Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems
| Autor: | Daniel Hutter, Benjamin Overton, Chris McLendon, Jennifer T Le, Steven A. Benner, Kevin M. Bradley, Zunyi Yang |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Computer science
allele-specific PCR Context (language use) Computational biology 010402 general chemistry 01 natural sciences General Biochemistry Genetics and Molecular Biology law.invention 03 medical and health sciences Molecular recognition law Primer dimer Multiplex polymerase chain reaction diagnostics SNP Polymerase Polymerase chain reaction 030304 developmental biology Methods Manuscript 0303 health sciences biology multiplex PCR 0104 chemical sciences SNP detection biology.protein General Agricultural and Biological Sciences Variants of PCR primer dimer |
| Zdroj: | Biology Methods & Protocols |
| ISSN: | 2396-8923 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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