Single enzyme-based stem-loop and linear primers co-mediated exponential amplification of short gene sequences

Isothermal DNA amplification only using a Bacillus stearothermophilus (Bst) DNA polymerase such as loop-mediated isothermal amplification typically entails multiple target sites for primer design and is thereby not suited for the amplification of short gene sequences, for example, the sequences with size below 200 nucleotides (nt). Here we present SLIMP, a novel single enzyme-based isothermal amplification of short gene sequence mediated by both stem-loop and linear primers. In SLIMP, a pair of stem-loop primers and a pair of linear primers are specifically designed to recognize only two target sites. Linear primers in SLIMP are similar as conventional PCR primers, but stem-loop primers are the modified linear primers through attaching a stem-loop structure at their 5′-ends. Attributed to this unique primer design, three basic reaction modes including linear-priming, single stem-loop-priming, and double stem-loop-priming amplifications co-mediate the SLIMP process under the function of Bst DNA polymerase. As a proof-of-concept assay, a synthetic 80 nt sequence from hepatitis B virus S gene was used as the template to develop SLIMP. On performance, SLIMP detection possesses high sensitivity and specificity, good selectivity, and the potential for analysing real sample. Therefore, SLIMP is expected as a novel alternative to amplify short gene sequences using a single enzyme.