Tuesday, 5 May 2015

Reverse transcription polymerase chain reaction (RT-PCR)...a primer

A DNA Down Under post
The polymerase chain reaction (PCR) is a technique for copying a chain of DNA as many as a billion times. It purpose is so that we can use some form of technology to detect what would otherwise be too little material to see in the first place. The process of PCR is covered on the PCR page so I won't repeat it all here.

Before PCR for virus detection from human samples, we usually prepare the nucleic acids with an extraction or purification step. The shorter this is the better when testing, or screening, a lot of samples.

PCR can be used to detect some viruses straight out of the box. In these cases, the viruses need to have genes or a genome that is made of DNA. But many viruses don't store their genetic code as DNA, they use RNA as the plan from which they make more of themselves and their viral proteins. In these cases, DNA only plays an intermediate role, if any.

DNA viruses include the adenoviruses, herpesviruses, HIV (an example which also has RNA stages), polyomaviruses, bocaviruses, parvoviruses, papillomaviruses, poxviruses, megaviruses and many others. PCR also works well for plasmids and human genes and other DNA fragments we want to work with in the lab for reasons other than a diagnosis where we ask if the virus is in the human or not.

RNA viruses include influenza viruses, parainfluenza viruses, rhinoviruses, enteroviruses, cosavirus, klasseviruses, parechoviruses, respiratory syncytial virus, coronaviruses, human metapneumovirus and also many others. We also look at gene activity which involves detecting and measuring gene/genome transcription via reverse transcriptase (RT) PCR-based quantification, usually of messenger RNA (mRNA). But because PCR is based around the use of a heat stable DNA-dependent DNA polymerase, we would need to first make that RNA into DNA so that the main enzyme can use it and duplicate it and make enough of it to detect or use in molecular biology...or whatever the downstream application may be.

To make a DNA copy of the RNA, we need to add in another enzyme and another step to the PCR process. That enzyme, the RT is used in a step called reverse transcription.

The addition of this step also changes the initialism of the technique to RT-PCR. This is not to be confused with real-time PCR which is shortened to rtPCR. So an RT-rtPCR, which we use when detecting or quantifying RNA viruses in human samples, is a reverse transcription real-time polymerase chain reaction.

A standard PCR then has added to it a 10-30min incubation at an appropriate temperature (40-50'C), a denaturation step to kill of the enzyme (and sometimes to active the DNA polymerase; 92-95'C for 2-15min) and separate any DNA that is double stranded, followed by the multi-cycle PCR amplification process which can use the new DNA strand as a template for exponential copying....the billion-fold amplification reaction.

Further reading..

  1. PCR primers...a primer!
  2. The mechanics of the polymerase chain reaction (PCR)...a primer
  3. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
  4. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
  5. Beld MGHM, Birch C, Cane PA, Carman W, Claas ECJ, Clewley JP, Domingo J, Druce J, Escarmis C, Fouchier RAM, Foulongne V, Ison MG, Jennings LC, Kaltenboeck B, Kay ID, Kubista M, Landt O, Mackay IM, Mackay J, Niesters HGM, Nissen MD, Palladino S, Papadopoulous NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schoildgen O, Scott GM, Segondy M, Seibl R, Sloots TP, Wang Y-W, Tellier R and Woo PCYl. Chapter 10:"Experts’ roundtable: Real-time PCR and microbiology”, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
  6. Mackay IM, Arden KE, Nissen MD and Sloots TP. Chapter 8. “Challenges facing real-time PCR characterisation of acute respiratory tract infections”, In: Real-Time PCR in Microbiology, Mackay IM (Editor). 2007. Caister Academic Press, Norfolk, UK. 269-317.
  7. Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: ”Real-time PCR; History and fluorogenic chemistries”, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
  8. Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5:”Quantification of microorganisms: not human, not simple, not quick”, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
  9. Mackay IM, Arden KE and Nitsche A. Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology, Microbial Imaging. (2005) Vol 34. Chapter 10.Elsevier. pp255-330.
  10. Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.