| Description |
Detection of specific sequences of DNA is invaluable for diagnosing the presence of a pathogen, as well as other areas of genetic analysis. A common way of detecting DNA is replicating it millions of times through Polymerase Chain Reaction (PCR) and using fluorescent molecules to detect those copies. This paper examines Cooperative Primers or CoPrimers through physical models. Primers are a new class of PCR Primer technology that virtually eliminates primer dimers (one of the problems inherent in PCR reactions) while maintaining the sensitivity and specificity required to detect the desired DNA sequence [1]. They consist of a primer and a capture sequence connected by an inert linker. When the capture sequence anneals, the primer is brought into artificially close proximity with its target, thus increasing the local concentration of the primer (Pl) by approximately 1,500X. This allows the primer to be synthesized with a very low binding affinity while still amplifying in PCR reactions. Cooperative Primers show promise in the fields of Single Nucleotide Polymorphism (SNP) detection, low-cost diagnostic PCR, and multiplex PCR [2]. However, little research has been done on their optimization. By accounting for additional effects caused by the flexibility of the target DNA sequence and asymmetries in the extension of the polymerase, we reformulated the equations used to maximize the Pl. Using these equations, we have changed the way we make Cooperative Primers to increase the Pl by 20,000X over original reaction concentrations - an order of magnitude improvement over previous design methods. Experimental results compare favorably to predictions - especially in relation to previous models. Future research avenues are proposed. |
