Massively parallel cloning of long target sequences has been achieved with a new kind of probe. The probe, called LASSO (for long adapter single-stranded oligonucleotide), can bind target genome regions for functional cloning and analysis. Collections of probes can be used to corral many DNA fragments in a single reaction. [Jennifer E. Fairman/Johns Hopkins University]
Massively parallel cloning of long target sequences has been achieved with a new kind of probe. The probe, called LASSO (for long adapter single-stranded oligonucleotide), can bind target genome regions for functional cloning and analysis. Collections of probes can be used to corral many DNA fragments in a single reaction. [Jennifer E. Fairman/Johns Hopkins University]

Stampeding DNA sequences can overwhelm clonehands who would like to capture not just individual sequences, but entire herds of them. The task is particularly daunting if the DNA sequences of interest include kilobase-sized fragments. Fortunately, there’s a new class of oligonucleotide probe in town. The probe, called LASSO (for long adapter single-stranded oligonucleotide), has demonstrated, in a proof-of-concept study, that it can achieve highly multiplexed cloning of long target sequences.

LASSO was developed by scientists at Johns Hopkins, Rutgers, the University of Trento, and Harvard Medical School. According to these scientists, LASSO can be used to isolate thousands of long DNA sequences at the same time, more than ever before possible. The new technology, they say, speeds up the creation of proteins, the final products of genes, and is likely to lead to far more rapid discovery of new medicines and biomarkers for scores of diseases.

LASSO’s gene-wrangling ways appeared June 26 in the journal Nature Biomedical Engineering, in an article entitled “Long-Adapter Single-Strand Oligonucleotide Probes for the Massively Multiplexed Cloning of Kilobase Genome Regions.” Besides describing how LASSO was used to corral over 3000 bacterial open reading frames (ORFs), the article suggested that LASSO could facilitate downstream sequencing or expression, helping to close the widening gap from “sequencing to significance.”

“Targets were enriched up to a median of around 60-fold compared with non-targeted genomic regions,” wrote the article’s authors. “At a cutoff of three times the median non-target reads per kilobase of genetic element per million reads, around 75% of the targeted ORFs were successfully captured.”

The article also described how LASSO probes were used to clone human ORFs from complementary DNA and to clone an ORF library from a human–microbiome sample.

Historically, figuring out what a gene does by cloning its DNA and expressing its protein was done one gene at a time. Approaches do exist, however, that can accomplish the parallel amplification of hundreds of DNA targets, provided that they are not too long. For example, a method called molecular inversion probes (MIPs) can capture hundreds of DNA targets in a single reaction, but only if the targets are no longer than about 200 bases of DNA.

With LASSO, each target gene sequence can be up to a few thousand DNA base pairs long, which is the typical size of a gene's protein-coding sequence. Also, collections of these LASSO probes can be used to grab desired DNA sequences—much like a rope lasso is used to capture cattle—but in this case thousands at a time in a single effort.

“Our goal is to make it cheap and easy for any researcher in any field to clone and express the entire set of proteins from any organism,” said Ben Larman, Ph.D., an assistant professor of pathology at the Johns Hopkins University School of Medicine and the study's co-senior author. “Until now, such a prospect was only realistic for high-powered research consortia studying model organisms like fruit flies or mice.”

Importantly, the researchers suggested in their paper, DNA sequences may be captured by LASSO in a way that permits scientists to analyze what the genes' proteins do, as demonstrated by conferring antibiotic resistance to an otherwise susceptible cell.

“We're very excited about all the potential applications for LASSO cloning,” added Dr. Larman. “Our hope is that by greatly expanding the number of proteins that can be expressed and screened in parallel, the road to interesting biology and new therapeutic biomolecules will be dramatically shortened for many researchers.”








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