Learn about cDNA synthesis.
cDNAs are synthesized using mature mRNA template in a catalytic reaction using the enzyme reverse transcriptase (RTase).
cDNA Synthesis Diagram
cDNA Synthesis Picture
Reverse transcriptase operates using a single strand of mRNA and generates the complementary DNA based on the pairing of RNA base pairs (A, U, G and C) and translates them to their DNA complements (T, A, C and G respectively).
To obtain eukaryotic cDNA whose introns have been spliced:
- Eukaryotic cells transcribe the DNA (from genes) into RNA (pre-mRNA) - this is the process of transcription.
- The pre-mRNA strand is then processed by splicing out introns
- A poly-A tail and 5’ Methyl-Guanine cap are also added to the mRNA.
- Amixture of mature mRNA strands are extracted from the cell.
- A poly-T oligonucleotide primer is hybridized onto the poly-A tail of the mature mRNA template, or random hexamer primers can be added which contain every possible 6 base single strand of DNA and can therefore hybridize anywhere on the RNA (Reverse transcriptase requires this double-
- stranded segment as a primer to start its operation.)
- Reverse transcriptase is added, along with deoxynucleotide triphosphates (A, T, G, C).
- the RNA strand is digested away using RNAseH
- PCR is used to make the single stranded cDNA double-stranded
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