Despite its usefulness for highly multiplex PCR based enrichment 3, this technique cannot be used for the amplification of full length DNA sequences because it relies on the presence of specific restriction enzymes sites in the target sequences. The parallel amplification of hundreds of DNA targets (~200 base pairs bp) in a single reaction using short selector oligonucleotides (~70bp) that act as a template for the circularization of specific target sequences has been shown 2. Traditional mutliplexed polymerase chain reaction (PCR) is generally not feasible for this purpose, due to the unpredictable interactions among large numbers of primer sets. Highly multiplexed cloning of long target sequences has not been previously demonstrated. Massively parallel technologies enabling the synthesis and cloning of long DNA sequences are therefore required to bridge the widening gap from sequence to significance. Consequently, a bottleneck has formed between our knowledge of DNA sequences and our understanding of their functional significance. synthesizing or cloning sequences of interest). With dramatic advances in parallelization, ‘reading’ DNA sequences has now become orders of magnitude more efficient and cost-effective than ‘writing’ them (i.e. LASSO probes could be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning.ĭNA sequencing has created an unprecedented wealth of biological information 1. We also show that LASSO probes can clone human ORFs from complementary DNA, and an ORF library from a human-microbiome sample. At a cutoff of 3 times the median non-target reads per kilobase of genetic element per million reads, ~75% of the targeted ORFs were successfully captured. Targets were enriched up to a median of ~60-fold compared to non-targeted genomic regions. coli genomic DNA (spanning 400–5,000 bp targets). As a proof-of-principle, we simultaneously cloned >3,000 bacterial open reading frames (ORFs) from E. Here, we show that long-adapter single-strand oligonucleotide (LASSO) probes can capture and clone thousands of kilobase DNA fragments in a single reaction. Current strategies to synthesize or clone complex libraries of DNA sequences are limited by the length of the DNA targets, throughput and cost. As the catalogue of sequenced genomes and metagenomes continues to grow, massively parallel approaches for the comprehensive and functional analysis of gene products and regulatory elements are becoming increasingly valuable.
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