 2 plasmids with EcoRI,
ligate and screen for E.coli clones that are resistant to both
antibiotics since they harbor the recombinant plasmids.
Patent 4,237,224, 1980
http://www.dnai.org/text/mediashowcase/index2.html?id=1186
1973 PNAS 70, 3240

5. RESTRICTION
ENZYMES
Restriction Enzymes (also called
Restriction Endonucleases) are proteins
that cleave DNA molecules at specific
sites, producing discrete fragments of
DNA.
Restriction Enzymes (RE) were first
isolated by Nathans and Smith in 1970.

6. WHYRESTRICTION
ENZYMES?
Why would bacterial cells contain
proteins that cleave DNA at specific
sequences?
Generally restriction enzymes are thought t
o
protect bacterial cells from phage (bacterial
virus) infection. Bacterial cells that contain
restriction enzymes can “cut up” invasive
viral DNA without damaging their own DNA.

7. ISOLATING
GENES
 Herbert Boyer and Stanley Cohen
built on the work of Berg, Nathans
and Smith to use restriction
enzymes to isolate a single gene and
ligate it to a plasmid.
 Bacterial cells were then transformed
with the recombinant plasmid.
.

8. THE BACTERIA HOST CELLS
REPLICATED THE PLASMID,
PRODUCING MANY COPIES OF THE
GENE, THUS AMPLIFYING IT.
The practical application was that expensive
human protein products, like insulin, which
were used to treat disease, could eventually
be produced from recombinant molecules in
the laboratory using bacteria or another host.
Human protein products like insulin could be
used in very large quantities from the
recombinant molecule. Patients no longer had
to use insulin isolated from pigs or cows.

9. PERFORMING THE
RESTRICTION DIGESTS
You will need to set up a restriction
digest of your plasmid vector and your
DNA of interest
Restriction enzymes all have specific
conditions under which they work best.
Some of the conditions that must be
considered when performing restriction
digest are: temperature, salt
concentration, and the purity of the DNA

10. PURIFY YOUR DNA
FRAGMENTS
 The insert of interest that you want to clone
into your plasmid needs to be separated from
the other DNA
 You can separate your fragment using Gel
Electrophoresis
 You can purify the DNA from the gel by cutting
the band out of the gel and then using a variety of
techniques to separate the DNA from the gel
matrix

11. Ligase mechanism:
DNA ligase forms activated
ligase-AMP (from ATP or
NAD), AMP links to 5’-P, 3’-
OH attacks forming new
phosphodiester bond, seal!
Ligation of the sticky ends:
two DNA molecules, cleaved
with EcoRI and ligate to form
recombinant molecules

12. Addition and cleavage of a chemically synthesized
linker. Producing sticky ends with adaptors.
Formation of cohesive ends

13. JOINING DNA
FRAGMENTS
In 1972, Paul Berg and colleagues made the first
“artificial” recombinant DNA molecule.
Demonstrated that the DNA of Simian virus 40 (SV40)
could be linearized by EcoRI
Created a circular DIMER of SV40 DNA by
joining two linearized fragments
Also inserted pieces of Lambda phage DNA into
linearize molecule.

14. Plasmid: circular, ds, extrachromosomal self-replicating DNA molecule occuring naturally in
bacteria, yeast and higher eukaryotic cells, either parasitic or symbiotic, ~kb to 100 kb, at least
one to the daughter cell (drug-resistance, transfer genes encoding proteins forming
macromolecular tube or pilus).
Plasmid and cloning vector
Cloning vector: engineered plasmids with reduced size (~3 kb), containing only ori, drug-
resistance gene, cloning site. Ampr encodes -lactamase which inactivates ampicillin.

15. LIGATION
Ligation is the process of joining two
pieces of DNA from different sources
together through the formation of a
covalent bond.
DNA ligase is the enzyme used to
catalyze this reaction.
DNA ligation requires ATP.

16. Ligase reaction: opposite to a restriction enzyme, requires 5’-P
(from DNA2) and 3’-OH (from DNA1).

17. PLASMID
VECTORS
Plasmids are circular pieces of DNA
found naturally in bacteria.
Plasmids can carry antibiotic resistance
genes, genes for receptors, toxins or
other proteins.
Plasmids replicate separately from the
genome of the organism.
Plasmids can be engineered to be
useful cloning vectors.

18. PLASMID VECTORS
(CONTINUED)
Plasmid vectors can be designed with a
variety of features:
Antibiotic resistance
Colorimetric “markers”
Strong or weak promoters for
driving expression of a protein

19. Antibiotic Resistance Markers
Antibiotic
Resistance
Gene

20. MULTIPLE CLONING
REGION
Multiple
Cloning
Region
The cloning marker for this plasmid is the lacZ gene.

21. CLONING A PIECE OF
DNA
AvaI
Cut plasmid vector
with AvaI
AvaI AvaI
5´ 3´
Excise DNA insert of interest from
source using Ava I
Ligate the insert of interest
into the cut plasmid

22. TRANSFORMING
BACTERIA
After you create your new plasmid construct that
contains your insert of interest, you will need to
place it into a bacterial host cell so that it can be
replicated.
The process of introducing the foreign DNA into
the bacterial cell is called transformation.

23. COMPETENT HOST
CELLS
Not every bacterial cell is able to take up
plasmid DNA.
Bacterial cells that can take up DNA
from the environment are said to be
competent.
Can treat cells (electrical
current/divalent cations) to increase the
likelihood that DNA will be taken up
Two methods for transforming: heat
shock and electroporation

24. SELECTING FOR
TRANSFORMANTS
The transformed bacteria cells are
grown on selective media (containing
antibiotic) to select for cells that took up
plasmid.
For blue/white selection to determine if
the plasmid contains an insert, the
transformants are grown on plates
containing X-Gal and IPTG. (See notes
for slide 11.)

