DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations

• Published in: Molecular biotechnology Vol. 41; no. 3; pp. 247 - 253
• Main Authors: Bogdanova, Ekaterina A, Shagina, Irina A, Mudrik, Elena, Ivanov, Igor, Amon, Peter, Vagner, Laura L, Lukyanov, Sergey A, Shagin, Dmitry A
• Format: Journal Article
• Language: English
• Published: New York New York : Humana Press Inc 01.03.2009; Humana Press Inc; Springer Nature B.V
• Subjects: Animals; Anomura - enzymology; Anomura - genetics; Anomura - metabolism; Anthozoa - enzymology; Anthozoa - genetics; Biochemistry; Biological Techniques; Biotechnology; Cell Biology; Chemistry; Chemistry and Materials Science; Cloning; Crustaceans; Decapoda; Deoxyribonucleases - genetics; Deoxyribonucleases - metabolism; Deoxyribonucleic acid; DNA; DNA, Complementary - genetics; DNA, Complementary - metabolism; Enzymes; Female; Gene expression; Gene Library; Human Genetics; Humans; Hybrids; Nucleic Acid Hybridization; Nucleic acids; Phosphoric Diester Hydrolases - genetics; Phosphoric Diester Hydrolases - metabolism; Placenta - enzymology; Placenta - metabolism; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Protein Science; Studies; Kamchatka crab; Expression cloning; Duplex-specific nuclease; Full-length cDNA; cDNA depletion
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-008-9131-y

A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.