In Vivo Cloning

The complementary bases can pair up forming hydrogen bonds but they are weak. For permanent adhesion of the DNA fragments, DNA ligase needs to be used to create phosphodiesterase bonds between the phosphate and sugar groups of the adjacent DNA nucleotides.

Fig. 1 - Restriction enzymes cut the DNA at specific locations with a palindromic sequence. This leads to formation of sticky ends

In vivo cloning

In both eukaryotic and prokaryotic systems, RNA polymerase initially binds to the regulatory regions of the DNA. This is called the promoter and is adjacent to the gene. Transcription factors also bind to the promoter regions and assist recruitment of the RNA polymerase to the promoter. Without the promoter region, the DNA cannot be transcribed. Therefore, before inserting the DNA fragments into a host cell, we need to add a promoter region to the start of the gene.

Genes in the cells also contain a terminator region at which the RNA polymerase dissociates from the DNA and ends the transcription. Hence, we need to add a terminator region to the end of the gene.

Insertion into a vector

So far we have learned how recombinant DNA is produced. To introduce the recombinant DNA into a cell, we first need to insert it into a vector. A vector is any carrier, usually a virus or plasmid, that is used to transport a desired piece of DNA into a host cell.

We use the same restriction endonucleases used for creating the DNA fragments to cut open the plasmid. This is due to the formation of complementary sticky ends in the plasmid and the desired DNA fragment which facilitates the incorporation of the DNA fragment into the plasmid. When the sticky ends of the plasmids and the DNA fragments are lined up, DNA ligase is used to join the sugar-phosphate backbone of the strands and make their adhesion permanent.

At the end of this process, plasmids would be containing recombinant DNA.

Fig. 2 - Insertion of the gene of interest into a bacterial plasmid (vector)

Transformation of host cells with the vector

Transformation involves the introduction of recombinant DNA into the host cells.

In the case of bacterial cells, transformation is the reintroduction of recombinant plasmids into bacterial cells. This process requires the presence of calcium ions and changes in the temperature to make the plasma membrane permeable to the passage of plasmids.

The process of transformation often does not have a high yield. This is for three reasons:

1. Not all bacterial cells successfully uptake the recombinant plasmids.
2. Some of the plasmids may have been closed up without the incorporation of the desired gene.
3. The sticky ends of some of the desired DNA fragments may have also paired up with each other without being incorporated into a plasmid.

Identification of cells with the plasmid

Since the transformation process does not have a high yield, it is important to have a strategy for selecting the host cells that have taken up the plasmids. This selection is achieved by using antibiotics. Plasmids usually contain the genes that confer resistance to antibiotics. These genes often code for enzymes that can break down the antibiotic molecules to prevent the destruction of bacterial cells.

All the bacteria are grown in a medium that contains an antibiotic to use antibiotic resistance to select for bacteria that have taken up plasmids. Bacterial cells that have taken up the plasmid are resistant to the antibiotic and will survive. Meanwhile, other bacteria that have failed to take up the plasmid remain susceptible to the antibiotic and will die.

Not all the cells that have acquired resistance to the antibiotic contain the desired gene. This is because some plasmids may have closed up without incorporating the gene. Therefore, there need to be some strategies for selecting the cells that have taken up the recombinant plasmids.

Selection of cells that have acquired the desired gene

Scientists commonly use marker genes for identifying the cells that have been successfully transformed with the desired gene since they are located on the same DNA fragment that the desired gene is.

We normally use three different types of marker genes for this purpose:

Antibiotic resistance genes

Plasmids may often contain different genes that confer resistance to two different antibiotics.

The restriction endonucleases used to cut open the plasmid are designed to have their restriction sites in the middle of an antibiotic resistance gene. Therefore, when the desired DNA fragment is successfully incorporated into the plasmid, it also results in the bacteria no longer being resistant to that antibiotic. For instance, if the restriction enzyme cuts the R-plasmid at the tetracycline resistance gene, the recombinant plasmid would no longer contain a functional tetracycline resistance gene. So, if the cells are grown on a medium that contains tetracycline, the cells with recombinant DNA will die.

This causes a problem since the cells that we are interested in would be dead. To tackle this problem, we use the replica plating technique.

Fig. 3 - The replica plating technique used for selection of transformed cells with the desired gene

Fluorescent markers

Fluorescent marker genes code for proteins that fluoresce and glow when they absorb light. One example of these is the green fluorescence protein (GFP) which was first identified in jellyfish.

When GFP is added to the DNA fragment that contains the desired gene, the cells that have successfully acquired the recombinant plasmid would then express the GFP. They would then appear green. Since there is no need for replica plating, fluorescent markers make the selection process easier.

Enzyme markers

Similar to GFP, we can use enzymes such as lactate to select recombinant cells. The lactase enzyme needs to be attached to the DNA fragments with the desired gene. The cells that have acquired the recombinant plasmid would then produce lactate. Lactate can turn a colourless substrate blue. Therefore, in presence of the recombinant cells, the substrate would turn blue.