cDNA cloning: Principle and steps involved in cDNA cloning

Principle of cDNA cloning:

Complementary DNA (cDNA) cloning is termed for the gene cloning (cloning of DNA fragments) obtained from cDNA.
The principle of cDNA cloning is that it involves the copying of mRNA transcripts into DNA, which are then inserted into bacterial plasmids and then placed into bacteria by transformation.
At this stage, it should be clear that mRNA used for cDNA preparation is a processed transcript and not the original one transcribed from DNA.
In-order to clone a DNA sequence that codes for a required gene product, the gene should be removed from the organism and cloned it in the vector molecule.
A gene library is a random collection of cloned fragments in an appropriate vector that particularly consists of all the genetic information about that species.
There are two methods for the formation of gene libraries. They are:
- Complementary DNA (cDNA)
- Genomic DNA libraries

A crude extract of the tissue with the gene of interest is prepared.
The extract must be free from proteins, polysaccharides and all other contaminants.
The technique of oligo-deoxythymine (oligo-dT) cellulose chromatography is used for the further purification of many eukaryotic mRNAs from the total or polysomal fraction.
mRNAs consist of poly A (adenosine residues) tail at their 3′ end.
Under favourable conditions, this tail will bind to a string of thymidine residues immobilized on cellulose and then poly (A)⁺fraction can be eluted.
Two or three passages of the poly (A)⁺ fraction through such a column produces a fraction highly enriched for mRNA.
This fraction includes different mRNA sequences, however certain techniques can be employed for extracting a particular mRNA species.
After the preparation of the fraction, it is essential to confirm if the extracted mRNA consists of the sequence of interest.
It is performed by translation of mRNA in vitro and identification of suitable polypeptides in the products obtained.

Reverse transcriptase is a RNA dependent DNA polymerase which is used to copy the mRNA fraction into the first strand of DNA.
This enzyme, like all other DNA polymerases, can only add residues at the 3′-OH group of an existing primer, which is base paired with the template.
The most commonly used primer is oligo-dT for cloning of cDNAs.
Oligo-dT primer is 12-18 nucleotides in length, that binds to the poly (A) tract at the 3′ end of mRNA molecules.
The RNA strand of the resulting RNA-DNA hybrid is destroyed prior to second strand synthesis through alkaline hydrolysis.

The second strand of cDNA can be synthesized by two techniques. They are:
i. Self-priming cDNA:
- In Self-priming, the mRNA hybrid obtained is denaturated for the synthesis of second strand on the single strand of cDNA by the klenow fragment of DNA polymerase I.
- The transitory hairpin structure at the 3′ end of single-stranded DNA can be used to prime the synthesis of second strand of cDNA by the klenow fragment of Escherichia coli DNA polymerase I.
- Single-strand specific S1 nuclease digests the hairpin loop and any single-stranded overhung at the other end.
- The ultimate product is a population of double-stranded, blunt-ended DNA molecules complementray to the original mRNA fraction.
ii. Replacement synthesis:
- In this method, the cDNA:mRNA hybrid works as a template for a nick translation reaction.
- In the mRNA strand of the hybrid, RNase H produces nicks and gaps, creating a series of RNA primers.
- These RNA primers are used by E. coli DNA polymerase I during the synthesis of second strand of cDNA.
- The advantages of this technique are:
  - – very efficient
  - – can be performed directly using the products of the first strand reaction
  - – eliminates the need to use nuclease S1 to cleave the single-stranded hairpin loop in the double stranded cDNA.

The most frequently used technique for cloning cDNAs involves the addition of complementary homopolymeric tracts to double stranded cDNA and to the plasmid vector.
To the cDNA, strings of cytosine residues are added using the enzyme terminal transferase to form oligo-dC tails on the 3′ ends.
Likewise, a plasmid is cut open at a unique restriction endonuclease site and tailed with oligo-dG.
Now, the vector and the double stranded cDNA are joined by hydrogen bonding between the complementary homopolymers.
It results in the formation of open circular hybrid molecules capable of transforming E. coli.

For the transforming of bacteria, the recombinant plasmids are used, usually the E. coli K-12 strain.
The uptake of plasmid molecules from the surrounding medium is performed by E. coli cells treated with calcium chloride.
Any gaps in the recombinant plasmid will be repaired by the host cells.
The transformed bacteria can be isolated from non-transformed ones on the basis of antibiotic resistance.
Majority of cloning plasmids contain two antibiotic resistance genes, one of which is destroyed during cloning.
For instance, in the case of pBR322, cloning into unique PstI site destroys ampicillin resistance but leaves tetracycline resistance intact.
Bacteria transformed with a recombinant plasmid will be sensitive to ampicillin but resistant to tetracycline.

The antibiotic resistance selection already performed has recognized which clones carry a recombinant plasmid, however there will be thousands of various inserts.
The cloning process generally commences with a whole population of mRNA sequences.
Selection of clones carrying the sequence of interest is the tough job.
If the gene is expressed, then the simplest selection is to screen for the presence of the protein.
It can be screened either by bacterial phenotype it produces or by the protein detection methods usually based on immunological or enzymological techniques.
If the protein is not expressed, then other methods such as nucleic acid hybridization are used.
Identification of the gene is discussed after the genomic DNA cloning.