title: Comparative assessment of 5' A/T-rich overhang sequences with optimal and sub-optimal primers to increase PCR yields and sensitivity.
abstract: Efficient PCR amplifications require precisely designed and optimized oligonucleotide primers, components, and cycling conditions. Despite recent software development and reaction improvement, primer design can still be enhanced. The aims of this research are to understand (1) the effect on PCR efficiency and DNA yields of primer thermodynamics parameters, and (2) the incorporation of 5' A/T-rich overhanging sequences (flaps) during primer design. Two primer sets, one optimal (ΔG = 0) and one sub-optimal (ΔG = 0.9), were designed using web interface software Primer3, BLASTn, and mFold to target a movement protein gene of Tobacco mosaic virus. The optimal primer set amplifies a product of 195 bp and supports higher PCR sensitivity and yields compared to the sub-optimal primer set, which amplifies a product of 192 bp. Greater fluorescence was obtained using optimal primers compared to that with sub-optimal primers. Primers designed with sub-optimal thermodynamics can be substantially improved by adding 5' flaps. Results indicate that even if the performance of some primers can be improved substantially by 5' flap addition, not all primers will be similarly improved. Optimal 5' flap sequences are dependent on the primer sequences, and alter the primer's T m value. The manipulation of this feature may enhance primer's efficiency to increase the PCR sensitivity and DNA yield.
authors.affiliation: National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, OK 74078, USA.
authors.forename: M
authors.initials: M
authors.lastname: Arif
authors.forename: F M
authors.initials: FM
authors.lastname: Ochoa-Corona
mesh_terms: D000465:Algorithms; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012984:Software; D013816:Thermodynamics; D014027:Tobacco Mosaic Virus / Q000737:chemistry
chemical_list: D017931:DNA Primers