Basics of Gene cloning

RECOMBINANT DNA TECHNOLOGY
 Recombinant DNA:
 DNA formed by combining DNA segments from two or more different
sources or different regions of a genome is termed as recombinant
DNA (rDNA).
 rDNA is any artificially created DNA molecule which brings together
DNA sequences that are not usually found together in nature.
 Crossing over produces rDNA under natural condition.
© Dr. Riddhi Datta

 Recombinant DNA Technology:
 A technology which involves joining together of DNA molecules
from two or more different sources that are inserted into a host
organism to produce new genetic combinations that are of
value to science, medicine, agriculture and industry
 This technology was invented in the early 1970s.
 It enables us to enhance the ability of an organism to:
 produce a particular chemical product (ex-penicillin from
fungus)
 prevent synthesis of a chemical product (ex- β-ODAP in
Lathyrus)
 enable an organism to produce an entirely different
product (ex- insulin in microorganism)
© Dr. Riddhi Datta

 Essential Components for cloning a gene:
 Enzyme of cutting DNA fragments:
Restriction endonuclease (RE)
 Enzyme of joining DNA fragments: DNA ligase
 Vehicles for introducing the recombinant
molecule into host cell: Vector
 DNA fragments: Gene libraries
 Selection: Selection of the transformed cells for
the presence of the rDNA
© Dr. Riddhi Datta

 Steps of cloning:
 Identification of the gene of interest (GOI).
 PCR amplification of the GOI with gene specific primer with specific RE
sites (Restriction endonuclease sites).
 Cutting the PCR product (insert DNA) using the specific Restriction
Endonuclease (RE).
 Selecting a cloning vector (a small molecule capable of self-replicating
inside host cells), and cutting the cloning vector with the same RE.
 Incubating the vector and insert DNA together to anneal and then
joining them using DNA ligase. The resultant DNA is called recombinant
DNA.
 Transferring the recombinant DNA to an appropriate host such as
bacteria, virus or yeast which will provide necessary bio-machinery for
DNA replication.
 Identifying the host cells that contain the recombinant DNA.
© Dr. Riddhi Datta

RESTRICTION ENDONUCLEASE (RE)
 Also called restriction enzymes
 1962: “Molecular scissors” discovered in bacteria
 E. coli bacteria have an enzymatic immune system that
recognizes and destroys foreign DNA
 3,000 enzymes have been identified, around 200 have
unique properties, many are purified and available
commercially
 RE: Molecular scissors that cut double stranded DNA
molecules at specific points.
 An important tool for manipulating DNA
© Dr. Riddhi Datta

Nomenclature:
Named for bacterial genus, species, strain, and type:
Example: HinDII (1st RE isolated)
Genus: Haemophilus
Species: influenzae
Strain: D
Order discovered: II
Example: EcoRI
Genus: Escherichia
Species: coli
Strain: R
Order discovered: I
Werner Arbor, Hamilton Smith and Daniel Nathans shared the
1978 Nobel prize for Medicine and Physiology for their
discovery of Restriction Enzymes.
© Dr. Riddhi Datta

 Biological Role:
 Most bacteria use RE as a defence against bacteriophages.
 REs prevent the replication of the phage by cleaving its DNA at
specific sites.
 Restriction Modification System: REs are paired with methylases.
 Methylases are enzymes that add methyl groups to specific
nucleotides within the recognition sequence. The methylation
prevents recognition by the RE. Therefore, the RE within a cell
doesn‟t destroy its own DNA.
© Dr. Riddhi Datta

 RE are bacterial enzymes that recognize specific 4-8 bp
sequences, called restriction sites and cleaves both the
DNA strands at this site (site specific).
 They cleave the DNA within the molecule, hence,
endonucleases.
 RE has 3 functions:
 Recognition
 Cleavage
 Modification
© Dr. Riddhi Datta

Type IIs: Separate endonuclease and methylase; recognition site is
asymmetrical; cleavage occurs on one side of the recognition
sequence upto 20 bp away
© Dr. Riddhi Datta

 Type II Restriction endonucleases:
 Simple enzymes having separate endonuclease and methylase
activities
 Recognise a specific nucleotide sequence and cut a DNA
molecule at this site and nowhere else
 Mostly recognise a hexanucleotide sequence
 Very stable and only require Mg2+ as cofactor
 Many REs make a simple double stranded cut in the middle of
the sequence and result in „blunt ends‟
 Other REs do not cut both strands of the DNA at the same
position and result in „staggered end‟ or „cohesive ends‟ or
„sticky ends‟. Base pairing between these ends can stick the
DNA fragments back together again.
 This type is used for gene cloning.
© Dr. Riddhi Datta

Enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky
ends”
EcoRI 5‟…GAATTC…3‟
3‟…CTTAAG…5‟
Some enzymes cut in a direct fashion – “blunt ends”
PvuII 5‟…CAGCTG…3‟
3‟…GTCGAC…5‟
© Dr. Riddhi Datta

 Uses for Restriction Enzymes:
 RFLP analysis (Restriction Fragment Length Polymorphism)
 DNA sequencing
 DNA storage – libraries
 Gene cloning & Transformation
 Large scale analysis – gene chips
© Dr. Riddhi Datta

DIGESTION CONDITIONS
 XbaI
 Buffer 2: (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT,
pH 7.9).
 100 μg/ml BSA (optional)
 1 Unit digest 1 μg DNA
 Incubate at 37°C for 1 hour
 Heat inactivate 65° for 20 min
20 μl reaction:
10 μl DNA (~1 μg total)
7 μl water
2 μl 10X reaction buffer
1 μl RE 10 units/μl
Incubate 1 hour at appropriate temperature
Note:
1. 10 fold excess enzyme ensures complete digestion.
2. Enzyme should never exceed 1/10th of reactionvolume.
3. BSA is often recommended because it stabilizes the enzyme.
© Dr. Riddhi Datta

Double Digestion for directional cloning
© Dr. Riddhi Datta

ISOSCHIZOMERS AND NEOCHISCHIZOMERS
 Restriction enzymes that have the same recognition sequence as
well as the same cleavage site are Isoschizomers.
 Eg: SphI (CGTAC/G) and BbuI (CGTAC/G) are
 Restriction enzymes that have the same recognition sequence but
cleave the DNA at a different site within that sequence are
Neochizomers.
 Eg:SmaI and XmaI
C C C G G G C C C G G G
G G G C C CG G G C C C
XmaI SmaI
© Dr. Riddhi Datta

Some restriction enzymes may cleave sequences other than their defined recognition
sequence under sub-optimal reaction conditions. In general, these conditions include
high salt concentration, presence of impurities, or excessive enzyme compared to
substrate DNA. This altered specificity is called star activity.
Star activity
Conditions that Contribute to Star
Activity
Steps that can be Taken to Inhibit Star Activity
High glycerol concentration (> 5% v/v) Restriction enzymes are stored in 50% glycerol, therefore the amount of
enzyme added should not exceed 10% of the total reaction volume. Use the
standard 50 µl reaction volume to reduce evaporation during incubation.
High concentration of enzyme/µg of DNA Use the fewest units possible to achieve digestion. This avoids overdigestion
ratio (varies with each enzyme, usually and reduces the final glycerol concentration in the reaction.
100 units/µg)
Non-optimal buffer Whenever possible, set up reactions in the recommended buffer. Buffers with
differing ionic strength and pH may contribute to star activity.
Prolonged reaction time Use the minimum reaction time required for complete digestion. Prolonged
incubation may result in increased star activity, as well as evaporation.
Presence of organic solvents [DMSO,
ethanol, ethylene glycol,
dimethylacetamide, dimethylformamide,
sulphalane]
Make sure the reaction is free of any organic solvents, such as alcohols, which
might be present in the DNA preparation.
Substitution of Mg
2+
with other divalent
cations (Mn
2+
, Cu
2+
, Co
2+
, Zn
2+
)
Use Mg
2+
as the divalent cation. Other divalent cations may not fit correctly
into the active site of the restriction enzyme, possibly interfering with proper
recognition.
© Dr. Riddhi Datta

DNA LIGASE
 During replication DNA ligase catalyses the formation of 3‟ – 5‟
phosphodiester bonds between the short fragments of the
lagging strand of DNA in the replication fork.
 In rDNA technology, purified DNA ligase is used to covalently
join the ends of the restriction fragments in vitro.
 This enzyme catalyzes the formation of 3‟ – 5‟ phosphodiester
bond between the 3‟OH– end of one restriction fragment and
the 5‟ phosphate end of another restriction fragment.
 The process is called ligation.
© Dr. Riddhi Datta

DNA Ligase – enzyme
catalysing formation of
phosphodiesteric bound
between 3‟-OH group of
one end of DNA molecule
and 5‟-phosphate group of
the second end of DNA
Ligase cofactors
1. ATP
• DNA ligases of bacteriophages (phage T4, T7)
• DNA ligases of mammals
2. NAD+
•DNA ligases of bacteria (Escherichia coli, Bacillus subtilis,
Salmonella typhimurium)
• T4 DNA ligase can ligate sticky as well as blunt ends
• E. coli DNA ligase can ligate only blunt ends
© Dr. Riddhi Datta

