Colony PCR is a rapid, high throughput PCR method to determine the presence or absence of the inserted DNA into plasmid directly from the bacterial colonies.
Molecular cloning is one of the most popular methods for DNA transformation since long. However, for determining the presence or absence of the DNA insert we have to perform transformation experiments.
Colony PCR is a novel method in which by designing the inserted DNA specific primers, we can identify whether our DNA of interest is inserted into the plasmid or not.
However, it is not as simple as we are discussing.
In this article, we will focus on the Colony PCR especially, the principle of the Colony PCR, its advantages and limitations.
For that, we have to understand several terms and topics. We will start our topic from the basics. The content of the article is,
- What is Plasmid?
- What is Colony PCR?
- Principle of Colony PCR
- Protocol of colony PCR
- Procedure of colony PCR
- Advantages of colony PCR
- Disadvantages of colony PCR
- Application of colony PCR
What is plasmid?
“A plasmid is the bacterial circular DNA which replicates independently from the bacterial chromosome and used in the gene manipulation and gene transfer.”
Genetic cloning is a traditional molecular genetic tool used since long in the laboratories. Briefly, in the gene cloning, the gene of our interest is inserted into the plasmid through artificial means. This DNA is replicated independently from the bacterial chromosome.
Plasmids are actually, used to generate many copies of short segments of DNA. Because bacteria replicates faster than any other organisms, we can generate many copies of the gene of our interest by inserting it into the bacterial plasmid.
F-plasmid, Col-plasmid, degradative- plasmid and resistance plasmid are several common types of plasmids found in bacteria.
Furthermore, the plasmid can work as a molecular carrier which transfers short DNA segments from one cell to another cell.
We have covered an amazing in-depth article on plasmid DNA. Read it here: Plasmid DNA- Structure, Function, Isolation And Applications.
Apart from bacteria several other prokaryotes also contain plasmid DNA. The main function of the plasmid in bacteria is for their survival in the harsh conditions.
As the plasmid transfers gene of our interest, it is very important to determine if our gene of interest is inserted or not into the plasmid.
For that, we can use several methods such as PCR and microbial culturing.
Platting the colonies take more time and the sensitivity of the method is also not good as well. The chance of the contamination is always high in bacterial culture methods.
So the results are not accurate.
Our PCR helps here as well. Using the colony PCR method, a DNA insert can be determined or identified.
What is Colony PCR?
Colony PCR is the modification of the conventional PCR in which the bacterial colonies are directly used as a PCR template.
The plasmid DNA which contains the DNA of our interest is amplified in the cyclic-temperature dependent conditions.
The graphical representation of the colony PCR is shown into the figure below,
Principle of Colony PCR:
The bacterial colony containing the plasmid can directly be amplified using two sets of primers. The insert specific primers which amplify the insertion sequence and vector-specific flanking primers, which amplifies the plasmid DNA other than the inserted DNA (flanking regions on both the side of insert).
By using the insert flanking primers (which amplifies the rest of the DNA) the size of our DNA insert can be determined.
A bacterial colony is picked and added directly into the mastermix containing all the PCR reagents. Additionally, by adding one initial heating step to the PCR, the plasmid DNA comes out from the bacteria cell and amplified in the reaction.
This is the basic principle of the colony PCR, However, it can be modified depending upon the requirements.
The protocol of colony PCR:
The colony PCR is one of the excellent modification of the conventional PCR. Instead of template DNA, the bacterial colonies are directly added to the reaction. Besides this, Taq DNA polymerase, primers, PCR reaction buffer and DD/W are added in the PCR reaction too.
Here in the colony PCR, the selection of primers is very important. Also, the selection of primers depends on the objective of our experiment.
What type of information do we want from our colony PCR experiment?
- Information about the presence or absence of the insert only.
- Information about the size of the insert.
- Information about the orientation of the insert.
Depending upon that different PCR primers are designed for the colony PCR.
The insert specific primers bind to the specific location on both the side of the inserted DNA of our interest. If it is transferred properly into the plasmid, these primers can bind to it otherwise it can not be able to bind.
This primer set provides information regarding the presence or absence of the insert.
Orientation-specific primers are unique primers in which one primer binds inside the insert and another primer binds to the plasmid DNA sequence (sequence other than the insert DNA).
This types of primer set provide information about the orientation of inserted DNA of our interest. If our insert DNA is not properly ligated into the vector, the primer specific to that side of the sequence cannot bind, and we will not get the amplification.
Plasmid specific primers are also as important as the orientation-specific primers. This set of primers are designed from the flanking region of the insert which binds to the outside of the DNA of our interest.
This set of primer helps to determine the size of the insert. It expands regions other than the insert DNA.
The PCR reaction for performing the colony PCR is as followed,
|Master mix (Special
for the Colony PCR)
|PCR reaction buffer
With 2mM MgCl2*
Procedure of colony PCR:
Well, the colony PCR does not need extracted DNA.
We are not extracting DNA here. Instead, several other methods are used for increasing the sensitivity of the reaction.
Ok, why we are not extracting DNA for the plasmid DNA?
Because the reason is simple, the cell membrane of the bacterial cell is very smooth.
We had already discussed about the cell membrane of the bacterial cell. Read it here: Different types of DNA extraction methods
A bacteria contains soft cell membrane which can easily be lysed by heating or centrifuging it at high speed.
