Thursday, 3 March 2016

Primers prime PCR unless past their prime...

The PCR is a tough old bird... 

The polymerase chain reaction (PCR) is a hugely powerful 
tool for detecting virus. It can still do this more quickly and sensitively, from any human specimen, than any other diagnostic method that came before it.

Amazingly, the method of DNA copying by extension hasn't changed so much since the 1980s. The major variations have been to the machines that control the temperatures, the wide range of kits and the way PCR product is detected.

In the 1990s this detection became about measuring fluorescence within the same tube as the reaction as it took place - in real-time. But prior to this method, detection of a successful PCR was about opening the tube after the run had finished and performing a multi-step lengthy process of identifying the successfully amplified DNA by electrophoresis - end-point detection. This change underpinned what became known as of real-time PCR (rtPCR).

Quite amazing to think that anything could remain so integral to virology - or anything - these days. Rather than go into the details of what real-time PCR is, was and could have been, you can get a pretty good background understanding in our 2002 open access review of it as used in virology, aptly named "Real-time PCR in virology".[1]


PCR in the laboratory with viruses...

Today, rtPCR is integral to detecting the presence of virus and a successful detection is usually taken to mean that the person who provided that sample had a current infection at the time of sampling. 

That's not technically a correct assumption, nonetheless decades of research and epidemiology have largely been based on it. It's a pretty safe bet that there is a good correlation - in my expert opinion - in most of the instances in which we use PCR in a diagnostic laboratory setting. Detection by PCR does not however, mean we have disease or that the signs and symptoms a patient may have at the time of sampling are caused by that virus. The laboratory results along with any relevant travel history, contact history, signs,  symptoms and other clinical investigations and background knowledge all combine to help a medical doctor make that call. It's a team effort but laboratory testing, including PCR, biochemistry and immunology, provide crucial information that very often cannot be supplanted by a diagnosis based on how a patient looks and feels.

When we use PCR we use it to either detect DNA from viruses that have a DNA-based genetic make-up, or RNA from those viruses with RNA-based genetic material. It is of course used for bacteria and fungi and humans and many other things too, but this is not "all manner of other things down under".

For a PCR to work on an RNA virus, given the enzymatic process that is at its core (a DNA dependent polymerase), we first have to make a DNA copy of the RNA - a process called "reverse transcription". This term comes from the need to copy RNA back into DNA  - transcription being the process of copying DNA into RNA which occurs in our cells and elsewhere.

What a primer looks like when you align it to a reference sequence..

This probably deserves its own explanatory blog but we'll see what we can get away with for now.

A nucleotide sequence alignment of Zika virus (ZIKV) sequences downloaded from GenBank aligned with a primer from a published RT-rtPCR assay.[2] 1-the region of a primer that, if poorly "matching" to a target region of viral sequence, will most likley negatively impact the efficiency of the resulting PCR amplification, possibly causing the PCR to perform poorly, which can be of concern when there is only lower amount of of virus present in a sample, or
 fail completely2-in this example,the primer from Lanciotti et al. does not match the target at these marked positions - there is one or more "mismatches" between the primer and some of the viruses represented here which will affect the stability of the primer's binding, or hybridisation, with the viral genetic material during the PCR. Generally-this primer binds very well to Asian lineage Zika viruses, but much less well to African lineage Zika viruses.
Alignment made using Geneious v 8.1.5. The primer, 911c is the reverse primer in a pair used for an RT-rtPCR described by Lanciotti et al.[2]
Click on image to enlarge.
In the figure above we can see a few important items to consider when looking at primers and how they may work when put it into use. I say 'may' because even rubbish primers can sometimes do the job in PCR'ville, while the best designs may sometimes fail. One has to test to know for sure whether a primer pair does or does not perform the way you intend. Much like in infectious disease'ville before you try and link an infection with a disease.

