Generation of cDNA

Topic: Recombinant Dna And Biotechnology

cDNA generation is a process of converting single-stranded mRNA molecules into complementary double-stranded DNA molecules.

In many cases, researchers need a DNA version of an mRNA molecule, which is a product of gene expression. The mRNA can act as a  template to quantify gene expression via the PCR technique. Or, another may clone cDNA into a bacterial plasmid to express it in bacteria and obtain its protein product. In all these cases, first, an enzyme named reverse transcriptase converts mRNA into cDNA (or complementary DNA). Particularly, this enzyme synthesizes a complementary DNA strand to RNA. In nature, viruses (e.g. HIV) use it to replicate their RNA genome in host cells.

Now we have a hybrid molecule with two strands, one strand of RNA and another of DNA. As a next step, an RNAse enzyme is used to degrade the RNA part. Then, a DNA polymerase synthesizes the second DNA strand. Reverse transcriptase has both RNAse and DNA polymerase activity.

At last, we have a double-stranded cDNA. As we have seen, one of these strands is complementary to RNA. Since cDNA originates from mRNA, it does not have introns. In that state, researchers can use it to express the gene in prokaryotes, which do not have splicing (intron removal) mechanisms. 

Practice Questions

Khan Academy

Molecular targets of antiretroviral therapies

MCAT Official Prep (AAMC)

Key Points

• cDNA is a complementary DNA molecule generated from mRNA.

• A reverse transcriptase enzyme is an essential tool in cDNA generation; it catalyzes the synthesis of complementary DNA strand from mRNA. 

• cDNA is a vital ingredient in several experiments such as quantification of gene expression or expressing genes in prokaryotes (e.g. bacteria).

Key Terms

mRNA: an RNA molecule that is complementary to one strand of DNA and a product of the transcription process.

plasmid: a circular DNA in bacteria and protozoa that is separate from the chromosomes.

DNA polymerase: an enzyme that produces DNA molecules from its building blocks – nucleotides. 

gene expression: synthesis of mRNA and proteins in the cell.

cDNA: DNA synthesized from a single-stranded RNA by the enzyme reverse transcriptase

reverse transcriptase: an enzyme used to generate complementary DNA (cDNA) from an RNA template

PCR: Polymerase chain reaction is a method used copies of a specific DNA sample rapidly

RNAse: an enzyme that catalyzes the degradation of RNA into smaller components

introns: non-coding sections of RNA or DNA

prokaryotes: organisms whose cells do not contain a distinct nucleus

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