Abstract
We present an overhang-based DNA block shuffling method to create a customized random DNA library with flexible sequence design and length. Our method enables the efficient and seamless assembly of short DNA blocks with dinucleotide overhangs through a simple ligation process. Next generation sequencing analysis of the assembled DNA library revealed that ligation was accurate, directional and unbiased. This straightforward DNA assembly method should fulfill the versatile needs of both in vivo and in vitro functional screening of random peptides and RNA created with a desired amino acid and nucleotide composition, as well as making highly repetitive gene constructs that are difficult to synthesize de novo.
Highlights
We present an overhang-based DNA block shuffling method to create a customized random DNA library with flexible sequence design and length
Generation sequencing analysis of the assembled DNA library revealed that ligation was accurate, directional and unbiased. This straightforward DNA assembly method should fulfill the versatile needs of both in vivo and in vitro functional screening of random peptides and RNA created with a desired amino acid and nucleotide composition, as well as making highly repetitive gene constructs that are difficult to synthesize de novo
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]
Summary
An overhang-based DNA block shuffling method for creating a customized random library. 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 The flexibility in both sequence design and length allows versatile downstream in vitro and in vivo analyses (Fig. 1a). By constructing a customized random DNA library that encode proteins consists of only ten amino acids, we achieve to demonstrate accurate, directional and unbiased ligation properties of our novel overhang-based DNA block shuffling method. Our approach maintains tight control over the amino acid representation in the library compared to the traditional poly-NNK method (Fig. 3) This method provides great flexibly for users to design original ‘‘DNA blocks’’ based on different applications (i.e., cell barcoding, ribozyme and peptide aptamer screening, constructing de novo proteins consist of limiting amino acid variation, etc.). Immediate applications could be the study of various repeat expansion disorders[13], as well as the production of industrially and biomedically important proteins with repetitive segments[14] such as spidroin (spider silk protein) and elastin-like proteins
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