Enzymatic Method for the Synthesis of Long DNA Sequences with Multiple Repeat Units

Abstract

A polymerase chain reaction (PCR) derived method for preparing long DNA, consisting of multiple repeat units of one to ten base pairs, is described. Two seeding oligodeoxynucleotides, so-called oligoseeds, which encode the repeat unit and produce a duplex with 5′-overhangs, are extended using a thermostable archaeal DNA polymerase. Multiple rounds of heat–cool extension cycles, akin to PCR, rapidly elongate the oligoseed. Twenty cycles produced long DNA with uniformly repeating sequences to over 20 kilobases (kb) in length. The polynucleotides prepared include [A]n/[T]n, [AG]n/[TC]n, [A2G]n/[T2C]n, [A3G]n/[T3C]n, [A4G]n/[T4C]n, [A9G]n/[T9C]n, [GATC]n/[CTAG]n, and [ACTGATCAGC]n/[TGACTAGTCG]n, indicating that the method is extremely flexible with regard to the repeat length and base sequence of the initial oligoseeds. DNA of this length (20 kb≈7 μm) with strictly defined base reiterations should find use in nanomaterial applications.