TOPO Cloning - TOPO-Blunt, TOPO-TA, TOPO-directional

Summary

This educational video script delves into TOPO cloning strategies, explaining three main types: Blunt TOPO, TOPO-TA, and directional TOPO. It simplifies complex molecular biology concepts by detailing how topoisomerase, specifically Vaccinia Topoisomerase, facilitates PCR product cloning without restriction enzymes or ligases. The script explores the enzyme's mechanism, including DNA nicking and religation, and addresses the nuances of each cloning strategy, such as the importance of 5' hydroxyl groups and the use of ccdB gene to prevent self-ligation. It also touches on expression vectors and the significance of promoters for gene expression, providing a comprehensive guide for cloning and genetic engineering.

Takeaways

• 🔬 There are three types of TOPO cloning strategies: Blunt TOPO, TOPO-TA, and directional TOPO cloning, each utilizing different DNA end characteristics.
• 🧬 TOPO cloning bypasses the need for restriction enzymes and ligases, simplifying the process of cloning PCR products.
• 🌟 The enzyme Vaccinia Topoisomerase, derived from an ancestor of the smallpox virus, is central to TOPO cloning due to its unique binding and cleaving sites.
• ✂️ TOPO enzymes create a nick in one DNA strand, unlike restriction enzymes that cut both strands, leading to DNA relaxation.
• 🔄 The nicking activity involves a nucleophilic substitution reaction where the topoisomerase's tyrosine amino acid attacks the DNA backbone.
• 🔗 The 3’ phosphate of the nicked DNA forms a temporary linkage with the topoisomerase, allowing for self-ligation without external ligases.
• 🏷️ Blunt TOPO vectors have the recognition sequence CCCTT at their ends and require dephosphorylated inserts for successful cloning.
• 🔄 TOPO TA vectors have sticky ends (T overhangs) and require inserts with complementary A overhangs, facilitating specific base pairing.
• 🧭 Directional TOPO vectors ensure the insert is cloned in a specific orientation, often used for gene expression, with one end blunt and the other sticky.
• 🌐 The use of ccdB gene in TOPO vectors helps prevent self-ligation by killing bacteria that have not incorporated an insert, thus selecting for successful clones.

Q & A

What are the three main types of TOPO-based cloning strategies mentioned in the script?

The three main types of TOPO-based cloning strategies are Blunt TOPO cloning, TOPO-TA cloning, and directional TOPO cloning.

What is the role of topoisomerase in the cloning process?

Topoisomerase, specifically Vaccinia Topoisomerase, plays a crucial role in TOPO cloning by binding to a supercoiled DNA, making a cut in one of the DNA strands, and subsequently re-ligating the cut, which allows for the cloning of PCR products without the need for restriction enzymes and ligases.

How does the topoisomerase create a nick in the DNA?

The topoisomerase creates a nick in the DNA by using a tyrosine amino acid at the 274th position from the N-terminal end to attack the DNA backbone between the T and N base in a nucleophilic substitution reaction.

What is the significance of the CCCTT sequence in TOPO cloning?

The CCCTT sequence is one of the two binding sites for the topoisomerase and is recognized by the enzyme, leading to a cut after the last Thymine in the sequence, which is essential for the cloning process.

Why is dephosphorylation of the insert necessary in TOPO cloning?

Dephosphorylation of the insert is necessary because the 5' end needs a free hydroxyl group to perform the nucleophilic substitution required for ligation to the vector.

How does the Blunt TOPO Cloning strategy prevent self-ligation of the vector?

Blunt TOPO Cloning prevents self-ligation by either removing hydroxyls from the ends of the vector or by placing the TOPO cloning sites within a gene called ccdB, which, if the vector self-ligated, would activate and kill the bacteria.

What is the difference between TOPO TA cloning and traditional TA cloning?

TOPO TA cloning uses a T-overhang vector and an A-overhang insert, allowing for nucleophilic substitution without the need for ligases, while traditional TA cloning relies on base pairing and the use of ligases to join the vector and insert.

How does the directional TOPO cloning strategy ensure the insert is cloned in a specific orientation?

