Comparative Assessment of 5′ A/T-Rich Overhang Sequences with Optimal and Sub-optimal Primers to Increase PCR Yields and Sensitivity


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.

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We thank Jacqueline Fletcher, Ulrich Melcher, Astri Wayadande, and Shefali Dobhal for reviewing this manuscript. This study was funded by the Oklahoma Agricultural Experiment Station (Project Numbers OKL02773). The mention of trade names or commercial products in this publication does not imply recommendation or endorsement by Oklahoma State University.

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Correspondence to F. M. Ochoa-Corona.

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Arif, M., Ochoa-Corona, F.M. Comparative Assessment of 5′ A/T-Rich Overhang Sequences with Optimal and Sub-optimal Primers to Increase PCR Yields and Sensitivity. Mol Biotechnol 55, 17–26 (2013).

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  • DNA amplification
  • Diagnostics
  • Primer design
  • qPCR
  • Tobacco mosaic virus
  • 5′ A/T-rich flap