PCR primers...a primer!

A DNA Down Under post

PCR (described here) functions mainly because of two components - a thermostable DNA polymerase and a pair of DNA 'primers'. Primers are short, made to order, stretches of oligonucleotides ('oligos' - from Greek meaning scanty or few). Modern oligos can be synthesized in lengths >100nt however the behaviour of oligonucleotides longer than 20nt is different from that of shorter oligos and different calculations are employed to determine their thermodynamic characteristics.

What is a primer...?

Primers, as their name may suggests, prime the nucleic acid template for the attachment of the polymerase. This is the first step towards duplicating that template. The primer directs the polymerase to move in a 5' to 3' direction (drawn left-to-right; Figure 1) because of the 'direction' of

DNA (See DNA Structure for more background).

Figure 1. DNA has direction. The polynucleotide chain shown 
above is 'read' in a 5' to 3' direction by the polymerase. This would 
be from the top to the bottom or from the phosphate group 
to the hydroxyl group.

Primer binding...

Primers hybridize at a temperature that is affected by their sequence, concentration, length and ionic environment. This annealing temperature is usually referred to as the T_M (melting temperature) but is in fact 5�10�C below the T_M. The term T_M describes the temperature at which 50% of the primer�target duplexes have formed.

Primer specificity...

PCR gleans its extreme specificity from the primers. At each and every position of a new primer, we have 4 nucleotides to choose from, dATP, dCTP, dGTP and dTTP. 

Figure 2. Deoxynucleotide triphosphates. Each of the five deoxynucleotides are shown.2'-deoxycytidine-5'-triphosphate (dCTP; C9H16N3O13P3, MW=467),2'-deoxyguanosine-5'-triphosphate (dGTP; C10H16N5O13P3, MW=507),2'-deoxyadenosine-5'-triphosphate (dATP; C10H16N5O12P3, MW=491),2'-deoxythymidine-5'-triphosphate (dTTP; C10H17N2O14P3, MW=482) and 2'-deoxyuridine-5'-triphosphate (dUTP; C9H15N2O14P3, MW=468). 

So, if we design a sequence-specific primer of 20-30nt nucleotides in length ('20-30mer'), the chance that that exact sequence will occur randomly in nature will be 1/4 x 1/4 x 1/4 etc, 20 or 30 times i.e.

That means a 1 in 10¹² to 10¹⁸ chance of a 100% homologous match to an unintended target. Or to put that in perspective, there are 2.85 x 10⁹ basepaired nucleotides in the entire human genome.

While that all sounds very convincing, in reality, primers designed to detect viruses often share significant amounts of homology with the human genome - sometimes resulting in false positive amplifications. Even when the homology is far from 100%, primers may still amplify an unintended target as shown below. This most likely reflects the co-evolution of many viruses with humans during which time they have "captured" bits of our genome and "deposited" bits of their own genome. 

Next we'll list a few of the problems we can encounter when using the PCR.

Primer dimer...

The first problem I'll discuss is the most common and the most difficult to avoid. Depending on your requirements, it may also be the least significant.

When a small amplicon results from the extension of self-annealed primers, you get primer-dimer (PD) i.e. a dimer of one (self-annealing) or both primers resulting in a template capable of being extended by the polymerase. PD formation is highly efficient because the primers are in vast excess compared to the amount of template or even to the number of amplicon molecules at the end of the PCR.

This excess drives the formation of PD. Two main concerns arise from PD formation.

1. Because PD formation is so efficient, it rapidly consumes dNTPs and primers and generates amplification inhibiting pyrophosphates. All of which can prematurely plateau the exponential accumulation of product.
2. If we using a dsDNA-associating fluorescent molecule to follow the PCR's progress during real-time PCR, then PD will also show up, and, at least during the kinetic portion of the assay, cannot be differentiated from the signal of specific amplicon accumulation.

Figure 3. Examples of how primer-dimer (PD) amplicon can be formed.
Ten examples are shown of sense and antisense primer interactions
resulting in an amplicon. Note that different length amplicons can be formed.
The largest PD would result from the hybridization of the smallest number of
nucleotides and would approach the length of the two primers added together.

Mispriming...

Mispriming is the result of a primer binding to an unintended template resulting in amplification. The amplicon (PCR product of a single species) can sometimes be the same size as the intended product, but is usually a different size when viewed following agarose gel electrophoresis.

Mispriming occurs because of poorly optimised conditions or because we haven't checked whether our sequence will inadvertently bind to an entirely different target entity e.g. a region of the human genome instead of the intended virus genome. Sometimes it just happens.

Mispriming can usually be avoided by more intensive comparison of the primer's sequence against the GenBank database using the Basic Local Alignment Search Tool (BLAST) at NCBI. Of course, a BLAST comparison will only find matches among those sequences housed in the database. When it comes to PCR where a single nucleotide mismatch can cause an amplification to fail, or at least perform with reduced efficiency, BLAST'ing primers can lead to a feeling of very false security.

In some instances the homology of the primer to its template may indicate a perfect match simply because viral variants have not yet been sequenced and submitted. Also, because there may be many undiscovered viruses and unsequenced non-viral genomes in the world, obviously none of which are represented on GenBank, a specific match, or a "no match", does not mean that you have exhausted your search for homologues. Take it all with a grain of salt. Designing two pairs of primers around the target region is a good place to start. This helps address the unexpected.

