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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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  • Lamia Chkaiban,
  • H. Benjamin Larman,
  • Jeffrey Rosenfeld,
  • Biju Parekkadan
Rutgers The State University of New Jersey

Corresponding Author:[email protected]

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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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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.
07 May 2021Submitted to Biotechnology Journal
07 May 2021Submission Checks Completed
07 May 2021Assigned to Editor
11 May 2021Reviewer(s) Assigned
01 Jul 2021Editorial Decision: Revise Major
29 Sep 20211st Revision Received
30 Sep 2021Assigned to Editor
30 Sep 2021Submission Checks Completed
30 Sep 2021Reviewer(s) Assigned
29 Oct 2021Editorial Decision: Revise Minor
05 Nov 20212nd Revision Received
05 Nov 2021Submission Checks Completed
05 Nov 2021Assigned to Editor
05 Nov 2021Reviewer(s) Assigned
05 Nov 2021Editorial Decision: Accept
Feb 2022Published in Biotechnology Journal volume 17 issue 2 on pages 2100240. 10.1002/biot.202100240