TA Cloning (PCR cloning)
Summary
TLDRThis video explains TA cloning, a fast and easy method for subcloning genes of interest without the need for restriction enzymes. It involves three stages: preparing the vector, the gene, and the cloning process itself. TA cloning, also known as PCR cloning, uses T4 polymerase to add adenines to the 3' ends of the target DNA, which then pair with thymines on a linearized T vector. DNA ligase is used to seal the nicks, creating a stable clone. The video highlights the simplicity and cost-effectiveness of TA cloning but notes its non-directional nature as a limitation.
Takeaways
- π TA cloning is a fast and easy method of subcloning DNA, also known as PCR cloning.
- π¬ It does not require restriction enzymes for the cloning process, simplifying the procedure.
- 𧬠The 'TA' in TA cloning refers to the thymine (T) and adenine (A) bases that are used for base pairing in the cloning process.
- π§ͺ The process involves three stages: preparation of the vector, preparation of the gene, and the cloning itself.
- π PCR is used to amplify the target gene, and Tth polymerase is used to add an extra adenine to the 3' end of the DNA.
- 𧬠T Vector is a specific type of vector used in TA cloning, linearized and treated with terminal deoxynucleotidyl transferase (TdT) to add thymine overhangs.
- π The target DNA with an extra adenine at the 3' end can bind to the thymine overhangs of the T Vector, forming a stable complex.
- 𧬠DNA ligase is used to seal the nicks between the target DNA and the T Vector, completing the cloning process.
- π« A major drawback of TA cloning is that it is non-directional, meaning there's a 50/50 chance of the gene being inserted in either orientation.
- π TA cloning is more convenient and less expensive compared to conventional subcloning methods, but it lacks the directional control.
Q & A
What is TA cloning?
-TA cloning is a fast and easy method of subcloning, also known as PCR cloning. It involves attaching a gene of interest to a T Vector without the need for restriction enzymes.
How does TA cloning differ from conventional subcloning?
-TA cloning is faster and simpler than conventional subcloning, requiring just one specific vector and no restriction enzymes for most steps. It also simplifies the process by combining PCR with cloning.
Why is it called TA cloning?
-TA cloning is named based on the exploitation of the AT complement nature of base pairing, where 'T' stands for thymine and 'A' stands for adenine, which pair with each other in the cloning process.
What is the role of TdT enzyme in TA cloning?
-The terminal deoxy nucleotid transferase (TdT) enzyme is used to attach multiple thymine (T) residues at the five prime terminal of the T Vector, preparing it for the cloning process.
What is the purpose of adding an extra adenine at the three prime site during PCR in TA cloning?
-Adding an extra adenine at the three prime site during PCR allows for the attachment of the target DNA to the T Vector through the AT pairing, facilitating the cloning process.
How is the T Vector prepared for TA cloning?
-The T Vector is prepared by linearizing a circular vector using restriction digestion to create blunt ends, followed by the addition of thymine residues at the five prime ends using TdT enzyme.
What is the function of DNA ligase in the TA cloning process?
-DNA ligase is used to seal the nicks between the target DNA and the T Vector, joining them together to form a stable DNA molecule.
What is a drawback of TA cloning compared to other cloning methods?
-A major drawback of TA cloning is that it is not directional, meaning there is a 50/50 chance of the gene being inserted in either orientation within the vector.
Why are primers designed differently for TA cloning compared to conventional subcloning?
-In TA cloning, primers do not require restriction endonuclease sites because the cloning process does not rely on these sites for the attachment of the target DNA to the vector.
What is the advantage of using T Vectors like pJET in TA cloning?
-T Vectors like pJET are specifically designed for TA cloning, simplifying the process and making it more efficient by combining the target DNA preparation with the PCR process using Taq polymerase.
Outlines
π Introduction to TA Cloning
The paragraph introduces TA cloning as a fast and easy method of subcloning compared to conventional methods. It explains that TA cloning, also known as PCR cloning, allows for the subcloning of specific genes of interest without the need for restriction enzymes. The process involves three stages: preparation of the vector, preparation of the gene, and the cloning stage itself. The name 'TA cloning' is derived from the exploitation of the AT complement nature of base pairing to attach the gene of interest to the T vector. The paragraph also mentions that PCR is involved in the process to amplify the target gene and that Taq polymerase is used for this purpose.
𧬠TA Cloning Process and Vector Preparation
This paragraph delves into the specifics of the TA cloning process, detailing the preparation of the target DNA through PCR reactions and the subsequent addition of an extra adenine at the three prime site. It then explains the preparation of the T vector, which involves linearizing a circular vector using restriction enzymes to create blunt ends. The vector is further processed with terminal deoxynucleotidyl transferase (TdT) to add thymine residues at the five prime ends. The paragraph concludes with a description of how the target DNA is attached to the T vector through AT pairing, and the use of DNA ligase to seal the nicks, thereby completing the cloning process.