T4 DNA ligase
• T4 DNA Ligase catalyzes the joining of
two strands of DNA between the 5´-
phosphate and the 3´- hydroxyl groups
of adjacent nucleotides in either a
cohesive-ended or blunt ended
configuration.
• The enzyme has also been shown to
catalyze the joining of RNA to either a
DNA or RNA strand in a duplex
molecule but will not join single
stranded nucleic acids.
Inactivation of T4 ligase:
Heat to 70°C for 10 minutes.
© Dr. Riddhi Datta

 Ligase of phage T4
 Requires ATP as a co-factor
 Optimal pH (7.2-7.8)
 Requires bivalent ions (Mg2+, Mn2+) and reducing factors (β
mercaptoethanol or ditiotreitol)
 Inhibitors: polyamines (spermin, spermidine), high
concentration of ions (Na+, K+, Li+, NH4+)
 Can connect both cohesive and blunt ends (but for blunt ends
reaction is slower and requires higher concentrations of enzyme)
Typical ligation reaction:
COMPONENT 20 μl REACTION
T4 DNA Ligase Buffer (10X)* 2 μl
Digested Vector DNA (4 kb) 50 ng (0.020 pmol)
Digested Insert DNA (1 kb) 37.5 ng (0.060 pmol)
Nuclease-free water to 20 μl
T4 DNA Ligase 1 μl
Incubate at 16°C overnight or room temperature for 2 hours
© Dr. Riddhi Datta

What should be optimized for a successful ligation:
1. The ratio of the molar concentration of vector to insert.
- Optimum ratios may vary from 8:1 to as high as 1:16 vector:insert,
though generally fall in the range of 3:1 to 1:3.
2. Amount of DNA.
- Usually 10-200 ng of plasmid is used for reaction.
3. Volume of reaction.
- Usually a minimal volume is recommended (e.g. 10 µl).
4. Amount of ligase.
- Each ligation reaction generally requires 1-10 units of high quality
ligase.
5. Incubation time and temperature.
The ligation incubation time and temperature may also need to be
optimized. In general:
- blunt-ended ligations are performed at 4°C overnight;
- - sticky-end ligations are performed for 1-3 hours (at 22ºC or 16ºC)
or overnight at 4°C.
- In general, ligation reactions performed at lower temperatures
require longer incubation times.
© Dr. Riddhi Datta

Recombinant DNA molecule
One possible combination
Restriction site
DNA
5
5
5
5
5
5
5
5
seals strands
5
5
3
3
3
3 3
3
3
3
533
3
3
2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
3 DNA ligase
1 Restriction enzyme
cuts sugar-phosphate
backbones.
5 Sticky 3
end
5
GAATTC
CTTAAG
35
G AATT C
C TTAA G
53
35
G AATT C
C TTAA G
© Dr. Riddhi Datta

CLONING VECTORS
 A self replicating DNA molecule attached with a foreign
DNA fragment to be introduced into a cell
 Has features that make it easier to insert DNA and selectfor
presence of vector in cell:
 Origin of replication
 Antibiotic resistance gene
 Cloning site (MCS)
 Promoters and terminators for expression of the cloned
gene
© Dr. Riddhi Datta

For expression
of the inserted
gene, it should
have suitable
control elements
like promoters
and terminators.
4
© Dr. Riddhi Datta

Types of cloning vectors:
1. Plasmids (about 10kb)
2. Bacteriophage (bacterial viruses), 30-50kb inserts
3. Cosmids (35-50kb insert)
4. Bacterial Artificial Chromosomes (BACs)
o Use fertility F plasmid
o 75-300kb inserts possible
o Developed during the human genome project
5. Yeast Artificial Chromosomes (YACs)
o Mimics yeast chromosome
o Contains all regions for replication (yeast ori
and centromere)
o 100-1000kb inserts poss.
o developed during the human genome project
© Dr. Riddhi Datta

Plasmid
 Plasmids are double stranded, closed, circular DNA molecules
which exist in the host cell as extrachromosomal units.
 They are self-replicating, can be single or multi-copy per cell.
 Some plasmids are under relaxed replication control thus
permitting accumulation of huge copy numbers (upto 1000 copies
per cell). These are preferred in cloning because of their high yeild.
 1-200 kb in size
 Depend on the host proteins for replication and maintenance.
 All naturally occurring plasmids do not always contain all the
essential properties of a suitable cloning vector.
© Dr. Riddhi Datta

