Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology.
Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology.
复制标题
DOI：
10.1002/biot.202100240
复制
发表时间：
2022-03
影响因子：
4.7
通讯作者：
Parekkadan B
中科院分区：
文献类型：
--
作者：
Tosi L;Chkaiban L;Larman BH;Rosenfeld JA;Parekkadan B
为了弥合 DNA 序列与生物功能之间不断扩大的差距，我们开发了一种组装长接头单链寡核苷酸 (LASSO) 探针库的新方法，该方法能够大规模多重捕获千碱基大小的 DNA 片段，用于下游长读 DNA 测序或表达。该方法使用短 DNA 寡核苷酸（前 LASSO 探针）和质粒载体，该质粒载体通过 Cre-LoxP 分子内重组为成熟 LASSO 探针提供接头序列。该策略生成高质量的 LASSO 探针库（约 46% 的正确探针）。我们对从 LASSO 捕获 3,087 个大肠杆菌 ORF（跨度从 400 到 5,000 bp）获得的 DNA 环的捕获后 PCR 扩增进行了 NGS 分析。所有靶向 ORF 与非靶向 ORF 的中值富集度为 30 倍。对于大小高达 1kb 的 ORF，目标 ORF 富集到中值 260 倍。在这里，我们表明以这种方式获得的 LASSO 探针能够从人类总 cDNA 中捕获全长开放阅读框。此外，我们表明 LASSO 捕获特异性和灵敏度足以从总人类基因组 DNA 模板中捕获目标。该技术可用于长读长测序文库的制备和人类序列的大规模多重克隆。 LASSO 探针具有高度特异性，因为它们本质上相当于分子设计中抗体的 DNA。该探针由单链 DNA 寡核苷酸组成，具有两个与特定 DNA 靶标互补的可变臂。 LASSO 探针上的这些互补区域是根据基因组序列知识设计的，具有特异性和高亲和力。在这项工作中，我们测试了改进的 LASSO 探针的捕获稳健性和效率，这些探针是使用依赖于 Cre 重组的新颖方法组装而成的。随着来自所有生命王国的新基因组列表呈指数级增长，这种方法可以快速且廉价地生产大量基因。这种遗传文库可以发现新的生物功能，这些功能可用于药物开发、生物发酵和生物燃料工业等不同领域。
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 linker sequence for the mature LASSO probe through Cre-LoxP intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of correct probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3,087 E. coli ORFs spanning from 400- to 5,000 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, were 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. LASSO probes are highly specific in that they are essentially the DNA equivalent of an antibody in molecular design. The probe consists of a single strand DNA oligonucleotide with two variable arms that are complementary to a specific DNA target. These complementary regions on the LASSO probes are designed with the knowledge of a genome sequence, to have specificity and high affinity. In this work, we tested the capture robustness and efficiency of improved LASSO probes that were assembled with a novel methodology that relies on Cre-recombination. With an exponentially growing list of new genomes from all kingdoms of life, this method can enable the rapid and inexpensive production of large collections of genes. Such genetic libraries can enable the discovery of novel biological functions that can be exploited in different fields such as drug development, biofermentation and biofuel industry.
登录
查看更多内容
影响因子：
14.9
作者：
Kim H;Han H;Ahn J;Lee J;Cho N;Jang H;Kim H;Kwon S;Bang D
通讯作者：
Bang D
影响因子：
14.8
作者：
Gasperini M;Starita L;Shendure J
通讯作者：
Shendure J
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
4.4
作者：
Inoue F;Ahituv N
通讯作者：
Ahituv N
影响因子：
7.2
作者：
Hughes, Randall A.;Ellington, Andrew D.
通讯作者：
Ellington, Andrew D.