Amplifying Trouble

How to get robust results from your dodgiest PCR

Josh P. Roberts
Apr 1, 2008
<figcaption> Credit: © Pascal Goetgheluck / Photo Researchers, Inc.</figcaption>
Credit: © Pascal Goetgheluck / Photo Researchers, Inc.

PCR, in one form or another, has become fairly routine. It's used for everything from cloning and mutating, to analyzing RNA expression, to identifying fossils and criminals. Tools for conducting standard PCR, real-time PCR (RT-PCR), and quantitative PCR (qPCR) have followed suit, and there are Web sites for designing and ordering primers or purchasing off-the-shelf kits. These days, it's just a matter of adding reagents and nucleic acids to a tube or plate, pushing "start," and checking the computer a little while later to look at your results.

Sometimes things don't go as smoothly as the protocols would suggest. Target sequences might be shorter than the protocols can handle. Primers can dimerize, or they might not amplify a unique product. The samples themselves might be difficult to isolate:...