Method for High-Throughput Assembly of Long DNA Fragments in vitro

Pharmaseq, Inc., Monmouth Junction NJ

Investigators

Abstract

SUMMARY The main project goal is to develop principles and demonstrate the feasibility of a fast in vitro method that will increase the length of synthetic double-stranded DNA from its current limit of about 10 kb to 100 kb. This, in turn, will facilitate the ability to more rapidly create artificial genes and chromosomes and thus become an important tool in synthetic biology. The proposed approach involves ligations of 10 kb DNA fragments (?Building Blocks?) into larger, approximately 100 kb DNAs, which are then transferred into larger structures by transformation into yeast cells. The key element facilitating ligations relies on stretching the DNA Building Blocks by first immobilizing the DNA on micro- or nanoparticles, and then subjecting the particles and DNA to either magnetic or electric field during the ligation reaction. Major tasks include: (1) Development of hardware suitable for a feasibility study. The system will include a custom instrument which generates and control the electric and magnetic fields. We will also build a special test tube (L-tube) for conducting ligations during which an electric and magnetic field are applied to the ligation reaction components in the new instrument. (2) Assembly a 100 kb DNA construct. Toward this goal, we will study the ligation efficiency in a special assay in which DNA fragments are fluorescently labeled-- the ligation efficiency is monitored by observing fluorescence. The DNA Building Blocks will be combined in vitro in a stepwise fashion to yield the final product. (3) Transfer of the 100 kb ligated DNA fragment into yeast by electroporation to confirm the integrity of the assembled DNA which will be established by biological selection and screening for proper biomarkers, as well as by q-PCR and DNA sequencing. The large assembled DNA fragments can be used in gene expression experiments to create novel biological products in different organisms.