CLONING
Components & Methods
CLONING: Components & Methods
Linkers
Adaptors
T-cloning
Topo-TA cloning
pEntr-Topo cloning
Gateway Technology
oining Of DNA Molecules Together By Ligation
LIGATION OF STICKY ENDS IS DESIRABLE.
What do we do when insert has blunt ends ?
How do we increase the efficiency of ligation in
such cases ?
Strategies for converting blunt ends into cohesive
ends:
9.Linkers
10.Adaptors
11.Homopolymer Tailing
12.TA cloning
LINKERS
tegy to add cohesive ends to a blunt-ended molecule
ially-synthesized short pieces of ds DNA of known nucleotide sequence
vantage: The restriction site
er is also present in the
fragment.
ADAPTORS
Short synthetic oligonucleotides with a cohesive end.
• Problem: Cohesive ends could base pair with each other to form
dimers. This results in a blunt-ended molecule which can still ligate
to the blunt-ended DNA molecule. However we need to digest this
to obtain a cohesive end molecule.
ADAPTORS (contd.)
ptors synthesized such that cohesive end is different.
P terminus is modified to 5’-OH
nus.
e pairing occurs but is not stabilized
on reaction.
ptors can not themselves ligate but
e blunt ended molecule.
er ligation to the DNA fragment, the
rmal 5’-OH terminus is treated with
Homopolymer Tailing
Another method of producing
cohesive ends on a blunt end DNA
molecule.
Deoxynucleotidyl transferase adds a series of
nucleotides onto the 3’-OH termini of a ds DNA
molecule.
Generally complementary homopolymer tails a
added to the vector as well as insert.
Due to the differences in the length of the pol
attached, a nick or discontinuities are produced
This is repaired by Klenow polymerase.
In case the homopolymers are more than 20 n
long, the base-paired associations are quite stro
These molecules are introduced into bacterial ce
and host cell polymerase and DNA ligase repair
recombinant DNA.
TA-cloning
merases possesses terminal transferase activity that adds a single dA-o
of PCR products.
ectors provide the corresponding T for base-pairing.
A PCR Product A
ges: Quick and efficient cloning method.
No need to dephosphorylate the vector
ntage: Restriction digested fragments and PCR products of proof-readin
u, Pwo or Tli DNA Pol. yield blunt-end products.
can be employed.
-TA cloning: more efficient method of clo
ADVANTAGES REQUIREMENTS
• Highly efficient
PCR products
• One-step cloning
A linearized activated vector with dT-overhangs at 3’end
• Saves time Activated vector: Topoisomerases covalently bound to the
TOPOISOMERASES
Source: Vaccinia virus
Action: The phospho-tyrosyl bond between the DNA and en
attacked by the 5’-hydroxyl and releases the topoisomera
Different Ways of TA-cloning
Different Ways of TA-cloning
GATEWAY CLONING
• Recombination-based cloning technology
• Replaces the use of restriction endonucleases and ligases with site
specific recombinases
• Universal system for cloning and subcloning DNA fragments
• Rapid and efficient technology
Basis of Gateway Cloning Technology
Recombination-based technology
Recombination of phage lambda in E. c
INTEGRATION
att P: Phage attachment site (245bp)
att B: Bacterial attachment site(25 bp)
Proteins Involved: Int (integrase) &
IHF(integration Host Factor)
EXCISION
att L: left attachment junction
(100 bp)
att R: right attachment
junction(168 bp)
Proteins Involved: Int IHF & Xis
attB x attP
(excisionase) attL x attR
Commercial Vectors have been modified for efficient cloning
ite (43 bases removed) modified to make excision reaction irreversible
efficient
ions introduced in core att sites to eliminate stop codons and ensure sp
ions in 5 bp regions flanking the 15 bp core region of attB sites to minim
ary structure formation.
