Ligation Based Assembly and Polymerase Chain Reaction-Based Assembly for Extraordinary Adenine/Thymine Rich DNA

Abstract

Extraordinary adenine/thymine rich DNA has a low complexity resulting in the occurrence of many short repetitive sequences or longer imperfect repeats, consequently hindering the gene synthesis process. This report describes the rapid synthesis of a DNA fragment with guanine/cytosine content of 11.8% using ligation based assembly and polymerase chain reaction-based assembly respectively, via the use of multiple strategies addressing the adenine/thymine rich nature of the fragment. Sequences can be simply chopped and assembled without Tm optimization. Smaller amount of ligation products as templates in Interference Free polymerase chain reaction yielded markedly more than or equal amount of subassembly products as using larger amount of ligation products, and longer extension time was required for successful subassembly. Longer time at low annealing temperature also had obvious effects on polymerase chain reaction amplifications. No full-length products could be generated without initial oligonucleotide sub-pooling for both approaches. The chemical synthesis of extraordinary adenine/thymine rich DNA could enable researchers to assemble synthetic modules, to study and to access the repetitive DNA such as heterochromatin regions which harbor important functional elements.