Genome DNA sequencing has become an affordable means to resolve questions about the genetic background of life. However, the biological functions of many DNA-encoded sequences are still relatively unknown. A highly scalable and cost-effective cloning method to select natural DNA targets from genomic templates is therefore urgently needed to enable rapid understanding of the biological products of genomes. One such method involves LASSO probes, which are long single-stranded DNA oligonucleotides designed with a universal adapter that is used to link two sequences that are complementary to a genomic target of interest. Through a pooled assembly method, LASSOs can be made for multiplex DNA capture. Herein, we describe a robust, efficient method to assemble LASSO probe libraries using a Cre-recombinase-mediated reaction and a protocol for multiplex genome target capture. The starting components are a pre-LASSO probe library comprising short DNA oligo pools designed in silico and an Escherichia coli plasmid (pLASSO) that incorporates the pre-LASSO library. Through internal recombination of pLASSO with its inserts, a mature LASSO library in final configuration can be made with high purity. Assembly of a LASSO probe library takes 4 days, and target capture can be performed in a single day. With an exponentially growing list of new genomes available for investigation, this method can enable the rapid production of ORFeome libraries for high-throughput screening to identify biological functions as a complementary approach to understand genome functional biology.
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.
High throughput techniques that can massively produce in parallel, longer DNA sequences of interest can accelerate the decoding of gene functions. LASSO probes are a molecular biology tool that can enrich for DNA targets in a genomic sample via a multiplexed, single-pot reaction for downstream sequencing and/or cloning. Here we have explored aspects of process development and the design of the probes that relate to binding thermodynamics to determine impact on cloned library sequences. Control of ligase concentration, polymerase type, and melting temperature of probe are critical when translating the use of LASSO probes for homogeneous and high fidelity DNA capture.
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