π Advantages and Limitations of TA Cloning
The final paragraph highlights the advantages of TA cloning, such as the absence of the need for restriction endonuclease sites in the primers, making the process simpler and less expensive. It contrasts this with conventional subcloning, which requires specific restriction sites in the primers. However, the paragraph also points out a significant limitation of TA cloning: it is not directional, meaning there is a 50/50 chance of the gene being inserted in either orientation within the vector. The paragraph ends with a call to action for viewers to like, share, and subscribe for more informative videos.
Mindmap
Keywords
π‘TA Cloning
π‘Subcloning
π‘PCR (Polymerase Chain Reaction)
π‘T Vector
π‘Thymine (T)
π‘Adenine (A)
π‘Terminal Deoxynucleotidyl Transferase (TdT)
π‘DNA Ligase
π‘Selectable Marker
π‘Directional Cloning
Highlights
TA cloning is a fast and easy method of subcloning compared to conventional methods.
TA cloning, also known as PCR cloning, does not require restriction enzymes for the cloning process.
The process involves just one vector and the gene segment of interest for cloning.
TA cloning exploits the AT complement nature of base pairing to attach the gene of interest to the T Vector.
PCR is involved in TA cloning to amplify the target gene and add an extra adenine at the three prime site.
The T Vector is prepared by linearizing it and adding thymine residues at the five prime ends using terminal deoxynucleotidyl transferase (TdT).
The target DNA with an extra adenine at the three prime end can bind to the T Vector with thymine residues at the five prime end.
DNA ligase is used to seal the nicks between the target DNA and the T Vector.
The pJET vector is an example of a T Vector specifically designed for TA cloning.
TA cloning does not require the addition of restriction endonuclease sites in the primer for PCR.
One drawback of TA cloning is that it cannot be directional, leading to a 50/50 chance of gene insertion orientation.
TA cloning is less expensive and simpler than conventional subcloning methods.
The process is suitable for quick and easy cloning without the complexities of other systems.
TA cloning is ideal for those who need a fast and straightforward cloning method without the need for extensive molecular biology techniques.
The video provides a detailed explanation of the TA cloning process, making it accessible for beginners.
The presenter emphasizes the practical applications and simplicity of TA cloning in molecular biology research.
Transcripts
welome again guys uh welcome to another
video
from ta cloning okay so let's do this ta
cloning what is TA cloning ta cloning is
a process of subcloning it's a method of
sub cloning compared with the uh
conventional sub cloning tier cloning is
very very fast and it's kind of uh very
easy to perform the other name of the ti
cloning is PCR cloning and in both the
cases we we use tier cloning uh compared
with I mean compiled with PCR reactions
uh to subclone specific Gene of our
interest very easily and very very fast
okay without using any restriction
enzyme that is the idea of T cloning we
don't need any restriction enzyme we
need uh not actually technically don't
need we need it in only one step but
it's not that much big we don't need uh
restriction enzymes in the cloning uh
process exactly and second thing is that
we don't require so much of other
complexity that we used to have with
other cloning systems and cloning
processes uh just one vector is required
a specific Vector designed for this and
we need to prepare uh the gene segment
and then the cloning can be done very
very fast without uh any kind of
problems uh most of the cases now the
scenario here is in this ta cloning
method there are two three different uh
stages of T cloning actually first stage
is known as the preparation of vector
second stage is the preparation of the
Gene and the third stage is the cloning
stage
itself so why the name is TA this is the
first mystery right why it is TA okay
now T stands for thyine a stands for
adenine so now we know what it means you
know we know that a and t pairs with
themselves so in this case we exploit
the at complement nature of base pairing
uh to attach Gene of interest to the T
Vector okay the cloning Vector that we
are going to use here so here what we do
I told you that in this case this is
also known as PCR cloning because we're
going to tag this cloning approach or
process with the PCR reaction itself so
why and when exactly PCR is involved the
scenario is we know in PCR we can
amplify the target Gene of our interest
now in subcloning approaches what we do
we first amplify the target Gene using
PCR then we need to purify those those
genes we need to take that out then we
need to have separate vectors and then
do the cloning but here we can do that
kind of simultaneously after the PCR
processing is done so what we do here in
the PCR process you know we required the
thermos aquaticus polymerous for PCR
because as the PCR requires the
polymerization of nucleotide sequences
at higher temperatures not all the type
of polymer can achieve that uh specific
polymerase can only do this
polymerization at high temperatures
those are known as tack polymerase right
T polymerase or thermos aquaticus
polymerase a polymerase deducted taken
from the thermos aquaticus bacteria so
we take this tack
polymerase and we allow this whole PCR
process to be done okay so once the
whole PCR process is achieved let's say
this is the target DNA and the target
DNA is completely made so say this is is
the five Prime 3 Prime and this is again
five Prime 3 Prime the target
DNA okay so we produce the target DNA
after the production of the target DNA
what we do is we simply add an adenine
at the three prime site okay we have an
adinin residue extra adinin residue at
the three prime site after the PCR okay
so through whole process of PCR we allow
those tack polymerase itself to bind and
attach one extra adenine re
at the three prime
ends so what it will look like it will
look something like
this and at the three prime we have
adinin attached okay here is the adenine
here is another adenine
attached or I can draw it something like
this so one extra Adin are attached at
the three prime
end okay this is uh the target DNA what
