What is dPCR, it is basically a means to do hundreds, thousands, and possibly millions of tiny PCR amplifications from a single sample.  Why would you want to do this, might be your first question?  The answer, because you want to get quantitative numbers for the copies of template (target DNA or RNA) in a tube.  Certainly, if you divide a 1 microliter sample into 10,000 different reactions and that small volume contains 1000 copies of template, statistically you would expect 1 in 10 reactions to produce an amplification product.   That assumes that the designed assay being used to detect the target is actually capable of amplifying as little as one copy of template if it present in the reaction tube.  This assumption is one that is made for a variety of qPCR techniques, including dPCR.

So let us assume that the PCR that you are performing CAN actually detect a single copy of target DNA or RNA if it is present in the reaction tube.  The question then becomes how many actual PCRs do you really need to quantify the template material in the tube accurately?  First of all , it bears mentioning, that what we are talking about here is the number of amplifiable copies of the target DNA or cDNA (which is a DNA copy of RNA used as target template in a separate [though possibly in the same reaction tube just separated out biochemically either by time or temperature or both]) not total DNA in a tube.  An amplifiable copy of DNA (or cDNA) must be a co-linear stretch of sequence that contains both the forward and reverse primer binding sites.    Any breaks between the primers will give great primer binding (and use of PCR reagents, specifically dNTPs) and will result in altered primer sequences (due to primer extension without product completion). Also, they need to be complete primer binding sites as well, not merely portions of the primer binding sites as any altered primer binding sites will give poorer primer binding, thus reducing the overall sensitivity of the PCR to something likely to be less than the single copy detection capabilities which we are assuming.

If we add to the above assumption that the target that we will be detecting will be bound to some sort of fluorescent probe, and not simply detected using a non-specific double strand DNA binding dye, then the probe binding site(s) must also be invariant to maintain the assumed single copy detection capability of the assay.

How, in light of these assumptions, is merely dividing the sample into multiple reactions going to improve on these assumptions?  Simply put, it is not.

So then you must ask yourself, why do I want to have real-time PCR machine that is dedicated to performing digital PCR when I can have one that can do the whole gamut of real-time PCR applications AND with not many more additional reactions (and no additional assumptions) also do a pared down version of dPCR?

Realistically, are you really going to use dPCR on a daily basis to quantify the ‘exact’ number of copies of template in a sample?  Probably not, you might need this occasionally for new target standardization so that when you assign numbers to your qPCR standard curve you are putting in some sort of absolute number per dilution.  Then you simply use the built in software, that comes with virtually all real-time PCR machines to store the standard curve.  Until you run out of stock template, or you develop a new target assay you are not going to need that level of quantitative precision again.

So how are you going to do these calculations without a dedicated dPCR instrument?  There are a number of quantitative packages out there on the web and available commerically.  Here I have listed two, one is BioSTAT 2009 and another is QUALITY.  Of course, I would be remiss if I didn’t mention that our staff at Fluoresentric has been doing this type of template quantification for years and we offer both a DNA and an RNA stock template quantification service for existing assays and for those that you don’t even have yet.  You can find out more on our store or by contacting us at Fluoresentric (info@fluoresentric.com) or simply call 800.808.0490 x 100.

In the next installment of this topic I will go into the numerical details of dPCR and qPCR.

You’ve probably done it yourself, dropped off your dog on your way out of town.  Sure most kennels insist on having your dog’s vaccination records.  That they do is terrific news for all you pet owners out there; however, wouldn’t it be terrific if a simple diagnostic test for bordetella brochiseptica, or kennel cough, were readily available right there on site at the kennel?

Signal Diagnostics has developed just such a test it is a molecular diagnostic disease test that takes only a few minutes to prepare a sample from your pet, and with just a few pieces of laboratory equipment, those found in most veterinary clinics, your pet can be easily tested for bordetella.

Think of it, you drop off your pet and when you pick it up you know that your companion hasn’t been hanging around with other animals that may be passing on a potentially deadly bacterial infection.  All that with a simple, easy-to-use, and on-site test.  Ask your veterinarian or boarding shelter next time you drop off your pet if they have this test available to ensure your pet’;s safety and health.

I bet, in part, that the answer is yes.

