Basics of Cloning

V. Dinesh Kumar
Principal Scientist (Biotechnology)
ICAR-Indian Institute of Oilseeds Research,
Rajendranagar, Hyderabad 30

Presentation on 09-10-2017 to the participants of the

Refresher Course on
“Techniques in Molecular Cloning” at ABF, Hyderabad
Outline of discussion
 What is cloning
Preparation of insert – types of insert
Preparation of vector- types of vector
Ligation- kinds
Transformation
Selection/screening for transformants
 Problems in conventional cloning
Twin PCR
 Ligase Independent cloning - Gateway cloning
 Problems in cloning
 Cloning - a definition
• From the Greek - klon, a twig
• An aggregate of the asexually produced progeny
of an individual; a group of replicas of all or part
of a macromolecule (such as DNA or an
antibody)
• An individual grown from a single somatic cell of
its parent & genetically identical to it

Clone: a collection of molecules or

cells, all identical to an original
molecule or cell
Clones:
Asexual progeny that are genetically
identical to the original individual.
colonies of cells
 groups of viruses
some plants can be cloned.
Identical twins constitute a natural clone
A sheep named Dolly was cloned in
Scotland.
BACTERIAL CLONING VS GENE CLONING
 What is Gene cloning?
• To "clone a gene" is to
make many copies of it

• Act of making many

identical copies of gene

• Gene can be an exact

copy of a natural gene

• Gene can be an altered

version of a natural gene
 Why Clone DNA?
• A particular gene can be isolated and its
nucleotide sequence determined
• Control sequences of DNA can be identified &
analyzed
• Protein/enzyme/RNA function can be
investigated
• Mutations can be identified, e.g. gene defects
related to specific diseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
PURPOSE OF GENE CLONING
 Vector prep,

Insert preparation
(PCR product,
Part of DNA/RNA+
BASIC

OUTLINE

OF

GENE

CLONING
Gene Cloning: Linking of DNA fragment
or genes to carrier DNA molecules and
inserting these recombinant molecules into
a suitable host, one can produce large
quantities of these genes in pure form.
Isolation of Desired DNA fragments.
Insertion into a Carrier molecule capable
of either replicating or expressing or both.
 RATIONALE
Stable propagation of DNA sequences
Massive amplification of DNA sequences
A single DNA molecule can be amplified –
Studied- Sequenced
Manipulated- Mutagenised or Engineered
Expressed – Generation of a Protein
 Isolation of the insert

Random Fragmentation
Specific Fragmentation
Through PCR
Existing Clones/Libraries
Artificial Synthesis
 Random Fragmentation
 Non Specific
 Mechanical Shearing
( high speed stirring)
 Sonication
 French Press
 300bp
 -8kb
 May or may not yield Desired fragments
 Position and size variation occurs.
 Specific Fragmentation
Achieved through Restriction
Endonucleases
 Restrict the entry of Foreign DNA.
Fragment the DNA by cleaving the
Phosphodiester bonds b/w specific
sequences present internally.
Recognition Sequence:
 Target Sequence
 Restriction Enzymes
Restriction Enzymes (also called
Restriction endonucleases) are proteins
that cleave DNA molecules at specific
sites, producing discrete fragments of
DNA. Why they are there in bacteria????

Restriction Enzymes (RE) were first

isolated by Nathans and Smith in 1970.
 Choice of Restriction Sites/Enzymes
Once you have decided on your gene/fragment of interest,
you need to design a way to get it into your vector

•Endonucleases (or restriction enzymes) are

enzymes which cut DNA at specific internal
recognition sequences
•Compare to exonucleases, which cut from one end
•You must choose restriction sites that are available
in the plasmid you are cloning into
•They must not appear in your gene (silent mutation
can remove unwanted sites in your designed
gene)
RESTRICTION FRAGMENTS
ISOSCHIZOMERS
Really Important Factors to Remember
When Choosing Restriction Enzymes
• Restriction sites must exist only once in your
plasmid
• They must be in the correct position relative to the
purification tag
• Restriction sites usually add extra residues to
your gene product; make sure they are
compatible with your peptide/protein
• Some restriction sites are sub-optimal for cloning
• Blunt end sites
• dam and dcm methylation-affected enzymes
Obtaining the insert
 PCR Amplification of Insert from an
Existing Gene
The most common method of insert synthesis
•Necessitates a pre-existing construct
•Extra restriction sites and/or amino acid residues
can be added on each side of the gene
•Internal mutations are more difficult
Insert
 PCR Synthesis of Insert
PCR amplification from overlapping primers
•No pre-existing construct is needed
•PCR products messy, possibly making subsequent rxns
difficult
•Good for inserts >150 bp
F1: 10x
5’ 3’ F2: 1x
5’ 3’ 5’
3’
R1: 1x 3’ 5’
R2: 10x