Directional TOPO cloning uses a combination of sticky and blunt ends on the vector and specific primer design to ensure that the insert can only ligate in one orientation, preventing the reverse orientation.

Why is the directional TOPO vector also considered an expression vector?

The directional TOPO vector is considered an expression vector because it contains promoters and other elements necessary for transcription and translation, allowing the cloned insert to be used as a template for making RNA or proteins.

What is the purpose of the CACC overhang added to the primer in directional TOPO cloning?

The CACC overhang on the primer in directional TOPO cloning is complementary to the GTGG overhang on the vector, allowing for strand invasion and bringing the hydroxyl group close enough to the topoisomerase for nucleophilic substitution, ensuring correct orientation of the insert.

Outlines

00:00

🔬 Understanding TOPO Cloning Basics

This paragraph introduces the three main types of TOPO cloning strategies: Blunt TOPO, TOPO-TA, and directional TOPO. TOPO cloning is a method for cloning PCR products that avoids the need for restriction enzymes and ligases. It relies on the enzyme topoisomerase, which binds to supercoiled DNA, makes a cut, and re-ligates it, causing DNA relaxation. The paragraph explains the role of Vaccinia Topoisomerase, a Type 1 beta class topoisomerase, and its binding and cleaving sites, particularly the CCCTT sequence. It also delves into the biochemical process of nicking, where the topoisomerase creates a DNA 3' phosphotyrosyl enzyme linkage, allowing the DNA to be ligated without the need for additional ligases.

05:06

🧬 Blunt TOPO Cloning and Its Challenges

The second paragraph focuses on Blunt TOPO Cloning, explaining that the vector has the topoisomerase recognition sequence CCCTT at its ends, which are not connected, making it linear. The insert DNA must also be blunt and dephosphorylated to allow for nucleophilic substitution. The process can result in two possible orientations of the insert, necessitating screening for the correct one. A key challenge is the possibility of the vector self-ligating, which can be mitigated by using a gene like ccdB that kills bacteria if the vector self-ligated. TOPO TA cloning is also introduced, which uses sticky ends and requires dephosphorylation, and can be achieved through specific restriction digests or by adding adenine overhangs to PCR products.

10:08

🧭 TOPO Directional Cloning for Specific Orientation

The final paragraph discusses TOPO directional cloning, which ensures the insert is cloned in a specific orientation. This method uses a linear vector with both blunt and sticky ends. The insert is typically blunt and can be obtained from PCR amplicons. To achieve directional cloning, one side of the insert is designed with a CACC overhang to pair with the GTGG sticky overhang on the vector, preventing self-ligation and ensuring the insert can only bind in one orientation. The paragraph also touches on the vector's role as an expression vector, containing promoters and other elements necessary for gene expression, making it suitable for cloning cDNA or genes for further use in genetic engineering.

Keywords

💡TOPO Cloning

TOPO cloning is a method of DNA cloning that utilizes the enzyme topoisomerase to insert DNA fragments into vectors. Unlike traditional cloning methods that require restriction enzymes and ligases, TOPO cloning simplifies the process by using the topoisomerase's ability to cut and re-ligate DNA strands. The video script explains three types of TOPO cloning: blunt ends, TOPO-TA (sticky ends), and directional TOPO. This method is central to the video's theme as it represents a modern and efficient approach to molecular cloning.

💡Topoisomerase

Topoisomerase is an enzyme that alters the topological state of DNA by cutting one or both strands, allowing the DNA to relax, and then re-ligating the strands. In the context of the video, topoisomerase is crucial for the TOPO cloning process, where it facilitates the insertion of DNA fragments into vectors by creating a nick in the DNA backbone. The script mentions Vaccinia Topoisomerase, a type 1 beta class topoisomerase, which is used in TOPO cloning.

💡Blunt Ends

Blunt ends refer to the double-stranded DNA fragments that have flush ends without overhangs. In the video, blunt TOPO cloning is discussed as a strategy where both the vector and the insert DNA have blunt ends, allowing them to be ligated together without the need for complementary overhangs. This is an example of how the script explains the specificity of DNA ends in different cloning strategies.