Here is brief list from just the last two weeks: HIV , E. coli, Fungi, Malaria,  Cancer, and the list goes on and keeps growing.

Signal Diagnostics, a spin out of Fluoresentric,  has developed a patented method to screen organisms and native genomic DNA for the presence of mutations that point to drug resistance, for antibiotics and anti-cancer therapeutic agents.

If you have plans to detect drug resistance in any organism or cancer cell, our patented technique is likely your best solution.

We aren’t prescient here at Signal Diagnostics , but we do follow science news and about 3 years ago while we were developing our now patented drug resistance screening method azole resistance was starting to show up in the literature.  Like all good organisms, fungi just have to evolve and adapt to their environment.  So, not surprisingly, more and more news is coming out about yeast and the other fungi developing drug resistance to the chemicals used to eliminate them, specifically azoles and azole like compounds.

Fortunately, Signal Diagnostics has developed a rapid yeast test for drug resistance diagnostic test for antibiotic resistance, in this case an anti-fungal agent.

While you are visting the Signal Diagnostics site check out our other drug resistance marker assays, for Staph MRSA (methicillin), and Staph vancomyocin resistance, or for Malaria chloroquine resitance, and our most popular tests for Tuberculosis (rifampicin, isoniazid, ethambutol, streptomycin, and pyrazinamide) drug resistance.

Oh, I know, it is so tremendously geeky to even try to draw the PCR component into everyday socializing..but, that is what we at Fluoresentric spend our days doing, and we know there are others out there doing the same.  We all  just need a place to talk about those science articles that really excite us.  It seemed that a specific biology blog, or chemistry blog, a physics blog, or even biotech and biopharma blogs just wouldn’t have the reach that is necessary to create that networking chain reaction that ensures people are online to share in your research excitement or debate you about some new biology news report, quantum physics discovery, or even an archeological discovery.

On the Personal Chain Reaction network, you can post your own articles for all to see and read, or search our classified ads for those special lab items you have been hunting for. You can just post to your friends or shout your science news to the world from atop our message feed system.

Also, one last little feature, that you won’t find on Facebook or Twitter, I thought hey we have these nice discounts arranged with several vendors and we are passing those discounts on to your our molecular science friends.  Go ahead, check out our links to many great DIY biology,  DIY chemistry, DIY Physics, and just about any bio-hacker, chem-hacker product you need.

In the last 10 years the real time pcr primer, and real time fluorescent probe market has expanded to a huge number of lower and lower priced vendors.  Each of them offer to their customers various degrees of documentation for the quality of the oligos that they are selling.  One service they fail to offer is a performance guarantee; sure many of them will replace a failed oligonucleotide, but a typical researcher or diagnostic lab will have run several rt-PCR or qPCR samples before the culprit oligonucleotide is identified.  The cost of such investigative work is not solely in the replacement cost of the oligonucleotides themselves, but actually in each of the following categories:

1. PCR wet reagent costs, as much as $2.00 per failed reaction, assume 20 reactions or $40.00
2. PCR hard plastics, as much as $1.00 per failed reaction, again assume 20 reactions or $20.00
3. Technician time, conservatively estimated to be 3 hours at $ 15.00/hr, or $45.00
4. Re-order time, and any delays in sample processing that come out of that… $$.

So from this very conservative breakdown, one failed oligonucleotide may cost a lab at least $100+. Add to this $100.00, for actual failed oligonucleotides, the real cost of having to perform the quality control for primers and probes BECAUSE they haven’t been quality assured, and even if the molecules perform well with the in-lab QC,  there is still nearly $100.00 cost in performing that QC as well as the added cost of ‘used’ oligonucleotides that could have been dedicated to generating revenue, say for a $20 test, that could be as much as $200 in lost revenue per oligonucleotide order.

So much for cheap oligonucleotides.