Structural problems...

This problem results from the way we design our primers. I am excluding self-annealing and secondary structures from here because we will deal with them specifically in the next section. --work in progress---

Further reading...

1. A crowd-sourced database of virus primers. www.virusprimers.org/
2. The mechanics of the polymerase chain reaction (PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/the-mechanics-of-polymerase-chain.html
3. Reverse transcription polymerase chain reaction (RT-PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/reverse-transcription-polymerase-chain.html
4. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
5. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.

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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!
   http://newsmedicalnet.blogspot.com.au/2015/05/pcr-primersa-primer.html
2. The mechanics of the polymerase chain reaction (PCR)...a primer
   http://newsmedicalnet.blogspot.com.au/2015/05/the-mechanics-of-polymerase-chain.html
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.

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Why only 181 nucleotides of T.perforatus MERS-CoV sequence?

In some of the many articles written about the new discovery this week, there were comments along the lines of  its amazing any sequence could be obtained from the samples cause they had sat for 48-hours at US customs and thawed. A more precise quote could be found here for example.

I have some thoughts on that - and these come from me, someone who has worked with a lot of clinical human specimens from which I've been able to amplify viral bits and pieces on a regular basis. Many small (200-600 basepairs[bp] fragments) but also longer pieces of >1,000bp, assembling small viral genomes from them. These samples may >10-years old, having been freeze-thawed numerous times after spending various amounts of time in courier vans, planes or sitting at room temperature before having nucleic acids extracted, tested and eventually (extracts may also sit around during testing and preparation and be freeze-thawed etc) frozen at -20�C or -80�C.

Keeping in mind that this issue of thawing might simply be a case of "hold your horses people". The EID paper was an early and quick report announcing the discovery of this MERS-CoV strain. So, my thoughts:

1. Because the materials that yielded the sequence (collected in October 2012) were described as "thawed" we can presume that the dry ice they were shipped with ran out during the transport to, or waiting time at, US customs. Once the refrigerant is all gone, the samples would come to room temperature as fast as the cardboard box and plastic receptacle it held, allowed. The publication described them as having been thawed for 48-hours.
2. How warm are we talking? The average temperature of Bisha (where the Taphozus perforatus bat was found, in an old date orchard outdoors) in October ranges from 15-20�C to 30-35�C. I don't know where the US customs site was so don't know that temp range - but expect it's less. So let's make some wholly unfounded assumptions:
• That this MERS-CoV strain can spread via the virus found in faecal pellets or other bat excreta. Perhaps as wind-blown dust or to other animals via a faecal-oral route. Even if the bats are hanging from a cave ceiling, but certainly when they are hanging outdoors, the virus must be capable of surviving in faecal pellets at a very high "room temperature" to complete a transmission event. If they can survive, that means intact virus - RNA genome + proteins + capsid + lipid envelope - the whole lot. For RT-PCR - you only need the RNA bit, not infectious virus. So, you're already lowering your expectations for what's required of a "successful" shipment.
• To confirm bat species, a genetic test was used which required the amplification of another piece of DNA - a region of the cytochrome B gene was amplified and sequenced. How large this fragment was, I'm not sure. However, a relatively large fragment of this gene can be used to differentiates bats, useful when you can't tell them apart by looking at physical features. Other work on opossums by the collaborator who helped sequence this region (Dr George Amato) in bats, employed >800bp of sequence. Why did this fragment amplify so well if the viral RNA did not? Perhaps because DNA is more hardy (various reasons) or because the bat blood or skin that it was amplified from, better protected the DNA from the thawing than bat faeces did for the viral RNA? Or...
3. Perhaps the primers used for other regions of the T.perforatus MES-CoV strain failed because the virus was too genetically distinct. I've had a look at the alignments and the primer binding sites can be found so it's probably not that. However, some of these primers that produce larger products are very degenerate (primers specially designed to account for nucleotide variation in a range of subtly different viruses or viral strains). 
• Degenerate PCR primers generally have much decreased sensitivity compared to 100% target-specific primers. This drop in ability to detect low amounts of RNA is the case even when using nested PCR - sorry if this has become to PCR technical! 
• The primers that did work for the T.perforatus bat MERS-CoV, Nested CII-MERS-RdRp, were much more target specific with only 1 degenerate base in 4 primers. That, combined with a drop in viral RNA amount, may well be why this 1 assay worked, worked where the others did not.
4. There was no mention in the EID paper of the use of an internal control RT-PCR target - a region of a gene in bat faeces (or blood or tissue depending on what was tested) that might allow some quality monitoring to see if there was truly decreased amounts of intact RNA in the October 2012 batch compared to that in the April 2013 batch of samples. That would be helpful to know which course to follow next.

So what does all this mean? Just me thinking in print I guess.

It's always important to maintain the cold chain from sample collection through to nucleic acid extraction and template addition to an RT-PCR/PCR tube. But I think we should look elsewhere for reasons why the T.perforatus MERS-CoV-positive sample has not yielded more than 1 fragment from the few assays used. 

I wouldn't be surprised if there was more sequence coming soon from this sample.

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