💡TOPO-TA Cloning

TOPO-TA cloning is a variation of TOPO cloning that uses sticky ends to facilitate the ligation of DNA fragments. The script explains that this method involves vectors with T overhangs and inserts with A overhangs, enabling them to anneal and be ligated together. This approach is highlighted as a modification to the classic TOPO cloning method, showcasing an adaptation that allows for sticky end cloning.

💡Directional TOPO

Directional TOPO cloning is a strategy that ensures the insert DNA is cloned in a specific orientation relative to the vector. The video script describes how this is achieved by using a combination of sticky and blunt ends on the vector, and by designing the insert with specific overhangs that control the orientation during ligation. This concept is key to understanding the precision that can be achieved in TOPO cloning.

💡Vaccinia Topoisomerase

Vaccinia Topoisomerase is a specific type of topoisomerase derived from the vaccinia virus, an ancestor of the smallpox virus. As detailed in the script, this enzyme is used in TOPO cloning due to its ability to recognize and cleave specific DNA sequences, such as CCCTT. Its role in the video is to demonstrate the specificity of the enzyme-DNA interaction, which is essential for the successful cloning process.

💡Nucleophilic Substitution

Nucleophilic substitution is a chemical reaction where a nucleophile replaces another atom or group in a molecule. In the video, this concept is used to describe how the topoisomerase enzyme cuts the DNA by attacking the DNA backbone, forming a covalent bond between the enzyme and the DNA. This reaction is central to the nicking activity of topoisomerase during the cloning process.

💡ccdB Gene

The ccdB gene is mentioned in the context of a strategy to prevent self-ligation of vectors in TOPO cloning. If a vector ligates itself without an insert, the ccdB gene is reconstructed and active, leading to the death of the bacteria hosting the vector. If an insert is present, the gene is inactive, allowing the bacteria to survive. This concept is crucial for understanding the selection process in TOPO cloning.

💡Expression Vector

An expression vector is a type of cloning vector designed to allow the expression of a cloned gene in a host organism. The video script explains that directional TOPO vectors are often used as expression vectors, containing promoters that drive the transcription of the inserted gene. This is important for applications where the goal is not just to clone but also to express the gene product, such as in protein production.

💡PCR Amplicons

PCR amplicons are the products of the polymerase chain reaction (PCR), which are DNA fragments amplified from a template. The script mentions that PCR amplicons, especially those generated by proof-reading polymerases, are often blunt-ended and thus suitable for TOPO cloning. Understanding the nature of PCR products is essential for grasping how they can be used in cloning strategies like TOPO.

Highlights

TOPO cloning is a popular strategy for cloning PCR products without the need for restriction enzymes and ligases.

TOPO stands for Topoisomerase, an enzyme that relaxes supercoiled DNA by making a cut and re-ligating it.

Vaccinia Topoisomerase, used in TOPO cloning, is a Type 1 beta class topoisomerase with two binding and cleaving sites.

The CCCTT sequence is a common binding site for Vaccinia Topoisomerase in TOPO cloning.

TOPO cloning involves a nucleophilic substitution reaction where the topoisomerase cuts the DNA at specific sites.

The topoisomerase creates a DNA 3' phosphotyrosyl enzyme linkage during the nicking process.

Blunt TOPO Cloning uses a linear vector with topoisomerase recognition sequences at the ends, requiring a blunt insert.

Dephosphorylation of the insert is necessary for the nucleophilic substitution to occur in Blunt TOPO Cloning.

TOPO TA Cloning uses a vector with sticky ends and an insert that has been dephosphorylated and tailed with adenines.

The TA cloning method involves base pairing between T and A overhangs for vector and insert ligation.

TOPO directional cloning allows for the insert to be cloned in a specific orientation using both sticky and blunt ends.

In directional TOPO cloning, the insert's 5' end is designed to be complementary to the vector's sticky end to facilitate ligation.

Directional vectors are often used for expression cloning and contain promoters for transcription.

The GTGG overhang on the PCR primer ensures that the insert is in frame with the vector for expression.

