Biotechnology Techniques 13: 643–646, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 643 Studies on primer-dimer formation in polymerase chain reaction (PCR) Simantini Das, Satish C. Mohapatra & James T. Hsu ∗ Department of Chemical Engineering, Lehigh University, Bethlehem, PA 18015, USA ∗ Author for correspondence: (Fax: (610) 758-5851; E-mail: jth0@lehigh.edu) Received: 29 June 1999; accepted: 29 July 1999 Key words: mechanistic, PCR, primer-dimer, Taq DNA polymerase enzyme Abstract A viable mechanism for primer-dimer formation in polymerase chain reaction (PCR) process has been proposed based on experimental results. This mechanism results in a kinetic description of the primer-dimer formation process with the Taq DNA polymerase enzyme, the two primers and the dNTPs as the starting materials. The possible impact of this primer-dimer formation on the selectivity and yield of PCR process is discussed. Introduction Polymerase chain reaction (PCR) is widely used in many areas of biological research to carry out am- plification of selected nucleotide (DNA or RNA) se- quences through a succession of incubation at different temperatures (Saiki et al. 1988). The three steps in this process are: (1) strand separation of DNA, (2) primer annealing to the single strands, and (3) extension of the primers by a polymerase enzyme to complete a DNA. In PCR, the amount of DNA doubles after each discrete cycle of amplification, but this exponen- tial accumulation of the product becomes a limited process towards the late PCR cycles. When there are more amount of primer-template accumulate than the amount of enzyme is present capable of extending in the allotted time, the amplification of DNA follows a linear trend rather than exponential (Saiki et al. 1988). Further increase in the number of cycles results in a plateau phase where the DNA product concentra- tion remains constant with the cycle number. After this phase, DNA concentration may start to decrease. This phenomenon has not been explained properly in the previous research papers. However, mathematical modeling of PCR has brought some understanding of this process (Hsu et al. 1997), especially in the earlier cycles when the DNA concentration increases approx- imately exponentially and then linearly, but there is still a significant work to be done to characterize the process completely. One of the primary factors that reduces the fi- nal yield of target DNA product is the formation of primer-dimer which is a product of duplex formation between two primers. Formation of these duplexes not only decreases the concentration of primers in the reaction mix but also initiates the formation of non- specific DNA products. Brownie et al. (1998) have attempted to produce primer-dimers from a variety of primers with differing types and extent of complemen- tarity. Their observations led to the hypothesis that a system could be developed whereby the accumulation of primer-dimer in a PCR may be eliminated. They demonstrated that the primer-dimer formation can be suppressed by adding a sequence of nucleotides at the 5 ′ -ends (inactive side) of the primers. Other inves- tigators have suggested the optimization of reaction conditions such as carrying out the annealing of the primer and synthesis at higher temperature to reduce the extent of primer-dimer formation (Wu et al. 1990, Rychlik et al. 1990). Despite all these research work, there is a need to determine the mechanism of primer- dimer formation and develop a mathematical model that will lead to a kinetic description of the process and possibly explain the plateau and the later phases in the PCR process. In this paper, several experiments are reported which would help in proposing a mechanism of