buttons to find out more about the step)
Step 3: Complementary DNA (cDNA) synthesis
Since PCR amplifies DNA, the viral RNA which has been extracted using the spin columns must first be converted to DNA before it can be analysed. To do this we use the enzyme Reverse Transcriptase, a polymerase that synthesizes DNA from RNA (analysis of viruses with a DNA genome does not include this Reverse Transcription step, as the viral DNA can be amplified directly by PCR).
There are several steps in the cDNA synthesis process:
- A mixture of viral RNA, primers (random hexamers) and dNTPs is made, and heated at 65 deg C to denature the secondary structure of viral RNA. The mixture is placed on ice for 5 minutes to allow the primers to anneal to the denatured RNA.
- Reverse Transcriptase is added to the reaction mix, along with MgCl2 and RNAase inhibitor.
- The reverse transcription reaction proceeds at 50 deg C for 1 hour.
- The reaction is terminated by placing samples at 85 deg C for 5 minutes.
- RNaseH is added to each sample and the reaction proceeds at 37 deg C for 20 minutes. RNaseH is an endoribonuclease that digests the RNA which remains attached to the newly synthesized DNA strand.
- The cDNA can be stored at -20 deg C or used for PCR immediately.
