Transcription activator-like effector assembly

摘要

Described herein are techniques for assembling a polynucleotide encoding a transcription activator-like effector nucleases (TALEN). The techniques ligate and digest necessary modules for a TALEN assembly in one reactor or system. Methods and Kits for generating a TALEN are also described.

主权项

1. A method for assembling a polynucleotide encoding a transcription activator-like effector (TALE), the method comprising:
generating multiple Deoxyribonucleic acid (DNA) segments, an individual DNA segment of the multiple DNA segments corresponding to a repeat sequence of the TALE that identifies a single nucleotide of a particular target polynucleotide or two contiguous nucleotides of a particular target polynucleotide, the multiple DNA segments being polymerase chain reaction (PCR) amplification products, a number of multiple DNA segments being greater than 14; mixing the multiple DNA segments with type II restriction enzymes, DNA ligases, and a TALE backbone vector to generate the polynucleotide encoding the TALE, the multiple DNA segments being assembled in a single cloning reaction; and purifying the polynucleotide using plasmid-safe Deoxyribonucleases (DNases).