This video illustrates a series of molecular biology laboratory practices
whose ultimate goal is to obtain a recombinant DNA.
A recombinant DNA is the result of the incorporation of a foreign DNA fragment
which we call insert,
in a small molecule capable of replicating itself
for example, a plasmid or bacterial vector.
The result of the union of the insert and the vector is what is called a recombinant DNA.
In this practice we will use a commercial vector already open.
Furthermore, the cloning site of the insert is disrupting the lacZ reporter gene,
which encodes the β-galactosidase enzyme.
We will explain the utility of lacZ later in this video.
In this practice,
the insert is a gene from a eukaryotic organism (a plant).
To obtain it, we will follow a series of steps that we show next.
The first step is to extract genomic DNA
from a certain amount of plant tissue.
The key of the protocol
is to keep in mind that DNA is soluble in aqueous substances
and insoluble in alcohols.
Every DNA extraction consists of different steps:
first: to break the cells to release their content.
This is done mechanically, grinding the tissue,
and also chemically,
causing cell lysis using buffers containing detergents.
In our case we will use the CTAB buffer.
Once added, we incubate the samples at 65°C for 30 minutes in a heat block.
Next, we must separate the DNA from other cellular components.
This step is performed using organic solvents.
We removed the sample from the heat block
and add a mixture of phenol:chloroform in equal volumes,
mix the sample using the vortex
and centrifuge for 10 minutes.
After centrifugation,
the DNA is found in the upper (aqueous) phase of the sample.
We transfer it to a new tube
and add an equivalent volume of chloroform.
Vortex again and centrifuge again.
Once more, after centrifugation,
the DNA remains in the upper phase.
We transfer it to a new tube, and to precipitate the DNA
we add cold absolute ethanol.
We mix the sample by inverting the tube
and let the DNA precipitate at -20°C for at least 10 minutes.
We centrifuge the samples to properly precipitate the DNA
and remove the alcohol very well to allow the precipitated DNA to dry completely.
This may take about 10 minutes.
Finally, we resuspend the DNA in water or an aqueous buffer, in our case TE.
DNA is quite stable but should be stored at 4°C if it will be used soon,
or at -20°C if it is not expected to be used for some time.
To amplify exclusively the gene of interest (insert) from genomic DNA,
we must perform the polymerase chain reaction
(PCR).
This technique makes it possible to obtain many copies of a given DNA fragment
through the action of the DNA Taq polymerase enzyme.
In the PCR we will add:
the template DNA,
We adjust the volumes using water.
the Taq polymerase and its buffer, which must contain magnesium.
free triphosphate nucleotides (called dNTPs),
the primers that will specifically recognize
the ends of the DNA sequence that we want to amplify.
When we prepare the PCR mix
we always start by adding the largest volumes, and end by adding the enzyme.
Once the PCR mixture is prepared,
we introduce the tubes that contain it in the thermal cycler,
which will allow us to make consecutive temperature changes
very quickly.
PCR consists of several cycles of different duration and temperature
that allow:
1) The denaturation of the two strands of DNA.
This step is performed at 95°C, a temperature at which
the hydrogen bonds that hold the double-stranded DNA together are broken.
2) The pairing of the primers with the complementary template sequence.
This step is done at temperatures around 55-60 °C.
Lowering the temperature from 95 to 55°C
allows the template DNA and primers to bind and form a double strand.
3) DNA extension.
Taq polymerase at 72 °C
is able to incorporate free nucleotides after the primers,
copying the sequence of the template chain.
We must maintain this temperature long enough
for the Taq polymerase to have time to copy the entire sequence that we want to amplify.
Many Taq polymerases are capable of copying 1,000 nucleotides in one minute.
These three temperature changes are repeated for about 30 cycles,
so that the amplification of the sequence of interest is done exponentially.
At the end of the PCR, each initial DNA molecule will have been copied million times.
To visualize the result of the PCR, we will do a DNA electrophoresis.
First, we prepare the gel tray with the comb
that forms the wells where we will load the samples.
The agarose gel is prepared with an aqueous buffer
that will be the same that will be used to fill the electrophoresis cell.
This buffer prevents changes in pH due to the electric field.
We mix the agarose and the buffer
and bring it to the boil to dissolve it completely.
