Recombinant DNA technology

1. Recombinant DNA Technology
Plasmids, Vectors, Cloning and Expression of genes
By,
Abhinava J V
University of Agricultural Sciences, Dharwad

2. Vectors
• Vector is an agent that can carry a DNA
fragment into a host cell in which it is
capable of replication.
• If it is used only for reproducing the DNA
fragment, it is called a cloning vector.
• If it is used for expression of foreign gene, it
is called an expression vector.

3. Properties of a good vector:
• It should be autonomously replicating i.e. it
should have ori region.
• It should contain at least one selectable marker
e. g. gene for antibiotic resistance.
• It should have unique restriction enzyme site
(only one site for one RE) for different REs to
insert foreign DNA.
• It should be preferably small in size for easy
handling.
• It should have relaxed control of replication so
that multiple copies can be obtained.

4. Types of vectors
• Plasmid vectors
• Bacteriophage vectors
• Cosmids
• BACs & YACs
• Mini chromosomes

5. Plasmids
• Plasmids are 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) .
• In their simplest form, plasmids contains a
bacterial origin of replication, an antibiotic
resistance gene, and at least one unique
restriction enzyme recognition site which helps in
cloning.

7. Advantages of Plasmids in Molecular Biology
• Easy to work with - Plasmids are a convenient size
(generally 1,000-20,000 base pairs).
• Self-replicating - Endless number of copies of the
plasmid was obtained by growing the plasmid in
bacteria.
• Stable - Plasmids are stable long-term either as
purified DNA or within bacteria (as glycerol stocks).
• Functional in many species and can useful for
a diverse set of applications - Plasmids can drive
gene expression in a wide variety of organisms,
including plants, worms, mice and even cultured
human cells.

8. Major Limitation of Cloning in Plasmids
 Upper limit for clone DNA size is 12 kb
 Requires the preparation of “competent” host
cells
 Inefficient for generating genomic libraries as
overlapping regions needed to place in proper
sequence
 Preference for smaller clones to be transformed
 If it is an expression vector there are often
limitations regarding eukaryotic protein
expression.

9. PUC 18/19
• It is a circular double stranded DNA and has
2686 base pairs and it includes:
1. A gene for antibiotic resistance to
Ampicillin (ampR).
2. A gene (and its promoter) for the enzyme
beta-galactosidase (lacZ).
3. The lacZ gene contains a polylinker region,
with a series of unique restriction sites called
as multiple cloning site (MCS) found in the
plasmid.

11. pBR322
• It is a circular double stranded DNA and has 4361
base pairs.
• pBR322 also contains the ampR gene (source
plasmid RSF2124)
• The tetR gene (source plasmid pSC101),
• the rop gene encoding a restrictor of plasmid copy
number.
• The plasmid has unique restriction sites for more than
40 restriction enzymes.
• 11 of these 40 sites lie within the tetR gene.
• There are 6 key restriction sites inside the ampR gene

13. Bacteriophage Vector
• 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
• Lambda viral genome: 48.5 kb linear DNA with a
12 base ssDNA "sticky end" at both ends; these ends
are complementary in sequence and can hybridize to
each other (this is the cos site: cohesive ends).
• Infection: lambda tail fibres adsorb to a cell
surface receptor, the tail contracts, and the DNA is
injected.
• The DNA circularizes at the cos site, and lambda
begins its life cycle in the E. coli host.

14.  Advantages:
 Useful for cloning large
DNA fragments
(10 - 23 kbp)
 Inherent size selection
for large inserts
 Disadvantages:
 Less easy to handle

15. Cosmids
• Cosmids - an extrachromosomal circular DNA
molecule that combines features of plasmids and
phage; cloning limit - 35-50 kb
• Advantages:
▫ Useful for cloning very large DNA fragments
(32 - 47 kbp)
▫ Inherent size selection for large inserts
▫ Handle like plasmids
• Disadvantages:
▫ Not easy to handle very large plasmids
▫ (~ 50 kbp)

17. BACs & YACs
• Bacterial Artificial Chromosomes (BAC) -
based on bacterial mini-F plasmids.
cloning limit: 75-300 kb
• Yeast Artificial Chromosomes (YAC) - an
artificial chromosome that contains telomeres,
origin of replication, a yeast centromere, & a
selectable marker for identification in yeast cells;
cloning limit: 100-1000 kb
• Advantages:
▫ Useful for cloning extremely large DNA fragments
(100 - 2,000 kbp)
▫ This is very important for genome sequencing
projects
• Disadvantages:
▫ Not easy to handle extremely large DNA molecules

19. YAC vector
telomere telomerecentromere
URA3ARS HIS3
replication
origin
markers
large
inserts

20. Mini chromosomes
• It is the next generation vector used in
Recombinant DNA Technology.
• A mini chromosome is a small
chromatin-like structure consisting of
centromeres, telomeres and replication
origins and little additional genetic
material.
• They replicate autonomously in the cell
during cellular division.
• Mini chromosome vector having a
cloning limit up to 10 Mb.

22. Cloning
• Molecular cloning is a set of techniques used to
insert recombinant DNA from a prokaryotic or
eukaryotic source into a replicating vehicle such as
plasmids or viral vectors.
• Cloning refers to making numerous copies of a DNA
fragment of interest, such as a gene.
• Cloning is the cutting a piece of DNA from one
organism and inserting it into a vector where it can be
replicated by a host organism.

23. Tools for cloning:
Restriction Enzymes
DNA ligase
Cloning Vectors

24. Restriction Enzymes
• Restriction Enzymes (also called Restriction
Endonucleases) are proteins that cleave DNA
molecules at specific sites, producing discrete
fragments of DNA.
• Each enzyme recognizes and cuts specific DNA
sequences.
• For example, BamHI recognizes the double
stranded sequence:
5'--GGATCC--3'
3'--CCTAGG--5'

25. Where do restriction enzymes come from?
• Restriction enzymes are found in bacteria. Bacteria
use restriction enzymes to kill viruses – the enzymes
attack the viral DNA and break it into useless
fragments.
• They are named after the genus and species of the
organism they were isolated from and are given a
number to indicate the order in which they were
found.
• For example, EcoRI was the first restriction enzyme
isolated from Escherichia coli strain RY13, whereas
HindIII was the third enzyme isolated from
Haemophilus influenzae strain R d.

26. • Restriction enzymes cut through both nucleotide
strands, breaking the DNA into fragments, but they
don’t always do this in the same way.
• Restriction enzyme that cuts straight through the
DNA strands, creating DNA fragments with a flat
or blunt end.
• Restriction enzymes, cut through the DNA strands
at nucleotides that are not exactly opposite each
other. This creates DNA fragments with one
nucleotide strand that overhangs at the end. This
overhanging nucleotide strand is called a sticky
end because it can easily bond with
complementary DNA fragments.

28. DNA ligase
• DNA ligase (molecular glue)-Joins the two pieces of
DNA from different sources together through the
formation of a covalent bond.
• Cells naturally carry out ligation during DNA
replication, when the Okazaki fragments are joined
together.
• Up to 1 million breaks can occur in the DNA of a single
human cell each day.
• Ligases can join any DNA fragments with ‘blunt’ ends.
They can also join DNA fragments with ‘sticky’ ends,
but only if the nucleotides on the strands are
complementary. To get complementary ‘sticky ends’
the DNA fragments to be joined must be cut with the
same restriction enzyme.

29. Cloning Vectors
• It should have unique restriction enzyme site
(only one site for one RE) for different REs to
insert foreign DNA.

30. " RE
Cut plasmid vector
with RE
RE RE
5´ 3´
Excise DNA insert of interest from
source using RE
Ligate the insert of interest
into the cut plasmid

31. General Steps of Cloning with Any
Vector
• Prepare the vector and DNA to be cloned by
digestion with restriction enzymes to generate
complementary ends.
• ligate the foreign DNA into the vector with the
enzyme DNA ligase
• Introduce the DNA into bacterial cells (or yeast
cells for YACs) by transformation
• Select cells containing foreign DNA by screening
for selectable.

32. Methods for gene insertion
Transformation Viruses based
Microinjection Agrobacterium

33. Screening of Recombinant Clones
Blue-White Screening

36. Expression of Gene
• Expression cloning is a technique in DNA cloning
that uses expression vectors to generate a library of
clones, with each clone expressing one protein.
• Gene expression involves:
a) The production of messenger RNA by copying of the
DNA template by RNA polymerase.
b) Translation of the message into protein by the
protein synthesis machinery.
• The transcriptional and translational signals may be
synthetically created to make the expression of the
gene of interest easier to regulate.

37. • After the insert of plasmid, 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.

39. promoter
1 Cauliflower mosaic virus (CaMV)
2 opine promoters
3 plant ubiquitin (Ubi),
4 rice actin 1 (Act-1)
5 maize alcohol dehydrogenase 1 (Adh-1)