A simple modification increases specificity and efficiency of asymmetric PCR

Zoltán Tolnai, Ákos Harkai, Zsuzsanna Szeitner, Éva Nagyné Scholz, Krisztina Percze, Anna Gyurkovics, Tamás Mészáros

Research output: Article

3 Citations (Scopus)

Abstract

Although various methods have been developed to suffice the oligonucleotide demand of molecular biology laboratories, in vitro production of high-purity ssDNAs remains to be a challenging task. We hypothesized that complementing the asymmetric PCR with 3’ phosphate blocked limiting primer decreases the mispriming thus reduces polymerisation of DNA by-products. The presented results attest our assumption that the primer blocked asymmetric PCR (PBA-PCR) selectively produces ssDNA of interest and is even suitable for effective amplification of DNA libraries of large sequence space. The high-throughput sequence analysis demonstrated that PBA-PCR also alleviates the PCR bias obstacle since it does not distort the sequence space. The practicability of the novel method was verified by monitoring the process of SELEX and screening of aptamer candidates using PBA-PCR produced ssDNAs in Amplified Luminescent Proximity Homogeneous Assay. In summary, we have developed a generally applicable method for straightforward, cost-effective production of ssDNA with on demand labelling.

Original languageEnglish
Pages (from-to)225-230
Number of pages6
JournalAnalytica Chimica Acta
Volume1047
DOIs
Publication statusPublished - jan. 24 2019

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ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy

Cite this

Tolnai, Z., Harkai, Á., Szeitner, Z., Scholz, É. N., Percze, K., Gyurkovics, A., & Mészáros, T. (2019). A simple modification increases specificity and efficiency of asymmetric PCR. Analytica Chimica Acta, 1047, 225-230. https://doi.org/10.1016/j.aca.2018.10.017