Abstract
AbstarctExisting isothermal nucleic acid amplification (INAA) relying on the strand displacement activity of DNA polymerase usually requires at least two primers. However, in this paper, we report an unusual isothermal multimerization and amplification (UIMA) which only needs one primer and is efficiently initiated by the strand-displacing DNA polymerases with reverse transcription activities. On electrophoresis, the products of UIMA present a cascade-shape band and they are confirmed to be multimeric DNAs with repeated target sequences. In contrast to current methods, UIMA is simple to product multimeric DNA, due to the independent of multiple primers and rolling circle structures. Through assaying the synthesized single-stranded DNA targets, UIMA performs high sensitivity and specificity, as well as the universality. In addition, a plausible mechanism of UIMA is proposed, involving short DNA bending, mismatch extension, and template slippage. UIMA is a good explanation for why nonspecific amplification easily happens in existing INAAs. As the simplest INAA till now, UIMA provides a new insight for deeply understanding INAA and opens a new avenue for thoroughly addressing nonspecific amplification.
Highlights
As point-of-care testing (POCT) techniques for molecular diagnosis[1,2,3,4], isothermal nucleic acid amplification (INAA) is advantageous over conventional PCR, due to its high specificity, efficiency, rapidity, and the independent of thermal cyclers[5,6,7,8]
We concentrate on linear target isothermal multimerization amplification (LIMA) and find that LIMA belongs to an unusual isothermal multimerization and amplification (UIMA) which only requires one primer
To investigate whether UIMA was a template-dependent reaction, a synthesized single-stranded DNA (ssDNA) with the primer-recognized site was used as the template and three control tests of no-template control (NTC), no-primer control (NPC), and no-enzyme control (NEC) were set up
Summary
As point-of-care testing (POCT) techniques for molecular diagnosis[1,2,3,4], isothermal nucleic acid amplification (INAA) is advantageous over conventional PCR, due to its high specificity, efficiency, rapidity, and the independent of thermal cyclers[5,6,7,8]. To achieve high sensitivity and specificity, these INAAs entail multiplex primers targeting multiple sequence sites, which inevitably enhances the probability of nonspecific amplification[12]. As alternatives to multiple primers-based INAAs, polymerase spiral reaction (PSR)[14] and linear target isothermal multimerization amplification (LIMA)[15] are preferable. They only require two primers and two target sites, lowering the difficult on primer design. Single crossing CPA should not produce DNA fragments with large sizes, but electrophoresis obviously indicates large by-products, leading to incomplete digestion of the products These resulting by-products are from the LIMAs caused by CPA’s primers of 4 s and 3a/2a/5a.
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