Error Analysis of Chemically Synthesized Polynucleotides

摘要: Two single-stranded polynucleotide constructs, 123 and 126 nucleotides in length, were chemically synthesized using standard phosphoramidite chemistry. Clonable, double-stranded DNA fragments about 100-bp long prepared from the polynucleotides by primer extension with a polymerase end-trimming two restriction endonucleases, then ligated into separate plasmids. Errors individual insert copies determined dideoxy sequencing after vivo amplification of Five ten inserts sequenced contained errors, including seven single-base-pair deletions, one four-base-pair deletion G→C transversion. The origins latter errors are unclear, but single-base deletions inconsistent polymerases; thus, most common sequence chemical synthesis mutations. Deletions likely to result incomplete capping or de-tritylation. observed error rate can become significant limiting factor appl...