Efficient Correction of DNA Hetero–Duplexes Formed During SELEX Procedure
DOI:
https://doi.org/10.51173/ijmhs.v1i1.16Keywords:
ssDNA Aptamer , PCR Artifacts , Hetero–Duplexes , PCR Artifact Correction , SELEXAbstract
Background: Aptamers, oligos-based (ssDNA or RNA) and peptide-based, are now emerging as the first-choice biomolecules in various applications in medicine and biology. DNA aptamers selection process includes repeatable steps of DNA amplification and digestion. The amplification process of the enriched DNA aptamers includes using PCR.
Objective of study: In this article, we sought to describe an efficient and robust method that corrects or permanently removes the PCR by–products during the cycles of DNA aptamers selection.
Materials and Methods: Our method is simple, efficient, and requires no enzymatic treatment. In this method, we applied repetitive cycles of thermal treatment to the enriched aptamers final products during the enrichment cycles. The procedure starts with loading the PCR-induced DNA on 1% agarose gel to evaluate the presence of ladder-like DNA or smearing band-like DNA. Next, we cut and purified these by–products and applied thermal treatment. The thermal treatment was by heating the DNA to 65ºC for 10 minutes and then cool them down to 20ºC for 1 minute.
Results: PCR artifacts evaluation has been revealed two types of unwanted DNA sequences; the ladder-like and the smearing bands. The next–Generation illume sequencing specified the presence of long and short sequences. Upon the thermal treatment of the PCR products, the next–generation illume sequencing method results showed that the longer sequences and the shorter sequences of the DNA have been reduced considerably and effectively in the DNA pool over the cycles of the systemic evolution of ligands by exponential enrichment (SELEX) procedure. Furthermore, we could reduce the formation of the hetero–duplexes in the next cycles of (SELEX) procedure.
Conclusion: This method suggests practical and routine application to prevent or reduce DNA aptamer by–products during the selection process. This treatment could reduce the mis-shaped or irrelevant non-functional aptamers in SELEX procedure
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