Insert
Full-length insert should still be the major product
 Klenow Extension of Overlapping Primers
•Two primers that are complimentary in their 3’
region are designed (overlap  15bp)
•Extended to full length by the Klenow fragment of
DNA Polymerase I
•Useful if insert is 50 to 150 bp
5’ 3’

3’ 5’
Insert

5’ 3’
3’ 5’

Klenow
Klenow fragment: retains 3’ to 5’ polymerase activity, but does
not have exonuclease activity
 Complementary Full-Length Primers

• The simplest approach

• Order two primers that complement each other
• Mix the two primers, heat, and anneal slowly (to
ensure proper base-pairing)
• Feasible if the total insert size is < 60 bp

5’ 3’ Anneal
3’ 5’ Insert
SIX DIFFERENT TYPES OF CLONING
VECTORS
1. Plasmid cloning vectors

2. Phage  cloning vectors

3. Cosmid cloning vectors

4. Shuttle vectors

5. Yeast artificial chromosomes (YACs)

6. Bacterial artificial chromosomes (BACs)

 Vectors-Features

 Carrier molecule is called as a Vector.

 Autonomously Replication.
Origin
 Capable of Expressing the insert DNA.
- Promoters & Terminators
 Its presence should be easily detectable.
- Selection markers.
 Accommodation sites
-Unique restriction sites(MCS)
 Typical components of a transformation
vector: making a construct

• Selectable marker cassette (with promoter

and terminator)
• Gene of interest cassette (with promoter
and terminator)
• Scorable marker cassette (with promoter
and terminator)
What happens if the promoter is missing?
Is there ever a time when a promoterless
construct is desirable?
 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.
 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
 Antibiotic Resistance Markers
Antibiotic
Resistance
Gene
Multiple Cloning Region

Multiple
Cloning
Region

The cloning marker for this plasmid is the lacZ gene.

Let us get to work…
 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
 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
Joining DNA
Fragments

http://upload.wikimedia.org/wikipedia/commons/thumb/6/66/Scissors.sv
g/540px-Scissors.svg.png
 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.

In 1972, Paul Berg and colleagues made the

first “artificial” recombinant DNA molecule
LIGATION
 Methods of ligation

 Sticky end ligation

 Blunt end ligation

 Homopolymeric tailing.
Sticky end ligation
Blunt end ligation
PREVENTION OF SELF LIGATION BY
ALKALINE PHOSPHOTASE TREATMENT
USAGE OF ADAPTORS
USAGE
OF
LINKERS
HOMO
POLYMERIC
TAILING
 LIGATION OF PCR PRODUCTS
Taq polymerase adds a single 3l A
overhang
to each end of the PCR product
End-filling needs to be performed.
Efficiency of Blunt end ligation is low.
HOW?
Solution to this problem is to use T/A
cloning.
PCR fragment is ligated to a vector DNA
molecule with a single 3′ deoxythymidylate
(dT) extension
PCR primer may be designed which,
includes an extra sequence at its 5′ end
The extra sequence does not participate in
the first hybridization step – only the 3′
portion of the primer hybridizes – but it
subsequently becomes incorporated into
the amplified DNA fragment
Incorporation of restriction sites at each
end of the amplified product
 Twin PCR
 TA cloning requires the use of Taq polymerase only
 Blunt end ligations are non-directional and
inefficient
 RE sites incorporated in primers are not always
efficiently cut and the increased primer length
complicates PCR
 RE site to be used for cloning is present within the
insert (when RE sites are limiting)
- - Twin PCR is a way out in such cases…
Xie and Xie. 2011. World Journal of Microbiology
and Biotechnology. 27: 2223-25
Twin PCR – Schematic

EcoRI – site at the 5’ end

Bgl II – site at the 3’ end

Xie and Xie. 2011. World Journal of Microbiology

and Biotechnology. 27: 2223-25
 Transforming Bacteria
• After you create your new plasmid
construct that contains your insert of
interest , you will need to insert 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.
 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
 What did the cells take up?
• Plasmid only
• Plasmid with insert cloned
• Foreign DNA from the environment
• Nothing
 Selecting theTransformants
• 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.
INSERTIONAL INACTIVATION & α-COMPLEMENTATION
BLUE WHITE SELECTION
 Design Overview
Steps to follow in designing your cloning
experiment:
•Design your insert
•Design your gene
•Pick your enzymes
•Check your design
Functional
•Recheck your design construct
 Design of the Gene
•If you are designing the gene from scratch,
keep in mind codon usage
•Not all codons are created equal
•Un-optimized codons could lead to lower
expression levels
•The codon usage reflects levels of tRNA
available in E. Coli

•Pay attention to the stop codons too (XL1-

Blues read through TAG {amber stop
codon} 20% of the time)
What if we don’t have the DNA sequence?
Design from scratch! (don’t forget about codon usage)
E. Coli Codon Usage
UUU F 0.59 UCU S 0.17 UAU Y 0.6 UGU C 0.47
UUC F 0.41 UCC S 0.15 UAC Y 0.4 UGC C 0.53
UUA L 0.15 UCA S 0.15 UAA * 0.6 UGA * 0.31
UUG L 0.13 UCG S 0.13 UAG * 0.09 UGG W 1

