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
- Isolation of mRNA
- Synthesis of first strand of cDNA
- Synthesis of second strand of cDNA
- Cloning of cDNA
- Introduction to host cells
- 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).
- 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.