TOPO Cloning - TOPO-Blunt, TOPO-TA, TOPO-directional
Summary
TLDRThis 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
🔬 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.
🧬 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.
🧭 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.
Mindmap
Keywords
💡TOPO Cloning
💡Topoisomerase
💡Blunt Ends
💡TOPO-TA Cloning
💡Directional TOPO
💡Vaccinia Topoisomerase
💡Nucleophilic Substitution
💡ccdB Gene
💡Expression Vector
💡PCR Amplicons
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.
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