CUU L 0.12 CCU P 0.19 CAU H 0.58 CGU R 0.35

CUC L 0.1 CCC P 0.13 CAC H 0.42 CGC R 0.34
CUA L 0.04 CCA P 0.21 CAA Q 0.34 CGA R 0.07
CUG L 0.46 CCG P 0.47 CAG Q 0.66 CGG R 0.12

AUU I 0.49 ACU T 0.19 AAU N 0.51 AGU S 0.16

AUC I 0.38 ACC T 0.38 AAC N 0.49 AGC S 0.23
AUA I 0.13 ACA T 0.19 AAA K 0.73 AGA R 0.08
AUG M 1 ACG T 0.24 AAG K 0.27 AGG R 0.05

GUU V 0.29 GCU A 0.19 GAU D 0.63 GGU G 0.34

GUC V 0.2 GCC A 0.26 GAC D 0.37 GGC G 0.36
GUA V 0.17 GCA A 0.24 GAA E 0.67 GGA G 0.14
GUG V 0.34 GCG A 0.31 GAG E 0.33 GGG G 0.16
or preferably…
http://www.bioinformatics.org/sms2/rev_trans.html
http://www.entelechon.com/index.php?id=tools/backtranslation&lang=eng
 Deciding the primer combinations

Primer pair Insert gene orientation in 5’ to 3’ Schematic of the

combination direction – double stranded kinds of insert ends

5’ side 3’ side overhang

overhang
FL + RS & FS + RL 5’ overhang 5’ overhang

3’ overhang 3’ overhang

FL + RL & FS + RS 5’ overhang 3’ overhang

3’ overhang 5’ overhang
Site-specific Recombination:
Gateway™ Cloning
 There are more issues to think when developing
expression cassettes……

 Different types of methods to develop multi-gene cassettes

 problems with cloning -
 single enzyme cloning
 cloning with incompatible enzymes
 when multiple cassettes are to be cloned..
 several components are to be cloned
 too large or too small fragments to be cloned
 cloning of small fragment into a big vector (difficulty in confirmation)
 problems of expression - increasing expression - transcriptional, post
transcriptional, translational, post translational enhancers
that are described so far.. (this can include codon optimization,
all the cis elements)
 targeted expression
 induced expression
To sum up about cloning
….
Gene for blue flowers Digest
vector DNA
How did you amplify with
this gene? restriction
enzyme
What are
essential
components
Plasmid Ligate
of vector
vector gene
DNAs?
into
vector

Extract plasmid DNA

Transform plant
Thank you
Bacteriophage λ
Bacteriophage λ
Figure 7.11
 Design of the Insert

•Once you have your restriction enzymes

chosen, it is time to design the final
complete gene

•The multiple cloning site (or whatever

plasmid you are cloning into) should
already have the 5’ portion of the
gene intact (i.e. RBS, spacer, Met)

• Sequences must be in frame

 Design of the Insert
Always check and re-check your sequence!

ATGGGCAGCA GCCATCACCA TCATCACCAC

AGCCAGGATCCGggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAA

Translate the whole gene

atgggcagcagccatcaccatcatcaccacagccaggatccgggctgcgacagggcgagc
M G S S H H H H H H S Q D P G C D R A S
ccgtactgcggttaactgcaggtcgacaa
P Y C G - L Q V D

Everything looks good: in frame the whole way!

 Design of the Insert
The wrong way to do it:
AGCCAGGATCC ggctgcgacagggcgagcccgtactgcggttaaCTGCAGGTCGACAAGCTT

The gene is just inserted after the restriction site, which is

out of frame with the plasmid-encoded start-codon/His-tag

atgggcagcagccatcaccatcatcaccacagccaggatccggctgcgacagggcgagcc
M G S S H H H H H H S Q D P A A T G R A
cgtactgcggttaactgcaggtcgacaagctt
R T A V N C R S T S
Frame shifted = garbage!

**Some plasmids, for whatever reason, have restriction

sites out of frame with the translated gene** - so one
has to be careful and cautious
 Design of the Primers
Once the insert is designed correctly, the next step is designing
primers to order from IDT, based on insert synthesis strategy
Three main strategies towards insert synthesis:
•PCR amplification
•Klenow extension of overlapping primers
•Complimentary full-length primers

+
Insert
Vector
 Design of the Gene
If you are cloning out of a known plasmid, just use the sequence that you
have

Example, the gene we want:

G C D R A S P Y C G

We got this from phage display:

ggctgcgacagggcgagcccgtactgcggt
G C D R A S P Y C G

Phage sequence

Final sequence for the gene of interest:

ggctgcgacagggcgagcccgtactgcggttaa
G C D R A S P Y C G *

Add a stop codon