Abstract
A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements.
Highlights
Background next-generation sequencing (NGS) is widely used at present, and has been used to assemble many genomes, DNA libraries still have irreplaceable roles
Based on the issues described above, the final solution we devised for our Dugesia japonica fosmid library screening work was a “colony multiplex quantitative PCR-based 3S3DBC screening strategy” (Fig. 1)
We first described general considerations regarding the detection method and pooling strategy for screening a DNA library, and detailed our rapid and low-cost new screening method—a colony multiplex quantitative 3S3DBC method, which is superior to the conventional 3D screening method (S2 Table)
Summary
Next-generation sequencing (NGS) is widely used at present, and has been used to assemble many genomes, DNA libraries still have irreplaceable roles. Even if a genome can be assembled from NGS data, there will still be gaps and uncertain DNA regions that need to be confirmed; screening a DNA library and sequencing targeted clones can help to achieve gap-closure and to evaluate and correct the assembled genome. Assembly of some complicated genomes (with too many repetitive sequences or a high rate of heterozygosity or other variability) is extremely hard to accomplish by NGS alone, and sequencing of DNA libraries is still usually an indispensable method for achieving whole-genome sequencing at present [1,2]. A DNA library is still a valuable resource for work such as molecular cloning, physical mapping of genes, and comparative genomics. Library screening was PLOS ONE | DOI:10.1371/journal.pone.0116997 February 3, 2015
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