Abstract
Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how “self-avoiding molecular recognition systems” (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer–primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.
Highlights
Despite its widespread value to molecular biologists, the polymerase chain reaction (PCR) [1, 2] has an “endless ability to confound” [3]
Introducing too few self-avoiding molecular recognition systems” (SAMRS) components does not completely prevent the formation of primer dimers and other products arising from undesired primer–primer interactions
It forms a stable pair with adenosine. Further development of this would need to understand the chemistry and enzymology of these species. These thermostability studies show that different SAMRS components contribute different levels of stability to SAMRS:standard pair
Summary
Despite its widespread value to molecular biologists, the polymerase chain reaction (PCR) [1, 2] has an “endless ability to confound” [3]. One goal of this work is to manage the wellrecognized problematic formation of primer dimers [8,9,10,11,12], which can consume PCR resources, including the polymerase, primers, and the triphosphates, as well as downstream sequencing resources. This consumption becomes worse as target molecules become more numerous and scarcer [13]. Because they capture only imperfectly the Received: 6 January 2020; Revised: 4 February 2020; Editorial Decision: 5 February 2020; Accepted: 7 February 2020
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