Some key things worth noting about the figure above:

  1. This is called a nucleotide sequence alignment - or just an alignment. In this version, I've used dots to highlight whenever one of my reference sequences has the same nucleotide - an A, C G or T - at the same position as the primer I randomly chose for this example.

    If there is an entire column of dots, then at that nucleotide position we have a perfect match between all the viruses I've used in this alignment and that primer. In theory the primer will bind to those viruses if the one of them is in the patient's samples and we've successfully purify the virus's genetic material away from the other unwanted stuff in the samples - like proteins, inhibitory chemicals and carbohydrates.

    I won't be covering this "extraction" or purification process here.
  2. The enzyme in the PCR that makes PCR work relies on the primer to be matched up with the viral genetic material - especially at one end - the end at which the enzyme is going to be extending. I've highlighted that direction with the green arrow (no, not the Green Arrow).

    As the enzyme moves, it adds new nucleotides which it pulls in from the PCR chemical soup we use (in kits these days). This extension eventually results in a copy of the virus sequence. The primer shown is the reverse primer. There is also a forward primer which makes a copy in the other direction of the newly made strand. There is a bit more to this strand thing, but also not covering that here.

    As the copies get copied we eventually end up with just the region spanned by the primer pair amplified up - cloned if you like. Within that region, copied millions of times, is a sequence to which we previously designed a probe labelled with fluorescence molecules that has also been added to the chemical soup.

    As the copies were being made, the probe bound to its target and was destroyed, results in the signal which allows us to see those clones pile up....in real time.
  3. The end of the primer from which the enzyme starts must be well bound, or hybridised, to the viral sequence or else the polymerase may just fall off and not have anything to copy. This is really important to the success of the PCR, and if it's not a stable hybridisation (As hybridise to Ts, Cs hybridise to Gs), then the PCR won't work, or will work poorly.

    If hybridisation is poor then the early cycles don't make those first few copies which act as the template for more copies, and it is these early cycles which make or break the success of a PCR.

    In the figure, the important end is highlighted by the orange pill shape.
  4. I've added some info to the sequence names used in the alignment above. This indicates where and when some of them came from.

    The Asian lineage (see the basic tree below-also made using a nucleotide sequence alignment as a starting point)  is the one currently circulating in the Americas and previously in the Yap Island outbreak in 2007 and in French Polynesia.

    Zika virus complete or near-complete genome sequences aligned using Geneious v8.1.5.
    Neighbor-joining p-distance tree mad in Mega v6; 500 bootstraps 

    The primer example in the  alignment does not have any, but sometimes a PCR primer can have one or more "degenerate" nucleotide positions in it. These introduce variations to the primer sequence to accommodate one or more different nucleotides in the viral genetic target sequence.

    Sometimes, one has no choice but to design the primer to a variable viral region and so a degenerate position or two is added into your primer to help the primer hybridise and cope with that.

    We use codes for these degenerate positions - see the Table below excerpted from Chapter 2, written by Andreas Nitsche in
    a book I edited about a decade ago and in an extended version of the International Union of Pure and Applied Chemistry (IUPAC) code.[3,4]


    This is all a bit of a misnomer though because it means that when we order a primer from a company, they actually make a
    mix of primers - some have the full sequence and one nucleotide of the degenerate position, and the rest of the primer with the full sequence and the other nucleotide at that position - up to 4 different primers in the mix if we use an "N" degenerate position.
So with all that in mind - the next post - because this one is now too long - will examine some of the PCR-based diagnostics for Zika virus (ZIKV).

References...
  1. Real-time PCR in virology
    http://nar.oxfordjournals.org/content/30/6/1292.full
  2. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007
    http://wwwnc.cdc.gov/eid/article/14/8/08-0287_article
  3. Real-Time PCR in Microbiology: From Diagnosis to Characterization
    http://www.horizonpress.com/rtmic
  4. An extended IUPAC nomenclature code for polymorphic nucleic acids
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865858/