TA Cloning (PCR cloning)

Summary

This 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

• 🌟 TA cloning is a fast and easy method of subcloning DNA, also known as PCR cloning.
• 🔬 It does not require restriction enzymes for the cloning process, simplifying the procedure.
• 🧬 The 'TA' in TA cloning refers to the thymine (T) and adenine (A) bases that are used for base pairing in the cloning process.
• 🧪 The process involves three stages: preparation of the vector, preparation of the gene, and the cloning itself.
• 📈 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.
• 🧬 T Vector is a specific type of vector used in TA cloning, linearized and treated with terminal deoxynucleotidyl transferase (TdT) to add thymine overhangs.
• 🔗 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.
• 🧬 DNA ligase is used to seal the nicks between the target DNA and the T Vector, completing the cloning process.
• 🚫 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.
• 📚 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

00:00

🌟 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.

05:00

🧬 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.

10:02

📚 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.

Keywords

💡TA Cloning

TA Cloning, also known as PCR Cloning, is a method of subcloning that is both fast and easy to perform compared to conventional subcloning techniques. It involves the use of a specific type of vector and the preparation of a gene segment for cloning without the need for restriction enzymes. The video emphasizes that TA cloning simplifies the cloning process by eliminating the need for complex steps and multiple enzymes, making it more accessible for those looking to clone specific genes of interest.

💡Subcloning

Subcloning refers to the process of transferring a gene or DNA fragment from one plasmid to another. In the context of the video, it is contrasted with TA cloning, highlighting the latter's efficiency and simplicity. The script mentions that TA cloning is a method of subcloning that is faster and easier, suggesting that traditional subcloning methods can be more cumbersome.

💡PCR (Polymerase Chain Reaction)

PCR is a technique used to amplify specific DNA sequences, creating multiple copies of a particular gene. The video explains that PCR is integral to TA cloning because it allows for the amplification of the target gene, which is then used in the cloning process. The script mentions that PCR is used to prepare the target DNA, which is a key step in TA cloning.

💡T Vector

A T Vector is a specific type of cloning vector used in TA cloning. The script describes how T Vectors are prepared by linearizing circular vectors and adding extra thymine residues at the five prime ends using terminal deoxynucleotidyl transferase (TdT). This preparation allows for the subsequent attachment of the target DNA, making the T Vector a crucial component in the TA cloning process.

💡Thymine (T)

Thymine, represented by the letter 'T', is one of the four nucleobases in DNA, pairing with adenine. In TA cloning, the video explains how thymine is used at the ends of the T Vector to pair with adenine residues added to the target DNA, facilitating the attachment of the gene of interest to the vector. This base pairing is central to the TA cloning technique.

💡Adenine (A)

Adenine, represented by the letter 'A', is another nucleobase in DNA that pairs with thymine. The script describes how an extra adenine residue is added to the three prime end of the target DNA during the PCR process. This addition is essential for the TA cloning process, as it allows the target DNA to pair with the T Vector through the complementary base pairing of adenine and thymine.

💡Terminal Deoxynucleotidyl Transferase (TdT)

TdT is an enzyme used in the preparation of the T Vector for TA cloning. As explained in the video, TdT adds extra thymine residues to the five prime ends of the linearized vector, creating overhangs that can pair with the adenine residues on the target DNA. This enzyme is crucial for the successful attachment of the target gene to the T Vector.

💡DNA Ligase

DNA Ligase is an enzyme mentioned in the video that is used to seal nicks in the DNA, joining the target DNA to the T Vector. After the target DNA is attached to the T Vector through complementary base pairing, DNA ligase strengthens this bond by joining the phosphodiester backbone, ensuring the stability of the cloned construct.

💡Selectable Marker

A selectable marker is a gene that allows for the identification and selection of successfully cloned cells. The video script mentions that while TA cloning does not require specific restriction sites in the primer, it does require a selectable marker in the vector to identify successful clones. This marker is essential for screening and isolating the cells that contain the desired gene.

💡Directional Cloning

Directional cloning refers to the ability to control the orientation of a gene within a vector. The video points out that TA cloning is not directional, meaning there is a 50/50 chance of the gene being inserted in either orientation. This lack of directionality is a drawback of TA cloning, as it can lead to the need for additional screening to identify clones with the gene in the correct orientation.

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 180°

if you flip the target DNA

180° 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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thank you

h