![]() ![]() We further extracted 449,748 DNA block sequences containing adapter sequences on both ends to validate the quality of concatemers that will be used in the downstream in vitro and in vivo analyses. Remaining 1,332,848 reads had an average Phred quality score peak around Q27 (99.8% accuracy), while quality across all bases have shown deterioration toward the end of the read ( Supplementary Fig. Out of 1,396,867 raw sequence reads, 64,019 sequences containing more than one unknown (N) base were discarded and low-quality ends of the reads were trimmed. PCR products were further processed using TruSeq DNA kit and was sequenced by Illumina Miseq with 150 bp paired-end output (Illumina, San Diego, CA, USA). The assembled gene library was further PCR amplified to extract and concentrate gene constructs that harbor 5′ and 3′ PCR adapters for downstream cloning. In order to create a combinatorial gene library, DNA blocks were phosphorylated, mixed with 5′ and 3′ dsDNA adapter sequences containing restriction sites and ligated in one-pot reaction ( Supplementary Fig. Degenerate oligonucleotides were designed by following the binary pattern rule 11 to code for different hexapeptide secondary structures and were annealed to create dsDNA with AG-TC overhangs ( Supplementary Table 2). These oligonucleotides encoded hexapeptides consisting of only ten amino acids: Ala, Asp, Glu, Gly, Ile, Leu, Pro, Ser, Thr and Val. In order to evaluate the performance of our assembly method, a total of 168 degenerate oligonucleotide pairs corresponding to 8288 DNA blocks were prepared. Ligation products were visually confirmed up to 1 kbp in length ( Fig. We also tested various oligo lengths ranging from 12 bp to 30 bp with overhang sequence fixed to an AG-TC dinucleotide pair. As anticipated, a single nucleotide A-T overhang failed to generate long ligation products, while a G-C pair resulted in concatemers of up to 13 blocks, similar to the efficiency of dinucleotide overhangs. Based on a visual examination of the electrophoretic analysis, all five dinucleotide overhang pairs displayed ladder-like ligated concatemers of up to 16 blocks and longer ( Fig. Palindromic dinucleotide pairs (i.e., GC-CG, AT-TA) were excluded to maintain directional ligation. We first designed 16 bp DNA blocks with various nucleotide overhangs and tested their efficiency during ligation. The flexibility in both sequence design and length allows versatile downstream in vitro and in vivo analyses ( Fig. In order to simplify the procedure and minimize the errors, we have developed an overhang-based DNA block shuffling method for combinatorial DNA library construction. Recent advances in ink-jet–based microarray printing can offer millions of DNA sequences up to 200 bp in length at a reasonable cost 10, but this method requires specialized instrumentation and the quantity and length of the constructs are insufficient for the functional assays that deal with sequences on the order of 10 10 to 10 13. Furthermore each additional PCR step increases mutation error, workload and cost of library preparation 9. Homologous recombination with PCR-based extension is another common assembly method but requires the preparation of long overlapping dsDNA or oligonucleotides that limits the flexibility of sequence design. Standard restriction enzyme leaves a scar sequence between each sequence, resulting in one or more fixed amino acids at every ligation site. The ordered assembly of multiple DNA building blocks gives flexibility in length but requires a successive restriction enzyme digestion and ligation 8. Traditional site saturation approaches using random poly-NNK (K: G or T) libraries avoids two out of the three stop codons while covering all twenty amino acid codons, but the length of the randomized region is short and a fixed length 7. Most of these are due to the biased representation of codons as well as the formation of unexpected restriction sites. Several methods have been developed for creating combinatorial DNA libraries with high complexity, but each method has drawbacks. As the complexity of a library increases, it remains expensive to fully synthesize each DNA sequence. Creating an accurate and flexible combinatorial DNA library is another key milestone for synthetic biology, given the need for screening functional DNA/RNA elements and protein aptamers using various in vitro selection methods 2, 3, 4, as well as in vivo studies on genomic sequence variants 5 and DNA barcoding of individual cells 6. Synthetic biologists have developed various DNA synthesis and assembly methods to generate large DNA constructs that can reach the size of a whole bacterial genome with high precision 1.
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