Bacterial flap endonucleases can cleave and seal the nicks in partially ligated vectors, completing the circularization.

TOPO cloning bypasses the need for screening for the correct orientation of the insert, unlike traditional cloning methods.

Transcripts

There are three main types of TOPO based cloning  strategies. The topo cloning that uses Blunt ends.  

The modification to classic TOPO is TOPO-TA, which  uses sticky ends. Third is the directional TOPO  

which uses both sticky and a blunt end. In case  you are not familiar, the TOPO is shorthand for  

the enzyme Topoisomerase. This is a very popular  strategy to clone PCR products and it bypasses  

the need of restriction enzymes and ligases  that we saw in the classic molecular cloning.

You will understand topo cloning  if you understand the basics of  

topoisomerase. In the most simple form, a  topoisomerase binds to a supercoiled DNA,  

makes a cut in one of the DNA strands, and  subsequently re-ligates the cut. Through  

this simple cut in the supercoiled DNA,  the DNA undergoes relaxation. Following  

the re-ligation of the DNA and relaxation  of the DNA, the topoisomerase walks away.

In TOPO cloning, the topoisomerase that is used  is called Vaccinia Topoisomerase. Vaccinia is  

an ancestor of the smallpox virus. You don’t  have to know this, but Vaccinia topo is a Type  

1 beta class topoisomerase. The important  thing to know is that it has 2 binding and  

cleaving sites. Think of them as DNA motifs that  attract the topoisomerase to the DNA. CCCTT is  

one of the two binding sites. If present in a  double stranded DNA context, the vaccinia topo  

will recognize this site and make a cut after  the last Thymine in the CCCTT sequence. The  

second sequence of specificity is TCCTT. Same  logic here as well. The Cut is made after the  

last T. If you recall our simple diagram here, I  did say that the cut is made only in one of the  

DNA strands. This cleaving activity is actually a  “nicking” activity, since only one of the strands  

is cut. This is very different from restriction  enzymes, where a DNA sequence is recognized but  

the cut is made on both DNA strands, which  actually splits the DNA into two parts.

Generally, the CCCTT sequence is most used in topo  cloning. So I will restrict our discussion to just  

this one. There is a little bit of biochemistry  you must understand regarding the nicking or  

binding/cleaving activity of the topoisomerase.  Let’s zoom into this nick location. Here is the  

top strand of the DNA with its backbone.  In the active site of the topoisomerase,  

there is a tyrosine amino acid. From the N  terminal end it is the 274th amino acid. The  

free hydroxyl of the tyrosine attacks the  DNA backbone between the T and N base. It  

specifically attacks the phosphate - this  is a nucleophilic substitution reaction.  

This is what cuts or nicks the DNA. When  the substitution reaction is complete,  

the phosphate gets connected to the tyrosine  274 of the topoisomerase. If you annotate the  

direction of this DNA, you notice it is a  3’ phosphate connected to a tyrosine. So,  

we call it a DNA 3’ phosphotyrosyl enzyme linkage.  This means that the next base, the base N, is cut  

off from the T base. And the 5’ end of the base  N has a hydroxyl because the phosphate is on the  

3’ end of the base T. In a way, this reaction has  dephosphorylated the base N during this cleaving  

step. Now if we revisit the cut and ligation step  of topoisomerase, we can add an intermediate step,  

containing a DNA enzyme linkage. But, why or  rather how is this step an intermediate step?  

If you look closely, the free hydroxyl of the 5’  end here is similar to the free hydroxyl on the  

topoisomerase. So this is also free to attack the  phosphate in this DNA enzyme linkage. This will  

be yet another nucleophilic substitution.  When this second reaction is complete,  

the two bases are re-ligated back through the  phosphodiester linkage. We have now come full  

circle to the original stage. Just to reiterate,  the first attack is from enzyme to DNA, this is  

a nucleophilic substitution. The second attack  is a DNA to DNA, which again is a nucleophilic  

substitution. And this way, the intermediate  stage resolves itself, and the DNA ends are  

able to ligate without any need of DNA ligases.  This intermediate step is actually everything.

