Molecular Biology Home

cDNA Synthesis

Written by Super User. Posted in DNA

Learn about cDNA synthesis.

Learn about cDNA synthesis.

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

cDNA synthesis

 

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

Related cDNA Articles:

See also:

cDNA

cDNA cloning

cDNA library display_block('cdna'); ?>