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-speci...
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| Veröffentlicht in: | Molecular biotechnology 2009-03, Vol.41 (3), p.247-253 |
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| creator | Bogdanova, Ekaterina A Shagina, Irina A Mudrik, Elena Ivanov, Igor Amon, Peter Vagner, Laura L Lukyanov, Sergey A Shagin, Dmitry A |
| description | 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. |
| doi_str_mv | 10.1007/s12033-008-9131-y |
| format | Article |
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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. 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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.</description><subject>Animals</subject><subject>Anomura - enzymology</subject><subject>Anomura - genetics</subject><subject>Anomura - metabolism</subject><subject>Anthozoa - enzymology</subject><subject>Anthozoa - genetics</subject><subject>Biochemistry</subject><subject>Biological Techniques</subject><subject>Biotechnology</subject><subject>Cell Biology</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Cloning</subject><subject>Crustaceans</subject><subject>Decapoda</subject><subject>Deoxyribonucleases - genetics</subject><subject>Deoxyribonucleases - metabolism</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA, Complementary - genetics</subject><subject>DNA, Complementary - metabolism</subject><subject>Enzymes</subject><subject>Female</subject><subject>Gene expression</subject><subject>Gene Library</subject><subject>Human Genetics</subject><subject>Humans</subject><subject>Hybrids</subject><subject>Nucleic Acid Hybridization</subject><subject>Nucleic acids</subject><subject>Phosphoric Diester Hydrolases - 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Academic</collection><jtitle>Molecular biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bogdanova, Ekaterina A</au><au>Shagina, Irina A</au><au>Mudrik, Elena</au><au>Ivanov, Igor</au><au>Amon, Peter</au><au>Vagner, Laura L</au><au>Lukyanov, Sergey A</au><au>Shagin, Dmitry A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations</atitle><jtitle>Molecular biotechnology</jtitle><stitle>Mol Biotechnol</stitle><addtitle>Mol Biotechnol</addtitle><date>2009-03-01</date><risdate>2009</risdate><volume>41</volume><issue>3</issue><spage>247</spage><epage>253</epage><pages>247-253</pages><issn>1073-6085</issn><eissn>1559-0305</eissn><abstract>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.</abstract><cop>New York</cop><pub>New York : Humana Press Inc</pub><pmid>19127453</pmid><doi>10.1007/s12033-008-9131-y</doi><tpages>7</tpages></addata></record> |
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| 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 |
| title | DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations |
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