Hopefully, you have understood the basics, so  let's get into TOPO cloning. First type is the  

Blunt TOPO Cloning. All TOPO cloning vectors rely  on stabilizing or artificially creating a stable  

intermediate stage. All TOPO vectors are linear.  The TOPO blunt vector is meant for cloning DNA.  

It is NOT an expression vector. The ends of  the TOPO blunt vector have the Topoisomerase  

recognition sequence CCCTT. This is one continuous  DNA but the ends are not connected - hence it is  

a linear vector. At this recognition site, the  intermediate stage is stabilized - meaning that  

the T is connected to the topo on both ends  of the vector. This is the 3’ phosphotyrosyl  

linkage we discussed earlier, which occurs  at the base T. That is the vector, but what  

about the insert? Note that the vector is blunt  - meaning that the insert must also be blunt. If  

you get your insert from a digest, you must then  dephosphorylate the insert using a phosphatase.  

You may be wondering why is dephosphorylation  required? If you recall the intermediate step,  

the 5’ end needs a hydroxyl to perform the  substitution. TOPO cloning is typically meant for  

PCR inserts - typically PCR amplicons are blunt if  you use some proof-reading DNA polymerase. Normal  

PCR primers are not phosphorylated, so the 5’ ends  of a blunt PCR have this same dephosphorylated  

biochemical structure. So here is the ideal form  of the insert - the ends have a free hydroxyl, no  

phosphate, and they are blunt. The free hydroxyl  can perform nucleophilic substitution by attacking  

the phosphate in the vector. The insert will  replace the enzyme linkage, and the phosphodiester  

bond is created at both 5’ ends of the insert. But  wait! We have these two 3’ ends and the 5’ ends of  

the vector. They are not yet ligated. So this is  a partially closed vector. It is circular but not  

fully closed. Once inside the bacteria and the  nick can be fixed. So this way, a linear vector  

can be turned into a circular vector. There is  a small catch. Say the insert has two locations,  

x and y. And it is also possible that this  hydroxyl attacks this phosphate and the other  

hydroxyl attacks this phosphate. Right? So in the  first case, the x to y orientation can be cloned.  

In the other case, the insert can be flipped and  y to x orientation gets inserted. So depending  

on which hydroxyl and phosphate combination is  used, you can get two types of final vectors.  

This is also the principle of blunt cloning, that  you have to then screen for your desired vector.

There is one problem with this. A high probability  problem. Both the 5’ ends of the vector also have  

free hydroxyls in them, which can also attack  the phosphate and when it happens you close  

the linear vector by itself. How do you deal  with this problem? The easiest way is to get  

rid of hydroxyls from the ends of the vector.  The alternative and this is the most widely  

used strategy, is to have the TOPO cloning sites  in the middle of a gene called ccdB. If a vector  

closes itself, the gene is reconstructed and  becomes active. Once this self-ligated vector  

is inside the bacteria, the active gene will kill  the bacteria. Alternatively, if you have inserted  

a piece of DNA in the cloning site then ccdB  is inactive and those bacteria will survive.

Second type is TOPO TA cloning. This vector has  sticky overhangs. It is missing the complementary  

3’ Adenine. So this is a T overhang vector.  What about the insert? Well it needs to be  

sticky. You can get them from a sticky digest  like XcmI or AdhI. Like we saw before, it then  

must be dephosphorylated for the nucleophilic  substitution to work. These sticky digests give  

you an A overhang. What about PCR amplicons?  If you do a PCR with a non-proof reading DNA  

polymerase like the Taq polymerase, they can  add an Adenine nucleotide at the end of your PCR  

product. This is a template independent activity  of most non-proofreading DNA polymerases. Now this  

insert also contains the A overhang. If you do  the PCR using a proof-reading polymerase, then you  

must perform Adenine tailing on your PCR product.  This is achieved using dATPs and a special Klenow  

that lacks the Exo activity. This tailing will  add an adenine to the 3’ end of the PCR product.  

This A-overhang insert is now ready with its 5’  hydroxyls to attack the phosphate on the vector.  

