cDNA cloning – Definition, Principle, Steps 

Complementary DNA or cDNA Definition

• In genetics, the term “complementary DNA” (cDNA) is the DNA made from a single-stranded RNA (e.g. messenger (mRNA) or microRNA (mRNA) (or microRNA (miRNA)) template during an enzyme reaction. enzyme known as reverse transcriptase.

Principle of cDNA cloning/cdna synthesis principle

• Complementary DNA (cDNA) Cloning is the term used as a gene that clones (cloning of DNA fragments) made from cDNA.
• The premise behind cDNA cloning can be explained as copying the mRNA transcripts into DNA that are then transferred into bacterial plasmids, and later put into bacteria through transformation.
• At this point it should be evident that the mRNA utilized for preparation of cDNA is processed transcript, not the original transcript that was transcribed from DNA.
• In order to create a clone of a DNA sequence which codes for a specific protein in the genome, it must remove itself from living organisms and then clone into the molecule of the vector.
• Gene libraries are a set of cloned pieces in an appropriate vector which includes all genetic information of the species.
• There are two approaches to the creation of gene libraries. These are:
  • Complementary DNA (cDNA)
  • Genomic DNA libraries

Steps of cDNA cloning

1. Isolation of mRNA
2. Synthesis of first strand of cDNA
3. Synthesis of second strand of cDNA
4. Cloning of cDNA
5. Introduction to host cells
6. Clone selection

cDNA cloning protocol

1. Isolation of mRNA

• A simple extract of the tissue that contains the gene of interest has been taken.
• The extract should be free of polysaccharides, protein, and other harmful substances.
• The method of oligodeoxythymine (oligo-dT) the cellulose chromatography technique is used to further purification of a variety of Eukaryotic mRNAs, which are derived from the polysomal or total fraction.
• The mRNAs comprise their poly A (adenosine the residues) end at the 3′-end.
• In the right conditions the tail can connect to a string of Thymidine residues immobilized onto cellulose. Then, in turn, the poly (A)+ the fraction will be eliminated.
• A few or three passes through the poly (A)+ fraction in this column produce the fraction that is highly enriched for the mRNA.
• This part includes various MRNA sequences, but specific methods can be used to determine a specific mRNA type.
• After the fraction has been prepared It is crucial to determine if the mRNA extracted consists of the sequence of importance.
• It is done by translating mRNA into vitro , and identifying suitable polypeptides from the products resulting.

2. Reverse transcriptases and first-strand cDNA synthesis

• Reverse transcriptase is a DNA polymerase that is RNA dependent and can be used for copying the mRNA portion into the DNA’s first strand.
• Like the other DNA polymerases can only add residues to the 3′-OH portion in an already existing primer which are base-paired in conjunction with the templates.
• The most frequently used primer is oligo DT for the cloning of DNA cDNAs.
• The Oligo-dT primer measures 12-18 nucleotides long that binds to poly (A) tract located at the 3′-end of the mRNA molecules.
• The RNA string of the hybrid will be destroyed prior to the second strand synthesis by alkaline hydrolysis.

3. Synthesis of Second-strand cDNA synthesis

Second strands of cDNA can be synthesized using two methods. These are:

a. Self-priming cDNA:

• When Self-priming is performed, the hybrid mRNA created is denatured for the synthesis of a second one DNA strand by the klenow DNA fragment polymerase I.
• The hairpin structure that is transitory at the 3′-end of single-stranded DNA could be used to trigger the second strand of cDNA through the klenow fragment from Escherichia Coli DNA polymerase I.
• Specific to single-strands S1 nuclease digests hairpin loop as well as any single-stranded hanging at the other end.
• The end result is a collection of double-stranded DNA molecules that are blunt-ended, which complement the mRNA fraction that was originally created.

b. Replacement synthesis:

• In this procedure the cDNA:mRNA hybrid acts as a template to perform the Nick translation reaction.
• In the mRNA-strand of this hybrid gene, RNase H creates gaps and nicks and creates a sequence of DNA primers.
• These RNA-specific primers are employed to aid E. coli DNA polymerase I to create the second strands of CDNA.
• The benefits of this method are:
  • Very efficient
  • It is possible to perform the procedure directly with the products of the first strand reaction.
  • It eliminates the need to employ nuclease S1 in order to cut the hairpin loop that is single-stranded inside the double stranded DNA.