Fluoresentric, a real-time PCR assays development company, recognizes that reagent costs are only a small piece of the diagnostic testing balance sheet and that both high throughput, and low throughput labs need guaranteed oligonucleotides that will perform the same today as they did 10 years ago.  To help those labs achieve that end, we offer an oligonucleotide QA/QC  service where they can get that guarantee.   To find out more you can check us out online at store.fluoresentric.com . Online you can get your complete oligonucleotide primers and  fluorescent probes ready-to-run for as low as $0.25 per reaction with our unconditional guarantee that they will perform in your real-time PCR, rt PCR, qPCR, genotyping, or whatever type of oligonucleotide application you have.  Think of it,  no more ordering a synthesis scale and hoping you get enough material to run your tests, no more quality testing required.  Just rehydrate and go.

You can check out our publication on this product on the Fluoresentric home page.

Over these past 10 years of being in the real-time PCR assay development business and having worked on projects from quantitiative PCR, to genotyping, and from 1-step reverse transcription PCR to self-contained sample preparation and amplification instruments with freeze dried reaction mixes we have found that our very simple buffer system works well, if not superior, for just about any reaction that we have tested it against.

Now, for less than four (4) cents a reaction you can have our buffer in your reaction.  When you order the buffer it comes with MgCl2 to enable full optimization of your reaction.  We will even pre-formulate our buffer into larger bulk sizes with the necessary MgCl2 concentration.  This may seem unimportant, at first, but for those of you PCR scientists out there that want to be able to add the maximum sample volume possible, every microliter does count and we would be happy to give you those extra microliters of sample volume you so desperately need for some of your high value tests.

Check it out online at store.fluoresentric.com, and don’t be shy if you have any questions we are always happy to help you plan your reactions and buffer transition strategies.

I certainly wouldn’t disagree with this notion, that would be foolish, it is true that a great SYBR Green I assay that doesn’t produce non-specific products is less expensive per reaction.  Though the reality of a double stranded DNA binding dye based assay that uses standard PCR thermal cycling conditions is going to end up producing non-specific products which are going to either have to be systematically ‘ignored’ during the amplification or they are ‘hidden’ by foreshortening the thermal cycling protocol to something less than 30 cycles (for a robust amplification protocol).  Either way the non-specific products are there they are just removed from the analysis.  This often results in those situtations where low copy number template containing reactions are missed and called either ‘unknown’ or worse yet ‘negative’.

In the summer of 2004 I set my mind to solving this problem with the intent of eliminating it altogether.  What I came up with, with no small effort from my laboratory staff was a technique of DNA and RNA amplification that we have dubbed Dynamic Flux Amplification (DFA).  It is just what the name says, a method of dynamically taking advantage of the nature ‘breathing’ , or flux, of nucleic acids.  That natural breathing opens and closes regions of the nucleic acid and we target those opened regions with target specific primers so that only those opened and single stranded regions of DNA are interrogated by the primers.  This, in turn, makes it so the amplification is wholly specific and subsequently the formation of non-specific products (NSP) is eliminated.

We have evaluated the DFA technique against a variety of DNA templates, as one critic asked if this were something that could only work with specific G+C content templates, and determined that even over a broad range of G+C content templates from 38-60% the DFA technique performed comparably in sensitivity to PCR and without the formation of NSPs.

The really wonderful feature of DFA that comes out of the elimination of NSPs is that a whole slew of detection techniques are opened up the diagnostician.  DFA has been adapted to the following detection techniques: real-time PCR (dsDNA binding dye), gel-electrophoretic, chemiluminescent, colorimetric, and ELISA.  The list is continuing to expand, and we at Signal Diagnostics are excited and thrilled to work with any customer that has a dsDNA detection chemistry that they want to test with DFA.

Give us a call at 801-580-0490 and ask for me, Brian Caplin.  I  want to hear from you and together we can bring a new and even less expensive diagnostic chemistry online for your DNA or RNA assay.

Either way you are going to get, with each course you register for, breakfast, lunch, dinner, and a one-night stay in beautiful Park City, Utah.  All of that at one low price that also just so happens to come with a full day of real-time PCR training. Oh yeah, you also go home with lots of great new real-time PCR knowledge and a beautiful Certificate for completed course work.

If you are a lab director, and have 3 or more team members that need some real-time PCR training, we’ll train them and you can come along at no cost to you.*  Give us a call at 800-808-0490 to make these arrangements.

*You gotta be their Supervisor, or something akin to that, we want you in the morning meeting and joining us so that we focus on the items that your laboratory personnel need to learn to do their job.