The A will base pair with T, and the nucleophilic  substitution will join the vector and insert. Once  

again, this is a circular final vector that is  partially ligated and the remaining nicks can  

be fixed once this vector is inside the bacteria.  In this vector, there is no self ligation issue  

because vector closing depends on the sticky base  pairing. T cannot pair with T. Let’s take a small  

detour. You can do this with restriction enzymes  as well. Essentially you need a T-overhang vector,  

and an A overhang insert. The T and A will base  pair and ligases can be used to ligate the insert  

and the vector. This is called TA cloning because  of the pairing between T and A bases. This is a  

non TOPO version of TA cloning. Hopefully, you  see why this is called TA cloning. The big issue  

here is that the product is symmetric in all TA  cloning methods. Meaning that you can flip the  

insert 180 degrees and there is a chance that  you insert the reverse orientation. So similar  

to blunt TOPO cloning you have to screen for  your desired vector in TOPO TA Cloning as well.

The last strategy is TOPO directional strategy.  This implies that insert can only be cloned in  

one specific orientation. Like other TOPO vectors  it is also linear but instead it is an expression  

vector. Typically you would use this for cDNA  or gene cloning. Note that I mentioned how this  

vector uses both sticky and blunt sites? Well  one of the vector ends is a blunt end. This  

other end has a sticky overhang GTGG. This insert  is typically blunt. You can get it from Type IIS  

digests but typically you don’t do that. Almost  100% of the time you have a cDNA amplified from  

PCR that needs to be cloned. And PCR amplicons  from proof-reading polymerases are blunt. Now,  

let’s say you want to insert this DNA in the  x to y orientation. Meaning that x corresponds  

to the sticky side, the y corresponds to the  blunt side of the vector. So when you do a PCR,  

you must add CACC as the 5’ overhang to the primer  on the x side of the insert. Here is the final  

corrected form of the insert. Note that this GTGG  in the vector is in the same direction as the GTGG  

on the insert. Alright, when you mix the vector  and insert together, the y side of the insert  

can attack the blunt y side of the vector but the  x side of the insert cannot attack the x side of  

the vector because there is a sticky overhang. So  somehow the hydroxyl needs to be brought closer to  

the Topo in this situation. How is this achieved?  Well, the CACC is complementary to the GTGG. This  

GTGG from vector can strand invade into the blunt  x side of the insert and try to base pair. As a  

result of this strand invasion, the GTGG of the  insert is kicked out. This brings the C and T  

close to each other. This allows the hydroxyl from  the C to attack the phosphate on the T and the  

two DNA ends are ligated. The other side is just  blunt, so ligation on the y side is not a problem.  

Now we have a partially ligated circular vector,  which also has a small flap on it. But again,  

this is not a problem, the bacteria has flap  endonucleases that can cleave the flap and  

seal the nicks. You may have seen this type of  flap cut and nick sealing in Okazaki fragment  

maturation in bacterial DNA replication. So  this way you can clone the inserts in a specific  

direction. I hope it is obvious that only one  side of the insert should contain GTGG. Also,  

it should be obvious that there is no  self-ligation issue in this vector.

One last point about what makes this directional  vector an expression vector. The vector contains  

some sort of promoter like SP6, T7 or maybe the  pBAD of the arabinose operon. This follows the  

ribosome binding site which lives next to ATG -  our first codon. In frame with the ATG codon is  

this CCC from the cloning site. The next codon  is then TTC and ACC. This means that the GTGG  

overhang of your PCR primer is already part of  a codon in frame with the vector. And this means  

that the first base of the insert - the N is the  start of a new codon, and this continues until the  

last nucleotide which ends in a full codon. After  the N you start with the AAG from the vector,  

which is a codon, and this continues till a stop  codon. In between you may have some purification  

FLAG tags or something else. Depending on the  promoter in the front, you may have specific  

terminators or polyA signals at the end of  this expression cassette. So this way you  

can use the cloned insert which is a template  for making RNA or even proteins from those RNA.

If you found this video useful, I  have many more videos on cloning  

and genetic engineering, in the  playlist linked in description.