Dr. Singh - The nested PCR was done as described in Lo et al, with one small modification. We used the dUTP-UNG system to prevent contamination of our lab with PCR products.
Dr. Singh is right adding the dUTP-UNG system is a good idea for any non real-time PCR, and especially for multistep PCRs like the nested PCR. UNG does not have a negative effect on the PCR but does reduce the risk of contamination a LOT. While I was at Arcxis, we ran into a number of labs that would not buy PCR tests unless they had UNG present. It had become a standard in US clinical practice. That being said, basic research often does not use UNG.
The basic principle is as follows. Each PCR produces around 100 billion copies of the amplified product. The PCR reaction takes place inside of a fluid in a plastic tube. In order to analyze the reaction (unless you are using real-time PCR), you have to open the top of the tube to get access to the fluid. The top often pops open, releasing a microscopic spray into the air, kind of like when you sneeze. This spray potentially contains billions of copies of DNA that can contaminate other PCR reactions.
It will not contaminate culture and it cannot contaminate antibody tests. But it will contaminate other PCR tests using the same or similar primers (ie looking at the same gene). As Dr. Singh said, once this type of contamination is in a lab, it is almost impossible to get rid of short of burning down the entire establishment.
So how does the dUTP-UNG system help safe guard against this type of contamination? UNG is an enzyme, a protein that performs a function. If you recall from biology class, there are four standard DNA bases: A, C, G, and T. The dUTP-UNG system substitutes the T for an RNA base, U (ie this type of PCR uses A, C, G and U instead of the normal four). At the end of the PCR, instead of having a true DNA product, you have a DNA product interspersed with the RNA base, U. The UNG is activated at the beginning of the PCR reaction. It recognizes the U base in the DNA product and destroys the DNA products with U bases present.
That means, even if you had contamination in the lab from a previous PCR reaction, the UNG will provide a lot of protection against this type of false positive. We played with UNG to try to quantify exactly how much protection it provides. We found it would prevent false positives if less than around 1,000,000 contaminating copies were present in the tube. That is a large number, meaning that it should prevent contaminationfrom prior PCR reactions in almost every case.
Someone called us to ask if we used UNG in our reactions. We do not. But we do not use a PCR that has to open the top risking contamination of the lab. The easiest way to prevent contamination in real-time PCR is to simply never open the top of the tube after the PCR is finished!
Dr. Singh- "Since nested PCRs involve opening of tubes containing a PCR product, the danger of this product contaminating other equipment, reagents and samples in the lab is very real. Once contamination has occurred, it is very difficult to get rid of it. Any reputable lab that uses PCR as a test on clinical samples, uses dUTP-UNG. It has been shown to not affect the PCR itself, and to provide a very real safeguard against contamination.
I do not know why Lo et al or Lombardi et al used a process prone to contamination like the nested PCR, and then did not use the dUTP-UNG system (youll have to ask them). But I could not risk contaminating my lab with PCR amplicons then every subsequent test we did would be suspect."
Lombardi et al clearly ran their PCR as a research test in the Science article. UNG would not necessarily be expected in a first paper. Their use of culture and antibody testing would have been done to address any risk of false positives arising from PCR carry over contamination.
But now with all the WPI data in question, the use of UNG in a nested PCR would be very important. Nested PCR, even more than single round PCR, is subject to contamination. It has a first round of PCR potentially creating billions of copies of DNA from as little as one starting copy. Then the top is opened (remember the sneeze effect) and some of the fluid containing those billion copies is placed into a second PCR to create even more copies. The second round of PCR compensates for any inadequacies in the design of the first round PCR. Then the top of that reaction is opened (a second sneeze) to analyze its contents.
While this clearly will not explain the antibody positives or culture positives, it would be a good practice to implement in this type of reaction. Of course, even the addition of UNG will not prevent other types of contamination, such as the presence of virus in the polymerases being used, or the presence of virus on lab equipment, etc.
