Brief Summary

Genome-wide primer design database, via webserver or GPU version, providing a thorough search for target locations and possible mispriming sites involving many arrangements of mismatches using single base bulges.

Problem Statement

Primer design tools use a thermodynamic approach to calculate the overall stability of mismatches and bulges rather than counting the number and orientation of single base bulges.

Technology Description

Good primer design is critical for robust and reproducible PCR. Many factors are considered in primer design, yet assessing primer specificity is crucial for creating assays that amplify the intended target efficiently while avoiding non-specific products. Computational techniques have been optimized to quickly search the entire genome for sites suitable for primer selection and extended with a cumbersome amount of parameters. Many consider primer-template mismatches, yet ignore the possibility of other structures such as single-base bulges. Recent studies have emphasized the importance of 3’ specificity of a primer rather than considering the entire primer sequence. A genomic database of 14bp fragments accompanied by positional information was developed and optimized for a straightforward and effective primer search process that includes exact, mismatch, and single-base bulges. Upon search completion, a simulated gel is created and displayed to the user. Products that are perfectly matched to the queried primers are displayed as the brightest bands and alternate products are displayed as silver bands. .

Stage of Development

Design & Development

Benefit

• Straightforward faster primer design that takes into account all possible bulges in the whole genome.
• Reduce non-specific amplification, which would reduce false positives, improve results and increase PCR efficiency.
• Reduce base pair deletions/insertions introduced due to bulges in primer design.
• Generates a dynamic in silico gel of expected PCR products.

Publications

Genomic Database for Assessing Specificity of Primers- https://dna-utah.org/Posters/AMP_2019_Dwight.pdf

Contact Info

Aaron Duffy
(801) 585-1377
aaron.duffy@utah.edu