cDNA Synthesis And Library Preparation

The Process of cDNA Synthesis and cDNA Library Preparation

“A cDNA/ complementary DNA is synthesized from the mRNA using the enzyme known as reverse transcriptase. The entire set of it is known as the library of cDNA.” The cDNA is synthesized artificially for doing reverse genetics.


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.

This means, that the total genomic DNA present in a nucleus of a cell has all the information of an organism to make proteins and to regulate gene expression. 

The genomic DNA coding sequences encode proteins and non-coding sequences regulate expression of genes, this sentence is very important, note it down. Contrary to this, the cDNA is complementary DNA synthesized from the mRNA which only has information to make proteins.

Now quickly rewind the “so-called” process of the central dogma of molecular biology. Using DNA polymerase, the DNA doubles in replication from which the mRNA transcript is formed during transcription. The introns are spliced out from a gene and exon forms the mRNA from it using RNA polymerase. 

Finally, a protein is manufactured from the translation of mRNA in the cell cytoplasm.

Note: prokaryotes do not have a splicing mechanism for removing introns. 

“The cDNA is used to clone eukaryotic gene into prokaryotes.”

Graphical illustration of the process of transcription and translation.
Graphical illustration of the process of transcription and translation. mRNA is formed from the DNA through transcription while a chain of amino acid is translated from the mRNA.

Contrary to the central dogma process, the cDNA synthesis process is reverse! a single-stranded DNA is formed from an mRNA instead of protein by the enzyme known as reverse transcriptase. Now this cDNA- complementary DNA is different from the gDNA because it only has the coding information in it (no introns). 

Using the normal DNA polymerase another strand from the cDNA is synthesized and used for the cloning and library preparation experiments. This process is known as second strand synthesis. 

More reads DNA story: The structure and function of DNA.

The present article is very interesting and more technical, here we are discussing how cDNA is formed and what is its important. We will also discuss the process of library preparation.

cDNA synthesis and library preparation:

An artificial cDNA plays an important role in clinical research and diagnosis. Here we have explained the process of cDNA synthesis.

Synthesis of cDNA: 

mRNA isolation: 

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 isolation kit. To isolate mRNA from the rest of the RNA, oligo dT containing column is used in the isolation process. 

Once the mRNA is transcribed, the poly-A tail is added during the process called post-transcriptional modifications. And this differentiates the mRNA from the rest of the single-stranded RNAs. 

The poly-A tail of the mRNA remains bounded with the oligo dT containing column. After each round of washing, all the RNAs are washed off only the mRNA remains in the column. 

In the final step, the mRNA is collected in another tube by using the elution buffer. 

mRNA purification: 

Purified mRNA must require 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 gently. The purified mRNA is used for the reverse transcriptase PCR. 

Selection of enzyme: 

A normal polymerase can’t synthesize DNA from RNA. We need another type of polymerase for that- a reverse transcriptase polymerase. 

A reverse transcriptase enzyme is a special type of polymerase isolated from the retroviruses having the power to synthesize cDNA from the mRNA. 

The Avian Myeloblastomis virus reverses transcriptase and Moloney Murine Leukemia Virus reverse transcriptase are two commercially available RTs used commonly in the cDNA library preparation. 

Reverse transcripase PCR: 

Normal PCR is used for the synthesis of DNA from DNA while the reverse transcriptase PCR is applicable for the synthesis of DNA from the RNA template using the reverse transcriptase enzyme. 

DNA is synthesized back from the transcript or mRNA but the steps of the RT-PCR are almost similar to normal 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 digestion method. 

The sticky ends are generated on a 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 types of plasmids are used for constructing different cDNA libraries. The plasmid with the cDNA is now inserted into the bacteria and grown using the nutrient media under aseptic conditions.

The graphical illustration of the process of cDNA synthesis and library preparation.
The graphical illustration of the process of cDNA synthesis and library preparation.

What we have now in our BAC (bacterial artificial chromosome) is known as a library of cDNA or cDNA library which has all the fragments of our interest. We can use it anytime. To use it in downstream applications, plasmid DNA is first isolated and processed pas per protocol.

Application of cDNA: 

cRNA contains a sequence of DNA only to code DNA. 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 cloning and transformation experiments. 

The cDNA library is also used to study the expression of eukaryotic DNA by inserting it into a prokaryotic cell. 

Heterologous expression: 

The cDNA synthesis is applicable in the process of heterologous expression in which a protein is expressed in a type of cell that is not able to synthesize 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.

Retroviruses: 

The retroviruses are the class of viruses having a different mechanism of replication. The retrovirus contains RNA as their genetic material, using the special type of polymerase- reverse transcriptase the cDNA is synthesized from the RNA. 

Then the virus inserts the cDNA into the host cell where it replicated and forms different viral proteins. 

cDNA Synthesis And Library Preparation

Conclusion: 

This is the general outline of cDNA library preparation, however, the process to make a cDNA library is a tedious one. 

Also for doing the reverse transcription PCR, the selection of primers is one of the important parameter- oligo (dT) primers, random primers and gene-specific primers are three types of primers used in it. 

Quiz:

Which enzyme is used in cDNA construction?

  1. DNA polymerase
  2. RNA polymerase
  3. Taq DNA polymerase
  4. reverse transcriptase

[epcl_toggle title=”Answer ” custom_class=”” show=”closed”]4. Reverse transcriptase. [/epcl_toggle]

Which type of primers can not be used in reverse transcriptase PCR?

  1. oligo (dT) primers
  2. Random primers
  3. RNA primers
  4. Gene-specific primers

[epcl_toggle title=”Answer ” custom_class=”” show=”closed”]3. RNA primers.[/epcl_toggle]

The cDNA is synthesized from?

  1. mRNA
  2. microRNA
  3. tRNA
  4. rRNA

[epcl_toggle title=”Answer ” custom_class=”” show=”closed”]1. mRNA.[/epcl_toggle]


Sources:

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.

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