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Massively parallel DNA target capture using Long Adapter Single Stranded Oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology
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  • LORENZO TOSI,
  • Lamia Chkaiban,
  • H. Benjamin Larman,
  • Jeffrey Rosenfeld,
  • Biju Parekkadan
LORENZO TOSI
Rutgers The State University of New Jersey
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Lamia Chkaiban
Rutgers The State University of New Jersey
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H. Benjamin Larman
Johns Hopkins University
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Jeffrey Rosenfeld
Robert Wood Johnson Medical School Department of Neuroscience and Cell Biology
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Biju Parekkadan
Rutgers The State University of New Jersey
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Abstract

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the backbone for the mature LASSO probe through Cre-Loxp intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 E.coli ORFs spanning from 400- to 4,000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, are able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

Peer review status:UNDER REVIEW

07 May 2021Submitted to Biotechnology Journal
07 May 2021Assigned to Editor
07 May 2021Submission Checks Completed
11 May 2021Reviewer(s) Assigned