 Derived from naturally occurring plasmids
 Altered features
 small size (removal of non-essential DNA) higher
transformation efficiency, manipulation and purification
easier
 unique restriction enzyme sites
 one or more selectable markers
 other features: promoters, etc.
 Can hold up to 10 kb fragments
Plasmid Cloning Vectors
© Dr. Riddhi Datta

pBR322
 One of the most widely used standard artificial cloning vector
 It is a 4.36 kb double stranded plasmid vector originating from
fragments of three naturally occurring plasmids
 It has genes for resistance against two antibiotics: tetracycline and
ampicilin
 It contains 20 RE sites, six of which are located within the gene
coding for tetracycline resistance, two sites lie within the
tetracycline promoter and three within β-lactamase gene
© Dr. Riddhi Datta

pUC vectors
 Its name originated from University of California from where it
was developed.
 2.7 kb in size and possess:
 Gene for ampicilin resistance
 lacZ gene (coding for β-galactosidase gene) for blue/white
selection
 Origin of replication from pBR322
 Within the lacZ region there is a polylinker having unique RE
sites
© Dr. Riddhi Datta

Shuttle vectors
A shuttle vector is a vector (usually a plasmid) that can propagate in two
different host species. Thus the DNA fragment inserted into it can be
manipulated or tested in two different cell types.
The main advantage of using such a vector is that it can be manipulated
in E. coli and then introduced into a system which is more difficult or
slower to use (ex- yeast)
Thus a shuttle vector can propagate in eukaryotic and prokaryotic hosts
and between different species of bacteria.
A shuttle vector is frequently used to make quickly multiple copies
(amplification) of the gene in E. coli. They can also be used for in vitro
experiments and modifications.
© Dr. Riddhi Datta

Yeast shuttle vector
Common yeast shuttle vectors contain:
 Bacterial origin of replication
 Bacterial selectable marker (antibiotic resistance)
 ARS (autonomously replicating sequence)
 Yeast selectable marker (LEU2)
 Yeast centromere
© Dr. Riddhi Datta

• Upper limit for insert 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 and post
translational modification
Major Limitation of Cloning in Plasmids
© Dr. Riddhi Datta

Phage Cloning Vectors
 Fragments up to 23 kb can be may be accommodated by a phage
vector
 Lambda is most common phage
 Lambda phage is a virus that infects bacteria (E. coli)
 In 1971 Alan Campbell showed that the central third of its genome
was not required for lytic growth.
 People started to replace it with E. coli DNA (stuffer DNA)
 The stuffer fragment keeps the vector at a correct size and carries
marker genes that are removed when foreign DNA is inserted into
the vector.
 Stuffer DNA contains lacZ gene. When intact, beta-galactosidase
reacts with X-gal and the colonies turn blue.
 When the DNA segment replaces the stuffer region, the lacZ gene
is missing, which codes for beta-galactosidase, no beta-
galactosidase is formed, and the colonies are white.
© Dr. Riddhi Datta

Bacteriophage lambda (λ)
 Lambda genome is
approximately 49 kb
in size.
 Only 30 kb is
required for lytic
growth.
 Thus, one could
clone 19 kb of
“foreign” DNA.
 Packaging efficiency
78%-100% of the
lambda genome.
 Cos site: At the ends
short (12bp) ss-
complementary
region “cohesive or
ends---
after
sticky”
circulation
infection
© Dr. Riddhi Datta

LAMBDA AS A CLONING VECTOR
 Insertional vectors:
 (clone into one or multiple restriction sites
 can only increase genome size by 5%
 size of foreign DNA insert depends on the original size of the phage
vector, about 5 to 11 kb
© Dr. Riddhi Datta

LAMBDA AS A CLONING VECTOR
 Replacement vectors (removing “stuffer”):
 can clone larger pieces of DNA, 8 to 24 kb (sufficient for many
eukaryotic genes)
© Dr. Riddhi Datta

Cosmid Cloning Vectors
 Cosmids are plasmids that can be packaged into λ phage and they
combine essential elements of a plasmid and λ systems (cos sites).
 Concatemer of unit length λ DNA molecules can be efficiently
packaged if cos sites are 37-54 kb apart.
 Fragments from 30 to 46 kb can be accommodated by a 5 kb cosmid
vector.
 Cosmids are extracted from bacteria and mixed with restriction
endonucleases.
 Cleaved cosmids are mixed with foreign DNA that has been cleaved
with the same endonuclease.
 Recombinant cosmids are packaged into lambda caspids.
 Recombinant cosmid is injected into the bacterial cell where the
rcosmid arranges into a circle and replicates as a plasmid. It can be
maintained and recovered just as plasmids.
© Dr. Riddhi Datta

