Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems

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.

Barcoding the Neotropical freshwater fish fauna using a new pair of universal COI primers with a discussion of primer dimers and M13 primer tails

Designing primers for DNA barcoding is a significant challenge for the rich Neotropical fish fauna, which is comprised of ∼6000 species. Previously, researchers required multiple pairs of PCR primers or primer cocktails to obtain standard COI (i.e., mitochondrial cytochrome c oxidase subunit I) barcode sequences from assemblages of freshwater fish in this region. To simplify DNA barcoding and metabarcoding studies of Neotropical freshwater fish, we present a new pair of COI primers, which have yielded high quality barcodes across six teleost orders—Characiformes, Cichliformes, Cyprinodontiformes, Gymnotiformes, Siluriformes, and Synbranchiformes—native to South America. Following previous fish barcoding studies, we also tailed our primers with M13 forward and reverse primers to facilitate the DNA sequencing process. Although this practice generates primer dimers, we obtained complete and high quality COI barcode sequences for all samples. We discuss the problem of primer dimers and suggest strategies for neutralizing their influence on data quality.

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

Bumping the priming sites for RT-LAMP based on thermodynamic predictions can dramatically reduce non-specific background amplification.