Real-Time PCR: the TaqMan Method

 

Introduction:

The advent of Polymerase Chain Reaction (PCR) by Kary B. Mullis in the mid-1980s revolutionized molecular biology as we know it. PCR is a fairly standard procedure now, and its use is extremely wide-ranging. At its most basic application, PCR can amplify a small amount of template DNA (or RNA) into large quantities in a few hours. This is performed by mixing the DNA with primers on either side of the DNA (forward and reverse), Taq polymerase (of the species Thermus aquaticus, a thermophile whose polymerase is able to withstand extremely high temperatures), free nucleotides (dNTPs for DNA, NTPs for RNA), and buffer. This movie shows PCR in action. The temperature is then alternated between hot and cold to denature and reanneal the DNA, with the polymerase adding new complementary strands each time. In addition to the basic use of PCR, specially designed primers can be made to ligate two different pieces of DNA together or add a restriction site, in addition to many other creative uses. Clearly, PCR is a procedure that is an integral addition to the molecular biologist’s toolbox, and the method has been continually improved upon over the years. (Purves, et al. 2001)

Fairly recently, a new method of PCR quantification has been invented. This is called “real-time PCR” because it allows the scientist to actually view the increase in the amount of DNA as it is amplified. Several different types of real-time PCR are being marketed to the scientific community at this time, each with their advantages. This web site will explore one of these types, TaqMan® real-time PCR, as well as give an overview of the other two types of real-time PCR, molecular beacon and SYBR® Green. ( www.ambion.com 2003)

How TaqMan® works:

TaqMan® utilizes a system that is fairly easy to grasp conceptually. First, we must take a look at the TaqMan® probe.

Figure 1. The Taqman probe. The red circle represents the quenching dye that disrupts the observable signal from the reporter dye (green circle) when it is within a short distance. Image created by Dan Pierce.