Assembly of Long-Adapter Single-Strand Oligonucleotide (LASSO) Probes for Massively Parallel Capture of Kilobase Size DNA Targets.
Assembly of Long-Adapter Single-Strand Oligonucleotide (LASSO) Probes for Massively Parallel Capture of Kilobase Size DNA Targets.
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DOI：
10.1002/cpz1.278
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发表时间：
2021-11
期刊：
影响因子：
--
通讯作者：
Parekkadan B
中科院分区：
文献类型：
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作者：
Chkaiban L;Tosi L;Parekkadan B
基因组DNA测序已经成为解决生命遗传背景的一种负担得起的商品。然而，DNA编码序列的生物学功能仍然相对未知。因此，从基因组模板中选择天然DNA靶标的高度可扩展且具有成本效益的克隆方法是快速了解基因组生物产物的迫切需要。LASSO探针是长ssDNA寡核苷酸，其设计有连接在与感兴趣的基因组靶标互补的两个序列之间的通用衔接子。通过合并组装方法，可以制备LASSO用于多重DNA捕获。在本文中，我们描述了一个强大的和有效的方法，使用Cre重组酶介导的反应，以及用于多重基因组靶捕获的协议的LASSO探针库的组装。起始组分是一个pre-LASSO探针文库，它是在计算机上设计的短DNA寡聚物池和一个E。大肠杆菌质粒（pLASSO），其并入了pre-LASSO文库。通过pLASSO与其插入片段的内部重组，可以制备高纯度的最终构型的成熟LASSO文库。LASSO探针库的组装需要四天时间，而靶捕获可以在一天内完成。随着新基因组列表呈指数级增长，这种方法可以快速生产ORFeome库，用于高通量筛选，以鉴定生物学功能，作为了解基因组功能生物学的补充方法。
Genome DNA sequencing has become an affordable commodity to resolve the genetic background of life. However, the biologic functions of DNA encoded sequences are still relatively unknown. A highly scalable and cost-effective cloning method for selecting natural DNA targets from genomic templates is, therefore, of urgent need to rapidly understand biological products of genomes. LASSO probes are long ssDNA oligonucleotides that are designed with a universal adapter that is linked between 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 and efficient method for the assembly of LASSO probes libraries using a Cre-recombinase-mediated reaction as well as a protocol for multiplex genome target capture. The starting components are a pre-LASSO probe library that are short DNA oligo pools designed in silico and an E. 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 four days and target capture can be performed in a single day. With an exponentially growing list of new genomes, this method can enable the rapid production of ORFeome libraries for high-throughput screening to identify biological functions as a complimentary approach to understand genome functional biology.