Also, we do not need the bacterial own DNA. The circulating circular plasmid is present into the cytoplasm of the bacteria Therefore additional purification steps are not required as well. By rupturing the cell membrane, our template DNA is ready for the amplification.
Ok, lets quickly go through the method for obtaining good plasmid DNA.
With the help of the sterile picker, pick several bacterial colonies and transfer it into the Eppendorf tube.
Now add TE buffer in it and mix it well. You can use D/W as well.
Heat the sample in the boiling water bath for 20 minutes.
Gently vertex it.
Centrifuge the sample at high speed for 2 minutes.
Transfer the supernatant to another tube and use it as a template DNA.
A 20μL sample is added into the reaction.
Why supernatant and not pellet?
DNA is a biomolecule of life. The plasmid DNA is even smaller than the bacterial nuclear DNA. It contains only several genes of up to 1000bp to 20,000bp.
Hence by only centrifuging it, the lighter plasmid DNA comes out from the cell and settled in the supernatant while the pellet contains proteins and nuclear DNA so we are not using it.
Now coming to the point.
Our plasmid is ready for amplification.
In another method,
Use the bacterial colony directly.
This method is a combination of Hotstart PCR and colony PCR.
The bacterial colonies are picked and added to the PCR reaction tube.
The tubes are placed into the PCR machine. One additional heating step is added.
By heating it 5 to 7 minutes, the plasmid DNA comes out from the cell.
Now the insert-specific primers amplify the DNA we inserted. And the flanking primers amplify the rest of the DNA.
The amplification is done for 20 to 25 cycles. The cycling conditions for the colony PCR is listed below,
|PCR Steps||Initial Denaturation||Denaturation||Annealing||Extension||Final extension|
|Temperature||95 ̊C||95 ̊C||55-65 ̊C||72 ̊C||72 ̊C|
|Time||3min||10 sec||45 sec||50 sec||5 min|
Read the interesting article on conventional PCR: A Complete Guide of the Polymerase Chain Reaction
Tips for improvement:
Use only a few colonies, as many colonies increase the chance of the non-specific bindings.
Use positive control and negative contol.
As a positive control used the flanking primer even if the insert is not present, the PCR reaction gives DNA band of the plasmid DNA which indicates that the reaction we prepared is correct.
As a negative control, use the untransformed plasmid (plasmid without insert DNA), this plasmid DNA is amplified only if the insert is present.
As an insert use short DNA sequences, longer DNA sequences increase the chance of non-specific bindings and PCR reaction failure.
Furthermore, use shorter PCR programs.
The main application of colony PCR is in the identification of correct ligation and insertion of insert DNA into bacteria as well as yeast plasmid.
After the completion of the colony PCR reaction, the PCR products are run on the 2% agarose gel. The results of the experiment are shown in the figure below,
Now carefully observe the results, the M is the 3000bp molecular DNA marker. Suppose, the DNA of our interest, “insert” is a 400bp fragment which is inserted into the plasmid.
See the lane 2: the 400 bp fragment of our insert.
We designed flanking primers 100bp away from both the side of the insert. If the flanking primer amplifies the DNA along with the insert the product is 600 bp, see the lane 3 (400 bp of insert DNA + 200 bp flanking region).
Now, see lane 1, it is a positive control without the insert or a normal plasmid without the transformed DNA. Hence the flanking primers only amplify 200 bp of DNA.
See lane 1, 200bp fragment of DNA without insert (positive control).
Now, observe lane 4. Lane 4 is the results of the orientation-specific primers. The orientation-specific primer is a combination of insert-specific primer and flanking region-specific primer.
One primer from insert DNA and one primer from the flanking region-specific primer are selected for orientation-specific primer amplification.
Therefore, 100 bp fragment from the flanking region primer and 400bp from the insert DNA is amplified and 500 bp fragment of DNA is observed in lane 4.
Lane 5 is the insert specific control which gives 400 bp DNA fragment.
Lane 6 is the negative control without the template. By using negative control any contamination can be identified. The reaction tube contains all the ingredients except the template. So ideally no DNA bands are present in this lane.
If a DNA band is observed, the sample is contaminated.
Advantages of colony PCR:
- The technique is rapid and cost-effective.
- Further, the accuracy and specificity of the technique are higher.
- The set up is simple just like the conventional PCR, DNA extraction and plasmid purification like laborious steps are not required.
- No need for restriction digestion for identification of the insert DNA.
- The whole experiment can be completed within 90 minutes.
Disadvantages of colony PCR:
- The method is cost-effective, fast and reliable, However, any mutation in the insert cannot be detected.
- Moreover, the sequence information can not be obtained by the colony PCR. we need to do sequencing for the confirmation of the DNA transformation.
- The chance of false-positive results is high.
After the completion of the experiment, the sample is sent for the sequencing where the DNA sequence of our interest can be determined.
we can even do multiplex PCR by combining both insert specific primers and plasmid-specific primers.
Although the colony PCR is the best choice for the identification of gene transfer, the only colony PCR technique is not sufficient to interpret the results. It might possible that some of the mutations present into the insert, that can not be detected by the PCR.
For confirming the results DNA sequencing is required. After determining the sequence order we can say whether our gene of interest is inserted correctly or not.