Purification and Sequencing of DNA Guides from Prokaryotic Argonaute

Abstract

Some proteins utilize nucleic acids to guide them to complementary nucleic acid targets. One example is prokaryotic Argonaute protein, which, binds small single stranded DNA molecules as guides (Swarts et al., 2014). This protocol describes a method to purify DNA guides from these proteins. It also describes a PCR-based method to enrich the guides by PCR amplification. This methods relies on addition of a poly-A tail at the 3’-end of the ssDNA molecules by Terminal Deoxynucleotidyl Transferase (TdT), followed by ligation of a oligonucleotide to the 5’-end of the ssDNA molecule using T4 RNA ligase, and amplification by PCR. The generated dsDNA products are suitable for traditional cloning and sequencing and high-throughput sequencing. Importantly, the information which strand matches the ssDNA molecule is not lost during this process.