Open AccessA Colony Multiplex Quantitative PCR-Based 3S3DBC Method and Variations of It for Screening DNA Libraries
Author(s) -
Yang An,
Atsushi Toyoda,
Chen Zhao,
Asao Fujiyama,
Kiyokazu Agata
Publication year - 2015
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0116997
Subject(s) - genomic library , fosmid , library , genomic dna , multiplex , computational biology , biology , sequencing by ligation , dna , dna nanoball sequencing , insert (composites) , genome , cloning (programming) , molecular cloning , genetics , microbiology and biotechnology , computer science , gene , complementary dna , base sequence , mechanical engineering , 16s ribosomal rna , engineering , programming language
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.
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