4. Cloning of cDNA

• The most commonly used method to clone cDNAs is the introduction of homopolymeric tracts with complementary homopolymer to double-stranded cDNA, and to the vector of the plasmid.
• To the cDNA chains of cytosine residues are inserted with the help of the terminal transferase enzyme to create oligo-dC tails at the 3″ ends.
• The same way, a plasmid can be opened at a specific restriction endonuclease location and tied with the oligo-dG.
• The vector and the double-stranded cDNA are joined by hydrogen bonds between the homopolymers with complementary.
• It leads to the formation of hybrids that are open that are capable of altering E. Coli.

5. Introduction to host cells

• In order to transform bacteria, recombinant plasmids are employed, typically used for the E. coli K-12 strain.
• The uptake of plasmids from the medium surrounding is done by E. Coli cells which are treated using calcium chloride.
• Any holes in the recombinant virus will be filled in by the host cells.
• The transformed bacteria are isolated from non-transformed strains due to resistance to antibiotics.
• The majority of cloning plasmids have two resistance genes to antibiotics One of which is destroyed during the process of cloning.
• In the case of pBR322, the cloning process into the unique PstI site will destroy ampicillin resistance, but it leaves the tetracycline resistance unaffected.
• Bacteria transformed using a recombinant plasmid will be susceptible to ampicillin, but intolerant to Tetracycline.

6. Clone selection

• The selection of antibiotic resistance that has been carried out has revealed the clones that carry a recombinant plasmid/cDNA Plasmid. However, there are hundreds of different inserts.
• The process of cloning generally begins with the whole set that contains mRNA.
• The selection of clones that carry an interest sequence is a difficult task.
• If the gene is expressed then the most straightforward option is to test to see if the gene is expressed.
• It can be detected through the bacterial phenotype that it generates or through the detection of protein methods that are typically built on enzymological or immunological methods.
• If the protein cannot be expressed, then alternative methods like nucleic acid hybridization is used.
• The identification of the gene is described following the cloning of genomic DNA.

Application of cDNA

• CDNA is commonly utilized to clone eukaryotic genes in prokaryotes. If scientists wish to express a particular protein in cells that do not normally express the protein (i.e. heterologous expression) they transfer the cDNA coding for the protein into the cell of the recipient.
• Molecular Biology cDNA may also be produced to examine transcriptomic profiles of the bulk tissue, single cells or nuclei in tests like microarrays or RNA-seq.
• CDNA can also be produced in the natural course of retroviruses (such as HIV-1 and HIV-2, simian immunodeficiency virus etc.) and later integrated in the genome of the recipient which creates an antivirus.
• The term cDNA is also used often in a bioinformatics context to describe the sequence of mRNA transcripts that is expressed in DNA base (deoxy-GCAT) instead of bases in RNA (GCAU).

FAQ

References

• Harbers, M. (2008). The current status of cDNA cloning. Genomics, 91(3), 232–242. doi:10.1016/j.ygeno.2007.11.004 
• Carninci, Piero & Kvam, Catrine & Kitamura, Akiko & Ohsumi, Tomoya & Okazaki, Yasushi & Itoh, Mitsuteru & Kamiya, Mamoru & Shibata, Kazuhiro & Sasaki, Nobuya & Izawa, Masaki & Muramatsu, Masami & Hayashizaki, Yoshihide & Schneider, Claudio. (1996). High-Efficiency Full-Length cDNA Cloning by Biotinylated CAP Trapper. Genomics. 37. 327-36. 10.1006/geno.1996.0567. 
• Tamme, Richard & Mills, K & Rainbird, Barry & Nornes, Svanhild & Lardelli, M. (2001). Simple, Directional cDNA Cloning for In Situ Transcript Hybridization Screens. BioTechniques. 31. 938-42, 944, 946. 10.2144/01314rr05. 
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