4.2: Creating Recombinant DNA

Preparation of the insert and plasmid for cloning

Both cloning and expression plasmids contain a specific region made up of multiple restriction sites. This region, the Multi-Cloning Site, or MCS, is the site of DNA insertion. In order to insert a piece of DNA into a plasmid (either for cloning or expression) both the DNA and plasmid must be "prepared" or digested with restriction enzymes. While not as important in a cloning plasmid, insertion of DNA into an expression plasmid must be done in a specific orientation so the DNA can be expressed from the nearby promoter. Thus, in both cloning and expression plasmids, insertion into a MCS is typically done using a "directional cloning" strategy where the plasmid and insert are cut with two different restriction enzymes. This type of restriction digest is called a double digest because it uses two different restriction enzymes. This digest will produce unique DNA single-stranded overhangs at each end of the insert. DNA overhangs, complementary to the ends of the insert, will also be produced in the plasmid. As a result, there will be only one way that the DNA can be inserted into the plasmid. To learn about the restriction digest, go to Chapter 13.4: Restriction digestion of DNA.

Figure \(\PageIndex{2}\) and Figure \(\PageIndex{3}\) below shows such a directional cloning strategy using the restriction enzymes HindIII and PstI. In the first part of the procedure shown in Figure \(\PageIndex{2}\), both the DNA insert and plasmid are digested with HindIII and PstI. Digestion of the DNA insert with Hind III produces a 5' overhang with the sequence "AGCTT". Digestion of the insert with PstI will produce a 3' overhang of "GGTAC". When the plasmid is digested with the same two enzymes, a HindIII overhang of "TTCGA" and a PstI overhang of "CATGG" is produced (the tiny bit of plasmid DNA found between these two overhangs will be removed).

These complementary ends are critical for the correct ligation of the insert and plasmid to form a new piece of recombinant DNA.This is because the DNA insert overhangs of AGCT and GTAC are complementary to the plasmid overhangs of TCGA and CATG (Figure \(\PageIndex{3}\)). As a result, there is only one way that the DNA insert can "fit" into the plasmid. Once the insert and plasmid are brought together, an enzyme called a DNA ligase will form the phosphodiester bonds required to join these pieces of DNA together. Following ligation, the HindIII and PstI restriction sites are regenerated and can be used to digest out the insert if desired.

While directional cloning is the preferred strategy, there are times when restriction enzymes that produce a "blunt" end must be used. This technique is called blunt end cloning. In blunt end cloning, the restriction enzyme cuts both strands of DNA at the same position within the restriction site. As a result, no overhangs are produced and the ends are said to be "blunt" (Figure \(\PageIndex{4}\)).

As with directional cloning, once the DNA insert and plasmid are digested with the restriction enzyme, the two are ligated to one another by the DNA ligase. Following ligation, the two EcoRV restriction sites are regenerated and can be used to digest out the insert if desired (Figure \(\PageIndex{5}\)).

Because there are no overhangs produced when using restriction enzymes that produce blunt ends (i.e., blunt end restriction enzymes), it is important that the DNA insert be ligated into the plasmid so that the restriction sites are regenerated. The regeneration of the restriction sites ensures the correct orientation of the insert so that it can be expressed, if desired. Figure \(\PageIndex{6}\) below shows the correct cloning orientation of a DNA insert.

In addition to this orientation, there are three other possibilities for cloning the DNA insert. However, none of these will regenerate the restrictions sites at either end of the DNA insert (Figure \(\PageIndex{7}\)). While these other orientations may permit gene expression, the protein made will be incorrect. Therefore, when using a blunt end strategy, it is vital to confirm the orientation of the insert within the plasmid. This is typically done through DNA sequencing.

Ligation of the insert and plasmid to produce the recombinant plasmid

Insertion of the DNA insert into the plasmid requires the formation of new phosphodiester bonds between the two pieces of DNA. This is done by a type of enzyme called a DNA ligase (or ligase). Ligation of the insert and plasmid will join these two fragments into one piece, restoring the circular nature of the plasmid and forming a piece of rDNA known as a recombinant plasmid (Figure \(\PageIndex{8}\)). As described above, ligation will also regenerate the restriction sites used in cloning so that the DNA insert can be cut out again, if desired. It is important to know that DNA ligation is not 100% effective. Some plasmids will resume their circular configuration without incorporating a DNA insert. These "false positives" will need to be identified. To learn about DNA ligation, go to Chapter 13.5: Ligation of insert DNA into a plasmid.

Transformation of host cells with the recombinant plasmid

Once the DNA insert has been ligated into the plasmid, the recombinant plasmid is then introduced into bacterial cells so that the bacterial host cell can replicate the recombinant plasmid along with its own DNA. This introduction is done through a technique called bacterial transformation, or transformation (Figure \(\PageIndex{9}\)). Transformation relies upon the finding that bacteria will take up "naked" DNA from its surroundings if exposed to specific environmental conditions. One common method of transformation is to weaken bacterial membranes through their exposure to ice-cold salt solutions. These weakened bacteria are then mixed with recombinant plasmids and then "heat shocked" at 42°C for a short period of time. In a mechanism not understood, this heat shock allows for the entry of the recombinant plasmids into the bacteria. To learn about bacterial transformation, go to Chapter 13.6: Bacterial transformation.

Transformation is very inefficient. The majority of bacteria will not incorporate recombinant plasmids. As such, a selection process for identifying those bacterial cells containing recombinant plasmids must be used to eliminate those bacterial cells that do not have any recombinant plasmids. This process relies upon the presence of selection markers in the recombinant plasmid. The most common type of selection marker is an antibiotic resistance gene - a gene that will confer resistance to specific antibiotics that bacteria are not normally resistant to. Common antibiotic resistance genes will provide resistance to ampicillin, tetracycline, or kanamycin. Bacteria that are transformed with recombinant plasmids are typically grown overnight on agar plates containing a specific antibiotic. Only those bacteria with recombinant plasmids should survive exposure to this antibiotic. These bacteria will propagate on the agar over night and form a bacterial colony.

As with many other molecular techniques, antibiotic selection is not foolproof. Some transformed bacteria will contain plasmids that do not contain an insert. Other bacteria may survive exposure to the antibiotic and will have no plasmid at all. As such, confirmation of the recombinant plasmid within the bacteria must be obtained. This confirmation will necessitate the isolation of the recombinant plasmid DNA from bacterial cells (Chapter 13.7: Isolation of DNA from bacteria (i.e., the “mini-prep”), followed by additional tests to confirm the presence of the insert, such as polymerase chain reaction/PCR (Chapter 13.2: Polymerase Chain Reaction (PCR) and agarose gel electrophoresis (Chapter 13.3: Agarose gel electrophoresis).

Recombinant plasmids can also be introduced into mammalian cells through a process known as transfection (Chapter 13.10: Liposomal transfection into mammalian cells). Transfection typically occurs after the recombinant plasmid has been introduced into bacteria, amplified, and the presence of the DNA insert inside the recombinant plasmid has been verified.

Creating Recombinant DNA

• Lab Technique: Amplification of DNA by PCR
• Lab Technique: Polymerase Chain Reaction (PCR)
• Lab Technique: Restriction digestion of DNA
• Lab Technique: Ligation of insert DNA into a plasmid

Transformation of Bacteria

• Lab Technique: Bacterial transformation
• Lab Technique: Isolation of DNA from bacteria (i.e., the “mini-prep”)
• Lab Technique: Agarose gel electrophoresis

Transfection of Eukaryotic Cells

• Lab Technique: Liposomal transfection into mammalian cells