Synthesis
Though there are several methods for doing so, cDNA is most often synthesized from mature (fully spliced) mRNA using the enzyme reverse transcriptase. This enzyme operates on a single strand of mRNA, generating its complementary DNA based on the pairing of RNA base pairs (A, U, G and C) to their DNA complements (T, A, C and G respectively).
To obtain eukaryotic cDNA whose introns have been removed:
- A eukaryotic cell transcribes the DNA (from genes) into RNA (pre-mRNA).
- The same cell processes the pre-mRNA strands by removing introns, and adding a poly-A tail and 5’ Methyl-Guanine cap.
- This mixture of mature mRNA strands is extracted from the cell. The Poly-A tail of the post transcription mRNA can be taken advantage of with oligo(dT) beads in an affinity chromatography assay.
- 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). This synthesizes one complementary strand of DNA hybridized to the original mRNA strand.
- To synthesize an additional DNA strand, you need to digest the RNA of the hybrid strand, using an enzyme like RNase H.
- After digestion of the RNA, a single stranded DNA (ssDNA) is left and because single stranded nucleic acids are hydrophobic, it tends to loop around itself. It is likely that the ssDNA forms a hairpin loop at the 3' end.
- From the hairpin loop, a DNA polymerase can then use it as a primer to transcribe a complementary sequence for the ss cDNA.
- Now, you should be left with a double stranded cDNA with identical sequence as the mRNA of interest.
The reverse transcriptase scans the mature mRNA and synthesizes a sequence of DNA that complements the mRNA template. This strand of DNA is complementary DNA.
Read more about this topic: Complementary DNA
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