We really treat the iCubate cassette like slaves: we tell them do many tedious and “labor intensive” things without giving them a break, and they are not allowed to make any mistakes.
One of the things we ask the cassette ( or more precisely, the pipette in the cassette) to do lately is to add polymerase into the reaction in aliquot, every few cycles!
In a typical PCR reaction, all the enzymes and dNTPs, were added into the reaction at the beginning. But at those early cycles, only a few copies of the targets are there for the enzyme to work on. So, during the first half of the PCR cycles, the enzyme molecules are either doing the wrong thing by generating background or getting “tired” by the repeated heating.
Taking advantages of the automated pipette, we are adding fresh enzymes and dNTPs every few cycles, guess what? the sensitivity is improved as expected, and the background decreased.
This feature is extremely helpful when we want to amplify tough targets like those from the TB bacteria. Because of the high CG content, partial denature makes amplification efficiency very low. Higher denature temperature would help but that will kill the enzyme prematurely. By adding fresh enzymes every few cycles, we can limit the wear and tear on the polymerase and increase overall assay sensitivity.
Genius, right?
That is only one of the little tricks this smart machine can do.