to Gateway® Cloning
Gateway® Cloning is a universal cloning
technique developed by Invitrogen life technologies.
Gateway® Cloning Technique allows transfer
of DNA fragments between different cloning
vectors while maintaining the reading frame.
It has effectively replaced the use of restriction
endonucleases and ligases. Using Gateway®,
one can clone/sub clone DNA segments for functional
(a)The BP Reaction (PCR fragment
+ Donor vector = Entry Clone)
The BP Reaction is a recombination reaction
which is explained in the following lines.
For the reaction to take place, the gene of
interest is amplified with the help of an
attB tagged primer pair. The donor vector
includes attP sites. The PCR product that
includes the attB sites combines with the
donor vector that includes the attP sites
resulting in the formation of an entry clone.
This integration reaction between the attB
and the attP sites forms the basis of this
reaction. The resulting entry clone contains
the gene of interest flanked by attL sites.
(b)The LR reaction (Entry Clone + Destination
Vector = Expression Clone)
The LR Reaction, again is a recombination
reaction between attL and attR sites. The
reaction generates an expression clone and
is catalyzed by recombinant proteins. The
entry clone generated from the BP reaction
includes the attL sites. The Destination vector
is designed to include the attR sites. The
LR reaction is carried out to transfer the
sequence of interest to one or more destination
vectors in simultaneous reactions, making
the technology high throughput. The entry
clone is mixed with the appropriate Gateway®
vector and Gateway® Clonase enzyme. Recombination
between these sites generates two molecules.
One molecule contains the DNA segment of interest,
the other molecule is a by-product.
- Allows subcloning from one vector
backbone to another.
- Every subcloning reaction maintains the
appropriate reading frame.
- Subcloning process is fast and facilitates
- Supports site specific recombination.
- Multiple genes can be transferred to one
or more vectors in one experiment.
Cloning with SimVector
|SimVector is a comprehensive
DNA sequence analysis tool that allows
you to plan and design cloning experiments.
you can perform both the BP and the LR reactions
that constitute Gateway®
Cloning. The donor vector and the expression
vector are required for the BP reaction and
for generating an entry clone. An entry vector
and a destination vector are required for
the LR reaction and for generating an expression
Techniques Performed by SimVector
TA Cloning: SimVector makes it easy to design TA cloning
experiments. The TA
cloning wizard accepts both commercial
T-vectors or restriction designed T-vectors
and combines them with the modified PCR product.
You can then generate a highly accurate
recombinant DNA plasmid map.
This cloning technique requires restriction
enzymes to cut the vector molecule and the
molecule to be cloned. SimVector performs
restriction enzyme analysis and allows you
to filter, annotate and map the restriction
enzymes on the desired sequences.
SimVector identifies restriction enzyme recognition
sites in DNA sequences for restriction site
mapping. A comprehensive database of over
1000 restriction enzymes is used for the restriction
analysis. Two modes of enzyme filtering are
provided to specify the parameters for restriction
analysis. Enzyme filtering enables you to
set the enzyme criteria to match your experimental
requirements. For convenience, enzyme sets
from more than 20 commercial providers are
SimVector is an
exceptional tool to draw publication quality plasmid
maps. The graphics can then be enhanced
with patterns, styles, lines, and colors.
SimVector generates plasmid map images in
vector graphic format for Adobe Illustrator
10 and Microsoft PowerPoint 2002.