Article

Comparative Assessment of 5' A/T-Rich Overhang Sequences with Optimal and Sub-optimal Primers to Increase PCR Yields and Sensitivity.

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, .
Molecular Biotechnology (Impact Factor: 2.28). 03/2013; 55(1):17-26. DOI: 10.1007/s12033-012-9617-5
Source: PubMed

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.

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Questions & Answers about this publication

  • Alejandro Olmedo added an answer in Touchdown PCR:
    What is the best way to conduct a PCR with forward and reverse primers that have very different Tm?

    I have two primers with very different Tms. One of them has a mean Tm of 57C and the other has a mean Tm of 43C. It is not possible to construct new primers as they are based on an amino acid sequences of a group of ssDNA viruses that have high degeneracy in this region. Is it possible at all to do a PCR when they are so different and get something? I have been considering touchdown PCR but am not sure how to design it exactly and if this will solve my problem of annealing temperature. Thanks.  

    Alejandro Olmedo · Escuela Politécnica del Ejército

    Why don't you try to add some non-complementary extensions to the 5' ends of each primer to balance the °Tm of both primers?

    I can reference this paper:

     https://www.researchgate.net/publication/232765452_Comparative_Assessment_of_5'_AT-Rich_Overhang_Sequences_with_Optimal_and_Sub-optimal_Primers_to_Increase_PCR_Yields_and_Sensitivity