We cool it to about 60°C
and add the intercalating agent that will make the DNA bands visible
when exposed to ultraviolet light.
Next, we pour the liquid agarose into the mold and let it cool down.
Once cold and solidified, we remove the tape from the sides of the gel tray and the comb,
introduce the gel into the electrophoresis cell and fill it with the buffer.
We proceed to load a part of the PCR product
and the other we keep it.
To load the samples we must add a dye,
which will allow us to visualize them,
and glycerol, which will give them density and will make them stay inside the wells.
Nucleic acid molecules, such as DNA,
are negatively charged,
and are separated by applying an electric field to move them through the agarose matrix.
We close the cuvette and connect it to the power supply.
We program the voltage and start the electrophoresis.
The migration of nucleic acids is determined by the charge and size of the molecule:
the molecules move towards the positive pole.
Small ones migrate faster than large ones.
Once the electrophoresis is finished, we remove the gel from the cell and observe it in an ultraviolet light transilluminator.
After confirming that we have amplified the insert of interest by PCR,
we can introduce it into the vector.
This reaction is called ligation.
In a tube we mix the vector,
the insert (in our case the rest of the PCR product that we have reserved and purified),
and the ligase enzyme, which catalyzes the union.
Therefore, we must also add it corresponding buffer corresponding.
Ligation will be performed overnight at 4°C.
The final product of ligation is a recombinant DNA.
Bacterial transformation consists of introducing recombinant DNA
into competent bacterial cells
that will allow it to replicate many times.
In our case we will use chemocompetent Escherichia coli cells,
which have the ability to acquire recombinant DNA
when we apply a heat shock to them.
This shock causes the permeabilization of the membrane for a few minutes,
which allows the entry of recombinant DNA without damaging the cells.
For transformation we add the ligation product
(i.e., the recombinant DNA)
to a tube containing competent cells.
We incubate the cells on ice for 30 min.
Next, we apply a heat shock of 42 °C for a minute and a half,
let them recover on ice for a couple of minutes
and add 1 ml of liquid culture medium without antibiotics.
We leave the tube at 37 °C for an hour.
We then plate the transformation on plates containing solid medium supplemented with the antibiotic
and the corresponding selection agents,
working under a laminar flow hood to avoid contaminations.
We incubate the plates at 37° overnight.
En el plàsmid pGEM, tenim dos sistemes de selecció
In the pGEM plasmid we have two selection systems to identify cells that have acquired the insert:
the antibiotic resistance gene ampicillin and the lac Z gene.
In the absence of an insert, the lac Z gene is uninterrupted,
and therefore it will give rise to the protein β-galactosidase.
In the presence of the insert, the lac Z gene is interrupted,
and will not produce any protein.
On our plates with the antibiotic ampicillin,
only colonies of bacteria containing the pGEM vector will have grown.
In the presence of the IPTG inducer and the X-Gal substrate in the medium,
β-galactosidase does metabolize it and gives rise to a blue precipitate.
Thus, the blue colonies are bacteria that have incorporated the vector without an insert,
while the white ones are bacteria that contain the vector with the insert
(that is, the recombinant DNA that we wanted to obtain).
To isolate the recombinant DNA from the white bacterial colonies that contain it,
we will inoculate with them liquid cultures
that will grow shaking overnight at 37°C.
The next day we will recover the bacterial cells by centrifugation
and this time we will extract the plasmid DNA,
(that is, recombinant) in a similar way to the protocol described to isolate genomic DNA.
We can verify that the isolated plasmid DNA is the one that contains our insert of interest
by PCR or digestion with specific restriction enzymes.
Finally we sequence of the recombinant DNA
using Sanger sequencing.
We check whether the sequence is correct using specific software
and comparing it with sequences
in public databases whenever possible.
Once verified, we can use this recombinant DNA with multiple purposes!
This video describes the different steps to obtain
and analyze recombinant DNA in a comprehensive manner.
It is necessary to take into account
that the protocol may vary depending on where we obtain the insert of interest from,
that DNA extractions can also be performed using commercial kits,
that a recombinant DNA can also be obtained by recombination
instead of the restriction-ligation system explained here,
and that the nomenclature used may vary slightly in different laboratories.
We hope that the video has helped you to understand
the basic principles of these molecular biology protocols,
and that this opens the doors
to multiple adaptations and applications!