Yeast Artificial Chromosomes (YACs)
YACs are vectors constructed from yeast (Saccharomyces cerevisiae)
chromosomes to clone large DNA fragments
They are constructed as circular DNA molecule by assembling the
essential functions of natural yeast chromosomes, then splicing in a
fragment of foreign DNA.
This engineered chromosome is reinserted into a yeast cell to produce
the YAC.
© Dr. Riddhi Datta

Specific sequences of YAC:
Telomeres: Located at the two ends of each chromosome. They have
evolved as a device to preserve the integrity of the ends of DNA
molecule, i.e. to protect the linear DNA from degradation by
nucleases
Centromere: Attachment site of mitotic spindle fibers. They pull
one copy of each duplicated chromosome into each new daughter
cell.
Origin of replication: Specific DNA sequence that allows DNA
replication machinery to assemble on the DNA.
Selectable markers: Allows easy selection of the yeast cells that
have taken up the YAC
RE sites: For insertion of the foreign DNA
© Dr. Riddhi Datta

Bacterial Artificial Chromosomes (BACs)
 BAC is a cloning vector in E. coli developed as an alternative to YAC
vector for mapping and analysis of complex genomes.
 BACs are maintained in E. coli as large single copy plasmid that
contains inserts of 50 – 350 kb with a high degree of stability.
 A number of human and plant BAC libraries have been constructed.
Ex- Human, Arabidopsis, Rice, etc.
 BAC system is based on the single copy sex factor F of E. coli (~100 kb
circular ds DNA)
 The synthetic BAC vectors (~7.5 kb, double stranded) contains
replication origin OriS and gene repB of F plasmid for the initiation
and proper orientation of replication of BAC vector.
 The parB and parB genes of F plasmid that ensure efficient segregation
of the F plasmid into the daughter E. coli cells after replication are also
incorporated into the BAC vector.
© Dr. Riddhi Datta

This vector also includes the following:
 λcosN (single stranded, complementary extensions of λ phage DNA for
packaging dependent cleavage)
 P1 lox P sites (recognized by phage dependent recombinants)
 2 cloning sites (HinDIII and BamHI)
 Several G+C RE sites
(SfiI, NotI, etc.) for
potential excision of
the inserts
 Cloning sites are also
flanked by T7 and SP6
promoters for
generating RNA probes
 Selectable marker
genes for antibiotic
resistance (CMR)
 lacZ gene (for
blue/white selection)
© Dr. Riddhi Datta

PAC:
 P1 Artificial Chromosome (derivative of bacteriophage P1)
 These vectors are constructed using DNA of P1 bacteriophages.
 Can carry inserts 80-kb to 100-kb
 These vectors contain essential replication components of P1phage
incorporated into plasmid.
 PAC was developed as a cloning vector by Nat Sternberg and
colleagues in the 1990s.
© Dr. Riddhi Datta

TA CLONING
Taq polymerases often add a single deoxyadenosine, in a template-independent
fashion, to the 3´-ends of the amplified fragments. The pre-linearized TA vector
contains 3´-T overhangs at the insertion site to provide a compatible overhang for
PCR products.
Reaction Component Standard
Reaction
2X Rapid Ligation Buffer, T4 DNA
Ligase
5µl
pGEM®-T or pGEM®-T Easy Vector
(50ng)
1µl
PCR product (insert) Xµl*
T4 DNA Ligase (3 Weiss units/µl) 1µ
nuclease-free water to a final volumeof 10µl
© Dr. Riddhi Datta

CLONING A GENE WITH KNOWN SEQUENCE:
A PRACTICAL EXAMPLE
 Select a pair of restriction enzymes: Present in the MCS of the
binary vector and not within the gene of interest
 Design gene specific primers: Manually or through software (Eg-
Primer3); Add selected RE sites in the 5’ end of the forward and
reverse primers
 PCR amplify the gene of interest.
 Run agarose gel and gel purify the desired band (insert DNA)
 TA cloning of the insert DNA into TA cloning vector
 Transform the ligation product into competent E. coli cells
 Screen for positive E. coli colonies that contains the TA clone
 Double digest the TA clone with the selected pair of REs and gel
purify the gene fragment (insert)
 Double digest the binary vector with the selected pair of REs and
gel purify the digested vector fragment
 Ligate the digested insert and vector with T4 DNA ligase
 Transform the ligation product into competent E. coli cells
 Screen for positive E. coli colonies that contains the final clone
 Confirm the clone by PCR, RE digestion and sequencing
 Maintain the E. coli cells containing the clone as glycerol stock© Dr. Riddhi Datta

Basics of Gene cloning

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Basics of Gene cloning