With the advancement in the field of genetic engineering, gene expression analysis has become an indispensable tool. Researchers are always keen to find out whether their gene of interest is expressing (turned on) or not (turned off). For this, the mRNA (messenger RNA) is located and quantified in the given sample. mRNAs carry the information coded by DNA and, thus, further gets translated to produce respective proteins.
RNAs are very unstable and fragile, and are very likely to degrade by the omnipresent RNases. In order to combat this, the biological informations encoded in mRNA are stored in more stable form of nucleic acid, i.e. DNA. Therefore, cDNA is prepared from RNA, which stores entire sequence of the mRNA. It is more convenient to work with cDNA as compared to mRNA. This cDNA can be further used for various subsequent molecular biology and genetic studies.
What is cDNA??
cDNA means complementary DNA or copy DNA. According to the central dogma of the molecular biology, DNA is transcribed into mRNA. Then mRNA gets translated to produce protein. Therefore, the flow of biological information is from DNA to RNA to protein.
However, sometimes the flow of information is from RNA to DNA (as in the case of some viruses, e.g. HIV). This conversion of RNA to DNA is aided by an enzyme known as Reverse Transcriptase (i.e. RNA-dependent DNA polymerase). The cDNA prepared can be single stranded or double stranded. Therefore, molecular biologists make use of reverse transcriptase to prepare cDNA from mRNA for the sake of convenience in the molecular studies.
Principle of cDNA synthesis
Mature (fully spliced) mRNA is used as a template for preparing cDNA. In fact, cDNA can be produced from any RNA molecule. This conversion is brought about by reverse transcriptase. cDNA can be obtained both from prokaryotes and eukaryotes.
Reverse transcriptase is a RNA-dependent DNA polymerase. It acts on a single strand of mRNA. Using mRNA as a template, reverse transcriptase produces its complementary DNA based on the pairing of RNA base pairs. This enzyme executes reactions in the same way as DNA polymerase. It also requires a primer with a free 3′-hydroxyl group. For transcribing RNA having secondary structures, a reverse transcriptase with high temperature performance is recommended.
Procedure of cDNA synthesis
First of all, good quality intact mRNA or total RNA is isolated. Then, you need a few more reagents to prepare cDNA: dNTPs (dATP, dTTP, dCTP, dGTP), primers and reverse transcriptase.
In case of eukaryotic mRNAs, a poly-A tail is present at their 3′-ends. Therefore, a poly-T oligonucleotide is used as a primer. But certain modifications are needed when you use other RNAs which lack poly-A tail, e.g. prokaryotic mRNA, rRNA, RNA virus genomes, etc. In such cases, a poly-A tail is added to the 3′-end of the RNA. This makes it analogous to the eukaryotic mRNA.
The primer gets annealed to the 3′-end of the mRNA. Now, the 3′-end of the primer is extended with the help of the reverse transcriptase using mRNA strand as a template. This is known as “first strand reaction”. As a result of this, RNA-DNA hybrid molecule is produced. By the use of RNase H or alkaline hydrolysis, the RNA strand of this RNA-DNA hybrid molecule is digested. Now, the single stranded cDNA becomes free.
The reverse transcriptase used (most commonly used is Moloney Murine Leukemia Virus Reverse Transcriptase, MMLV RT) displays terminal transferase activity on reaching the end of the RNA template. It adds 3-5 residues (usually dC) to the 3′-terminal of the first strand cDNA. An oligo containing a stretch of G residues is used. This oligo gets annealed to the dC rich cDNA tail and serves as an extended template for reverse transcriptase. Now, the synthesis of the complementary strand of the first strand cDNA begins. This is called “second strand reaction”. Finally, a regular double stranded DNA is produced.
Types of primers used
Various types of primers can be used, in accordance to the requirements, to synthesize cDNA.
1) Oligo-dT primer- It is used when the mRNAs have poly-A tail, as in the case of eukaryotic mRNAs; or when a poly-A tail is attached to the existing RNA. Oligo-dT primer anneals to all the mRNAs simultaneously.
2) Sequence-specific primer- If you wish to generate cDNA from a particular population of mRNA among all the mRNAs, then sequence-specific primer is used. It will bind to a particular mRNA sequence only. This will give rise to a pure cDNA population generated from the desired mRNA. For designing sequence-specific primer, you must know the sequence of the mRNA of interest. Generally, the 3′-terminal sequence is preferred.
3) Random primer- A random primer cocktail is used to produce cDNA from all the mRNAs. The cDNAs produced are not full length. Random primer is extremely useful if production of the shorter cDNA fragments is desirable. Its use increases the probability of converting the entire 5′-end of the mRNA into the cDNA. In case of long mRNAs, reverse transcriptase is usually not able to reach the 5′-end. Therefore, random primer proves to be extremely advantageous in such cases.
Types of cDNA
cDNAs can be single stranded or double stranded. After the first strand reaction, cDNA obtained is single stranded. This single stranded cDNA can be converted to the double stranded form by second strand reaction. On the basis of the applications, single or double stranded form of the cDNA is used.
Applications of single stranded cDNA
1) Single stranded cDNA is most commonly used for RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). RT-PCR is done for gene expression studies. It determines whether the gene of interest is expressed or not, and the level of its expression.
2) It is also used to amplify particular gene of interest. For this, sequence-specific primers are used.
3) Real-time PCR (also known as quantitative RT-PCR, qRT-PCR) also makes use of single stranded cDNA. It is done for performing gene expression analysis. As the amplification progresses, the amplicons can be visualized with the help of a fluorescent reporter molecular. It is highly sensitive and effective as compared to RT-PCR.
Applications of double stranded cDNA
1) Double stranded cDNAs are used to clone them into the appropriate vector to prepare libraries of cDNA (i.e. cDNA libraries). These libraries contain all the mRNA sequences in the form of cDNA, which are all expressed in a cell.
2) Double stranded form of a particular cDNA of interest can be cloned. Then, expression of the desired genes is allowed at the RNA and protein level for further study.
3) Sequencing of the double stranded cDNA is carried out to obtain the expressed sequence tags (ESTs).
4) They are also used for doing microarray for analysing global gene expression.
5) Suppression subtractive hybridization (SSH) is also performed with double stranded cDNA. It is done to find out differential gene expression.
No comments:
Post a Comment