Investigations of a bio-synthetic approach to access large padlock as a part of a new concept for detecting DNA on a microarray

Abstract

Padlock probes are effective tools for specific and sensitive detection of DNA. The main hurdle restricting their popularity is the difficulties in synthesizing them. This study was aimed to develop a protocol based on ligation and gene synthesis methods to achieve: Firstly, generation of padlocks much longer than 100 bases to enable incorporation of new functions, secondly simple and cost-effective protocol, thirdly high yield and fourth opportunity to incorporate repetitive and other functional sequences using module-like building blocks. 120 bases of padlocks in extremely high yield, phosphorylated and with sequence specific ends were successfully synthesized using ligation and polymerase reactions.Repeated sequences were introduced to realize the use of padlocks as a new DNA amplification system. Difficulties in synthesizing padlocks with repetitive sequences were confirmed as several modifications of the protocols did not lead to the desired result. But finally, hybridization and ligation reactions were used to construct 205 bases long padlock with a threefold repetitive sequence inserted in to the non-target specific part of the probe.