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.
Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology