Polymerase chain reaction-based biochemical logic gate coupled with cell-free transcription-translation of green fluorescent protein as a report gate

摘要

Polymerase chain reaction-based biochemical logic gates were designed for AND, OR, NOT, and AND-NOT operations, whose output signal is reported by coupled cell-free transcription-translation of green fluorescent protein.rnRecently, much attention has been paid to designing a bio-molecular system that responds to chemical inputs. From this standpoint, polymerase chain reaction (PCR) is a model reaction of a 2-input AND logic gate because it responds to the input of two kinds of primers. In the reaction, DNA-amplification responds to an output signal, which is usually reported by fluorescence enhancement based on TaqMan chemistry or Molecular Beacon, or by the appearance of an electrophoretic band. Since PCR features a precision molecular recognition between DNA strands and reversible duplex formation, it is intriguing to attempt to develop PCR-based logic gates representing not only an AND gate but also OR or NOT gates. In this report, designing the templates appropriately and combining two or three primers, we constructed PCR-based AND, OR, NOT, and AND-NOT logic gates. The logic gate is a PCR reaction mixture containing a linear DNA template (logic gate template) coding a green fluorescent protein (GFP) and a single stranded oligodeoxy-ribonucleotide (ODN) that hybridizes with the logic gate template (pre-mixed primer). Fig. 1 illustrates the DNA logic gates designed and constructed in this study. The logic gate operation is executed by the addition of primer A and/or primer B as an input signal.