Introduction
to TA Cloning
TA Cloning is one of the most
popular methods of cloning the amplified
PCR product using Taq and other polymerases.
These polymerases lack 5'-3' proofreading
activity and are capable of adding adenosine
triphosphate residue to the 3' ends
of the double stranded PCR product.
Such PCR amplified product can be cloned
in a linearized vector with complementary
3' T overhangs.
TA Cloning Functioning
TA cloning is brought about by the terminal
transferase activity of certain type
of DNA polymerase such as the Taq polymerase.
This enzyme adds a single, 3'-A overhang
to each end of the PCR product. As a
result, the PCR product can be directly
cloned into a linearized cloning vector
that have single base 3'-T overhangs
on each end. Such vectors are called
T- vectors. The PCR product with A overhang,
is mixed with this vector in high proportion.
The complementary overhangs of a "T"
vector and the PCR product hybridize.
The result is a recombinant DNA, the
recombination being brought about by
DNA ligase.
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SimVector makes it so easy to design TA
cloning experiments. The TA cloning
wizard accepts both commercial T-vectors
or a restriction designed T-vector to
combine them with the modified PCR product
and generates a highly accurate recombinant
DNA plasmid map.
Other
techniques performed by SimVector:
Gateway®
Cloning: Using SimVector you can perform both
BP and LR reactions of Gateway®
Cloning. A donor and an expression
vector are required for the BP reaction
and for generating an entry clone. An
entry and a destination vector are required
for the LR reaction and for generating
an expression vector.
Restriction
Cloning: 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 included.
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. |
