Designer DNA for sensing and nanomaterials

Abstract

The Whitfield enzymatic extension method was explored for the development of three key concepts; i) amplification of size specific designer DNA (improved yield), ii) the synthesis of known spatial binding sites (novel nanomaterials) and iii) extended DNA brush synthesis (sensing applications). The size recovery and amplification of specific lengths of designer DNA products were investigated for use in DNA nanomaterials. Initially, oligoseeds, [AT]10/[TA]10 and [GC]10/[GC]10, were enzymatically extended using the Whitfield method to give large distributions of DNA of up to 20,000 bp. Product concentrations were 71.4 ng/µL for [AT]n and 135 ng/µL for [GC]n. Size recovery by removing aliquots of size specific DNA during gel electrophoresis afforded DNA aliquots of narrow size and in low concentration, < 5 ng/µL, thus amplification by ligation and qPCR was used to increase the concentration of the final product to ~ 30 ng/µL. In addition, the synthesis of long DNA products containing 3 multi-base sequences, ABC was developed. This approach afforded the incorporation of a 4th sequence, D. The ABCD-DNA method produced various sequences and lengths of novel designer DNA ranging from 500 - 5,000 bp. The new sequence D, not in the original oligoseed, allowed for the successful inclusion of spatially separated sequences of phosphorothioate bases into ssDNA or dsDNA of 500 - 1,500 bp. 3 nm gold nanoparticles bound specifically to the phosphorothioate regions as seen by AFM. Enzymatic extension of surface immobilised oligoseeds was used to grow long repeat sequence DNA brushes for enhanced sensing. The initial 20 bp oligoseed surface gave an enhanced fluorescent signal from the PicoGreen fluorescent indicator due to the multiplexed repeat sequences upon extension. The surface extension of the micro-satellite BAT-25 sequence demonstrated that small numbers of base mis-matches could be distinguished by comparison of their hybridised fluorescent signals.