25. DNA Libraries in  phage and other
cloning vectors
• Cloning all of the genomic DNA of higher organisms
into plasmid vectors is not practical due to the
relatively low transformation efficiency of E. coli and
the small number of transformed colonies that can be
grown on a typical culture plate
• Cloning vectors derived from bacteriophage do not
suffer from such limitations
• A collection of clones that includes all the DNA
sequences of a given species is called a genomic
library
• A genomic library can be screened for clones
containing a sequence of interest

26. (i)EM of bacteriophage  virion.
(ii)Simplified view of bacteriophage genome:about 60 genes, replaceable region
not essential and can be replaced by foreign DNA up to 25 kb. Large segment of
the 48kb DNA of the  phage are not essential for productive infection and can be
replaced with inserts.
(iii)Assembly of bacteriophage  virion: during late stage of  infection
concatomers are formed, Nu 1 and A protein push 1 copy of  from concatomer
into the head, then tail added and they are ready to go to the war.

27. CLONING

28. Charon phage 4 takes 20 kb,
remove the stuffer,
ligate the insert,
mix with the in vitro
packaging extract,
infect cells,
the insert has to be between
12 kb and 20 kb.
 phage vector

29. Alternative infection modes for  phage:
lytic or lysogenic pathway

30. A set of lambda (or plasmid)
clones that collectively
contain every DNA
sequence in the genome of
a particular organism.
Nearly complete genomic
libraries of higher
organisms can be prepared
by lambda cloning.

31. Genomic Library
(i)A set of lambda (or plasmid) clones that
collectively contain every DNA sequence in
the genome of a particular organism. Nearly
complete genomic libraries of higher
organisms can be prepared by lambda
cloning.
(ii)Construction of a genomic library:
Sau3A and BamH1 generate
complementary sticky ends.

32. (iii) Plaque hybridization: selection of positive clones,
filter replicates the dish, plaque containing phage DNA
denatured by base, block the filter with non- specific
DNA or protein, hybridize with specific gene probe,
autoradiography to select the target .gene.
(iv) The most common approach to identifying a
specific clone involves screening a library by
hybridization with radioactively labeled DNA or
RNA probes. Identification of a specific clone
from a l phage library by membrane
hybridization. Identifying, analyzing, and
sequencing cloned DNA

33. 


Larger DNA fragments up to 45
kb can be cloned in cosmids and
other vectors,
cosmid has cohesive ends for
packaging and plasmid ori for
replication as plasmids in
bacteria.
cosmids cannot replicate as
phages but they are still
infectious.
COSMID VECTOR

34. M13 Phage: a filamentous
virus 900 nm long and 9 nm
wide, single strand 6.4kb
circle DNA protected by 2710
identical proteins, gets into E.
coli via sex pilus, replicate to
RF, only (+) is packed into
virus particle. Useful for
sequencing.
M13 Phage

35. oligo-dT column to purify mRNA
Complementary DNA (cDNA) libraries
are prepared from isolated
mRNAs
Complementary DNA

36. Complementary DNA (cDNA) libraries
Formation of cDNA duplex: RT, alkali digestion, oligo(dG) tailing, oligo(dC) primer

37. Preparation of a cDNA library

38. Making a cDNA Library: Reverse
Transcriptase, RNase H (degrade RNA in
RNA-DNA hybrid) , DNA polymerase I (using
RNA as primer and perform nick translation),
Terminal deoxynucleotidyl transferase (adding
dCTP to the cDNA duplex, and dGTP to the
vector), Ligase and pol I in the host perform
ligation, RNA removal, and sealing.
Making a cDNA Library

39. Use RT-PCR to clone a
single cDNA if the sequence
of mRNA is known.

40. pBS or pBluescript, MCS inserted into lacZ’, ori of the ss phage f1, T3 and T7 phage
RNA polymerase promoters.
Phagemid

41. (i) Forming fusion protein in plasmid vector: pUC and pBS vectors place inserted
DNA under the control of the lac promoter. If the DNA is in frame with the lacZ’
gene, a fusion protein will be expressed.
The expression vector contains a fragment of the E. coli chromosome containing the
lac promoter and the neighboring lacZ gene. In the presence of the lactose analog
IPTG, RNA polnormally transcribes the lacZ gene, producing lacZ mRNA, which is
translated to the encoded protein, -galactosidase.
Expression vector

42. The lacZ gene can be cut out of the expression vector with restriction enzymes
and replaced by the G-CSF cDNA. After transformation, IPTG will induce the
expression of G-CSF protein.
E. coli expression systems can
produce full-length proteins:
Producing high levels of proteins
from cloned cDNAs. Many
proteins are normally expressed at
very low concentrations within
cells, which makes isolation of
sufficient amounts for analysis
difficult. To overcome this problem,
DNA expression vectors can be
used to produce large amounts of
full length proteins. Lac promoter.

43. Even larger amounts of a desired protein can be expressed with a two-step
system, 10-70% of the total protein synthesized by these cells after IPTG is the
protein of interest.
Two-step system : T7 RNA polymerase and T7 late promoter

44. Forming fusion protein in phage vector: g11with lac control region followed by lacZ gene,
cloning sites are located within the lacZ gene, direct detection of protein with antibody.

45. Degenerate probe: screen for the correct cDNA or genomic
clone from the library.

46. EXPRESSING YOUR CLONED
GENE
Even if your plasmid contains insert, it
may not be able to generate functional
protein from your cloned DNA.
The gene may not be intact, or mutations
could have been introduced that disrupt it.
The protein encoded by the gene may
require post-translational modifications (i.e.,
glycosylation or cleavage) to function.
Also, some enzymes are a complex of
peptides expressed from separate genes.