Bad Boys Bad Boys Whacha Gonna Do When They Come For You?
How to Design a Properly Controlled Experiment
The heart and soul of scientific method is the controlled experiment. This is an experiment conducted with things that should work (positive control) and things that we know won’t work (negative control) to ensure that the results from our samples are real.
Rule #1- Scientists are way too tired and way too busy to be clever…
The names of things are often exactly what they mean.
A positive control is a control that is positive….It should have worked in the experiment.
A negative control is a control that is negative….It should not have worked in the experiment.
A false positive is a positive that is false….It should not have worked but it did.
A false negative is a negative that is false….It should have worked but it did not.
Rule #2- Controls Can Have Real Life Consequences…
So who cares about controls?
Let’s think of a real life scenario. In fact DNA technology (especially polymerase chain reaction or PCR) is used in court cases. The details of how PCR works you will learn later. All you need to know right now is the experiments are set-up in tubes and run in a machine called a thermocycler. The samples are then run though a gelatin-like substance called a gel using an electrical current. You will learn to do all these things in your research! A positive result in this type of PCR is seeing a band on the gel and a negative result is no band seen.
You have been accused of murder by the local district attorney!
Off you go in handcuffs!
The press following around as you are escorted to the paddy wagon!
Forensics has isolated DNA from blood stains found at the crime site and this will determine whether or not you go to jail maybe for decades!
So you hire the best defense lawyer you can afford. And it comes down to the PCR testing of DNA at the crime scene.
(http://www.bing.com/images/search?q=arrest+cartoon
&view=detail&id=D2D2BE77F9BF5564D39B7C0F78E3D89
960D9C12E&first=1)
The district attorney is concerned about false negatives (negatives that are false). In other words, he believes you are guilty and wants you in jail. If the PCR does not work, it might be because the technician messed-up the experiment by leaving something out or adding too much of something. The district attorney would want to know if that happened, because these experiments can mean you are convicted or not, right? And he really wants you in jail. So to be safe, a sample of your DNA is taken by swabbing the inside of your mouth to serve as a positive control.
This positive control is something that should work, and it is there to keep the district attorney happy. It will yield a positive result. In the case of PCR a band will be seen.
On the other hand, the defense attorney is concerned about false positives (a positive that is false). I mean… what if the murderer works in the lab doing the DNA testing? What if some of the murderer’s skin cells contaminated the reagents used for PCR (and thus his/her DNA)? You could easily get a false positive in PCR with reagents contaminated by the murderer’s DNA, right? You and the defense attorney would not want that!
So the forensic scientist would have a tube in her/his PCR experiment that does not have any DNA and thus no band will be seen in that sample, to keep you and the defense attorney happy.
So the experiment the forensic scientist sets up should have a positive control to protect against false negatives (your DNA). It should also have a negative control to protect against false positives (a PCR reaction without DNA in the tube).
How to Interpret the Results
If you positive control is negative (indicating a false negative) OR if your negative control is positive (indicating a false positive) you cannot use the results!
This is true even if your test samples worked or did not work! The experiment cannot be interpreted.
Negative Controls and PCR
It is possible to do work on something where a positive control is not available (e.g. doing PCR to detect a rare virus). The most important thing in PCR is that a negative control is negative. PCR is a very sensitive technique as you will learn and any contamination in the reagents can lead to a false positive.
So it is critical that you work almost like you are doing surgery!
Rule #3- EVERY PCR Experiment MUST HAVE A NEGATIVE CONTROL!