Gateway Technology is Flexible
ccdB ccdB
attL1 attL2 attR1 attR2 LR clonase attB1 attB2 attP1 attP2
Entry
Clone + Destination
Vector
Expression
Clone + Donor
Vector
BP clonase
KanR AmpR AmpR KanR
Requirements for Gateway System
REQUIREMENTS
Entry clone
Destination Vector
LR clonase enzyme (int, IHF & xis)
TYPES OF SELECTION
Positive Selection: Antibiotic resistance gen
Negative Selection: ccdB gene (interferes w
DNA gyrase gene)
Vector has ccdB gene and thus propagated i
E. coli strain DB3.1.
gyrA462
No degradatio
(DB3.1)
ccdB (toxin)
Chromosome is
degraded
ccdA Rejoining by
(antidote) DNA Gyrase
Different ways to generate the
entry clone
L1 L2
P1 ccdB P2
Donor Vector + B1 Gene B2
TOPO-Activated
Entry Vector
+ Gene
attB PCR Product PCR Product
1. BP Cloning 1. TOPO® Cloning
BP Clonase™ TOPO®
L1 Gene L2
Entry Clone
Ligase
1. Restriction/Ligase Cloning 4. Pre-made entry clone
5. Custom-made entry clone
L1 ORF L2
L1 L2
digested Entry Vector + B1 Gene
ORF Collection
digested DNA Fragment
1.BP Cloning
gene
a ttL 1 a ttL 2
ccd B E n tr y
C lo n e
a ttP 1 a ttP 2
a ttB 1 gen e a ttB 2 a ttR 1 ccdB a ttR 2
+ Donor
V e c to r + K an R
K an R
BP Clonase™
1. BP Cloning - Primer Design for PCR 90-99% correct clones
on Kan plates
• GGGG and the attB1 sequence must be added to the 5’-primer
(sense)
• GGGG and the attB2 sequence must be added
Geneto the 3’-primer
Specific
(antisense) Primer Sequence
attB1
5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN…
attB2
2. Restriction/Ligase cloning
Use when there are convenient sites
to cut insert out of another plasmid
Must cut out ccdB gene by using one
of four RE sites flanking the ccdB
Reading frame of insert must be
considered, as well as downstream
expression elements
Various reading frames of pENTR
vectors are available
3. TOPO Cloning
ectional and faster cloning by pEntr-D Topo reaction of the PCR product.
ward primer should contain CACC sequence
e 5’ end.
ures directional cloning.
y efficient.
ry clone ready for LR reaction with the
nation vectors.
ecombination Forms a Cointegrate Molec
mbination of Entry vector with a Destination vector
irements:
ry clone with the GOI between
and L2 site
stination vector with attR1 and
ites & ccdB gene
clonase enzyme
mation of Cointegrate molecule
ntegrate molecule resolves through a second
tion into two daughter molecules.
ardless of which pair of sites
and att R1 or
and att R2
to first from an integrate.
Types of Destination Vectors
Destination vectors available for expression in E. coli
Yeast
Baculovirus
Insect cells
Mammals
Plants
des the available vectors, any vector can be converte
Gateway vector
Conversion of a vector into Gateway vect
available which can be
vector by restriction
n.
emember:
ion vector ligation mix
propagated in DB3.1 cells.
ing a protein fusion vector, ensure the reading frame of the protein w.r
assettes A-C are blunt-ended molecules they may be inserted in either d
ck for the orientation of the inserted cassette.
confirm the type of cassette by restriction digestion.
l clones should be selected on chloramphenicol+ antibiotic resistance o
ateway cloning system is superior system
ecombination-based cloning.
xtremely rapid.
ery simple to perform.
ghly efficient cloning as 99% clones are
ect.
aithful maintenance and orientation of
GOI.
arallel cloning can be easily carried out.
ighly versatile system.
ny vector can be converted into GW system.
ectors available for many biological systems.
ultisite gateway systems are now available.
Suggested Reading
Gene Cloning and DNA Analysis: T. A. Brown
Gateway Manual: www.invitrogen.com
