“A DNA synthesised from the mRNA using the enzyme reverse transcriptase enzyme- only having the coding (exons) in it is called a cDNA/ complementary DNA.”
The cDNA is synthesised artifically for doing reverse genetics. In the present article we will understand the cDNA, how it is synthesised and what are the applications of it.
But before doing that we must have to know what exactly the cDNA is and how it is different than gDNA.
A gDNA is a total genomic DNA of us containing coding, non-coding and other junk DNA sequences.
Which means that the total genomic DNA present in a nucleus of a cell have all the information of an organism for coding genes and expressing it.
Note it down, the sensetence is very important, the genomics DNA coding sequencs encodes protiens and non-coding sequences regulates expression of genes.
Contrary to this, the cDNA is complementary DNA synthesised from the mRNA only have information for encoding protein.
Now quickly rewind the “so called” process of central dogma of molecular biology.
Using DNA polymerase, the DNA doubles in replication from which the mRNA transcript is formed during transcription.
The intrones are spliced out from a gene and exon forms the mRNA from it using RNA polymerase.
Note: prokaryotics do not have splicing mechanism for removing intrones.
Now the mRNA translated protien from it. This is the simple mechanism of the central dogma.
“The cDNA is used to clone eukaryotic gene into prokaryotes.”
On the other side, the cDNA is reverse process in which from the mRNA (transcribed from a gene), instead of protein translation, a sigle-stranded DNA is synthesised back using the enzyme, reverse transcriptase.
Now this cDNA, complementary DNA is different from the gDNA because it only have the coding information in it (no intrones).
Using the normal DNA polymerase another strand from the cDNA is synthesised and used for the clonning and library preparation experiments.
More reads: DNA story: The structure and function of DNA.
cDNA synthesis and library preparation:
The cDNA is applicable in clinical research and diagnosis field, thus it is very important to synthesise the cDNA artificially.
Steps in synthesis of cDNA:
The cDNA is only the coding sequences complementary to the mRNA transcript of our cell or gene of our interest, henceforth, we have to isolate mRNA first for the synthesis of cDNA.
The mRNA can be isolated using the ready to use mRNA islation kit. For that, first the mRNA is extracted.
Here, one of the unique thing is used for isolation the mRNA- oligo dT containing column.
Once the mRNA is transcribed, the poly-A tail is added during the process called post-transcriptional modifications.
And this will differentiate the mRNA from rest of single-stranded RNAs.
The poly- A tail of the mRNA remains bounded with the oligo dT containing column.
After reach round of washing, all the RNAs are washed off only the mRNA remains in the column.
In the fnal step, the mRNA is collected in another tube by using the elution buffer.
Purified mRNA must required for the next step, for that, the mRNA is purified using the purification kit and the oligo dT complementary nucleotides are removed from the poly- A tail by heating it gentally.
The purified mRNA is used for the reverse transcriptase PCR.
Selection of enzyme:
A normal polymerase can not synthesise DNA from RNA. we need another type of polymerase for that- a reverse tarnscriptase polymerase.
A reverse transcriptase enzyme is a special type of polymerase isolated from the retroviruses having a power to synthesise cDNA from the mRNA.
The Avian Myeloblastomis virus reverse transcriptase and Moloney Murine Leukemia virus reverse transcriptase are two commerically available RTs used commonly in the cDNA library preparation.
Reverse transcripase PCR:
Normal PCR is used for synthesis of DNA from DNA while the reverse transcripase PCR is applicable for synthesis of DNA from the RNA template using the reverse transcriptase enzyme.
Using the total mRNA, a DNA strand from the transcript is constructed, called the cDNA. However, the process of PCR is alomost similar to the conventional PCR.
Read more on reverse transcriptase PCR: Reverse transcription PCR: Principle, Procedure, Applications, Advantages and Disadvantages.
Construction of library:
Now we have the amplicons of a cDNA, the cDNA is now inserted into the plasmid using restriction digenstion method.
The sticky ends are generated on plasmid which binds complementary to the sticky ends of cDNA, these sticky ends are generated using the restriction digestion method.
Depending upon the size and type of cDNA, different type of plasmid are used for constructing different cDNAs.
The plasmid with the cDNA is now inserted into the bacteria and grown using the nutrient media under aseptic conditions.
Now we have the library of the cDNA we can use it in different molecular biology application.
For that, the DNA is extracted from the plasmid and used for downstream applications.
Application of cDNA:
The cDNA contains sequence only for coding protein.
Using the reverse transcriptase quantification PCR, the amount of the particular transcript or mRNA or the gene of our interest can be estimated.
It is also used for gene clonning and transformation experiments.
The cDNA library is also used in the expression of eukaryotic DNA into prokaryotes.
The cDNA synthesis is applicable in the process of heterologous expression in which the a protein is expressed a type of cells unable to synthesise that protein naturally.
One of the important use of constructing cDNA is to clone low copy number genes.
More to read: 50 Powerful Applications Of PCR.
The retorviruses are the class of virurese having a different mechanism of replication. The retro contains RNA as their genetic material, using the special type of polymerase- reverse transcriptase the cDNA is synthesised from the RNA.
Then the viruse inserts the cDNA into the host cell at where it replicated and forms different viral proteins.
This is the general ouline of cDNA library preparation, different tedious process are required for constructing it.
Also for doing the reverse transcription PCR, the selection of primer is one of the important parameter- oligo (dT) primers, random primers and gene-specific primers are three types of primers used in it.
Which enzyme is used in cDNA construction?
- DNA polymerase
- RNA polymerase
- Taq DNA polymerase
- reverse transcriptase
Which type of primers can not be used in reverse transcriptase PCR?
- oligo (dT) primers
- Random primers
- RNA primers
- Gene-specific primers
The cDNA is synthesised from?
L. B. Klickstein, R. L. Neve, E. A. Golemis, J. Gyuris. Conversion of mRNA into double-stranded cDNA. Curr Protoc Mol Biol. 2001 May; Chapter: Unit5.5.