we prepare okay the target DNA that that
we prepare so once we prepar the target
DNA this is the first stage the second
stage will be the preparation of vector
or vector
DNA this is the first stage and we
completed that stage second stage is the
preparation of vector how we prepare the
vector in this case we are going to have
a specific type of vector the vector is
known as T
Vector okay T Vector molecule now the T
Vector molecule does not have so much
complexity it's very simple very easy
kind of vector and the vector is
linearized and actually we make them
linearized using a restriction digestion
remember I told you we don't require
restriction digestion at the attachment
or cloning phase but we require it to
produce T Vector now normally the vector
is circular but what we do we treat it
with the Restriction enzyme to make it
linearized and once we make it
linearized what we have we have a blunt
end at both the terminal so let's assume
this is our T Vector this is our vector
and we cleave it from here so once we
cleave it from here we have a blunt end
DNA okay this is the blunt end Vector
DNA these are the blunt ends right blunt
end means no overhang at the end okay so
once we prepare this blunt and
DNA okay blun and DNA then after that we
use a specific enzyme which is called as
terminal deoxy nucleotid transfer is
okay a terminal nucleotid transferase
tdt it's known as tdt terminal deoxy
nucleotid transfer this enzyme can pair
or
attach T
residues it can actually attach multiple
multiple nucleotide residues but here it
attaches T
residues at the five Prime Terminal
remember sorry let's draw the five Prime
this way
they attach T residues at the five Prime
Terminal extra one extra T residues at
the both five Prime this is due to this
tdt enzyme okay so we produce the vector
this is the second stage so we have our
Target DNA produced com combined with
PCR reactions so you don't require any
other extra ST the PCR St and the T
polymeris can give us this target DNA
the second phase we can produce the
vector T Vector easily by using deoxy
nucleotid transfer as enzyme or terminal
transfer as enzymes so once we prepare
both of them then we can simply attach
our Target DNA with the vector this is
known as T Vector right so how can you
do that just
assume this is the double standard
structure of the DNA okay and at the end
let's say this and
this extra T and this is also T so
thyine is present right there five Prime
ends these are and now if we add this
one it's going to bind how with the
addin
in
okay and rest of the
DNA sequence like that so it is attached
now okay let's draw the double standard
DNA like that and also this is the whole
double
standard
DNA like that okay so now our Target DNA
can easily fit into the T Vector
properly this is the cloning stage now
require this in this cloning stage we
don't require any restriction indon
nucleus enzyme right do we we don't
require any we simply can add them but
remember after this addition they can
pair with only help of one adenine and
thyine interaction each side it's not
very strong interaction we know at any
time interaction is not that strong so
and also have some breaks some Nicks
right there is a Nick and there is a
Nick so we need to fill this Nick also
right how to fill this Nick we need to
use an enzyme the name is DNA liase so
we use the DNA lias
enzyme to fill this Nick to join the
Nick not actually filling because Nick
does not require to be filled uh there
is nothing in the Gap just we need to uh
attach the phospher bond so we need to
join the Nick right using DNA lias
enzyme so you have DN Li which will seal
the Nick so Nick sealing is done then we
have our Vector with the target GNA
inside okay this is how the whole
process is done now in the T Vector
there is multiple examples many
different multiple companies are
processing one of them example is
pjm T pjt is an example of a t Vector is
specifically designed for this ta
cloning okay so that's why it's known as
ta cloning and PCR cloning because the
process of the target DNA preparation is
combined with the process of uh the PCR
using Tac polymerous enzyme okay so in
both this way once we prepare we get the
Clone product okay and also in this uh
in this whole cloning system we we
obviously we require selectable marker
definitely present in the vector but we
don't require all the Restriction in
indon nucleous sites of the multiple
cloning sites now what happens in uh in
normal type of uh in con conventional
type of subcloning experiments we need
to add uh specific restriction
endonuclear sites in the primer itself
in the primer region while doing the PCR
because if we need to take all the
products from the PCR and do the cloning
then and there we need to use uh the
Restriction endonuclear sites should be
present in the both terminal part of our
Target DNA and for that we need to add
the restriction uclear site in our
primer sequence so when we are designing
primers for the subcloning experiments
we need to design it in such a way so
that it contains the Restriction indon
nucleus side okay that is the thing that
is a problem right but in this case we
don't require to do that we don't
require any of the Restriction of
nucleus side to be present in our primer
we can design the primer without all
these complexions right so it's very
easy fairly easy to do and also less
expensive uh the primers will be less
expensive also but the only draw about
which is a big drawback by the way uh
about this ta cloning is that this
cloning cannot be directional in nature
because this cloning can be a single
Direction you cannot do it I mean it can
happen in both the directions like if
you imagine this you if you flip it 180Β°
if you flip the target DNA
180Β° it is also going to bind to the
vector it will not change it will bind
to the vector anyhow okay so uh the
direction cloning is not possible so
there is always a 50/50 chance of where
exactly and how exactly your jeans going
to be inserted inside the vector that's
one major drawback with the TA cloning
otherwise it's a very good approach for
the conven compared with the
conventional sub cloning systems so
that's for the te cloning if you like
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h
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