DSN depletion is a simple method to remove selected transcripts from cDNA populations.
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.
Bogdanova
Ekaterina A
EA
Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia. katya@ibch.ru
Shagina
Irina A
IA
Mudrik
Elena
E
Ivanov
Igor
I
Amon
Peter
P
Vagner
Laura L
LL
Lukyanov
Sergey A
SA
Shagin
Dmitry A
DA
D000818:Animals; D033381:Anomura / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D037421:Anthozoa / Q000201:enzymology / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015723:Gene Library; D006801:Humans; D009693:Nucleic Acid Hybridization; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D010920:Placenta / Q000201:enzymology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
D018076:DNA, Complementary; D003851:Deoxyribonucleases; D010727:Phosphoric Diester Hydrolases