Recent developments in DNA synthesis and sequencing allowed the construction of comprehensive gene variant libraries and their functional analysis. Achieving high-replication and thorough mutation characterization remains technically and financially challenging for long genes. Here, we developed an efficient, affordable, and scalable library construction approach that relies on low-cost DNA synthesis and standard cloning technologies, which will increase accessibility to mutational studies and help advance the field of protein science. Each degenerate codon variant is physically associated with multiple DNA barcodes during synthesis, which overcomes the need for long-read sequencing for linking variants to barcodes. We demonstrate the scalability of our approach by constructing a complete library for PDR1 , a 3.2 kb multidrug resistance gene encoding a pleiotropic transcription factor in the yeast Saccharomyces cerevisiae . We demonstrate a near-perfect correspondence in the measurement of amino acid variants impact when assessed by barcode sequencing and direct sequencing of the mutated coding sequence.
Jann et al. (Wed,) studied this question.
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