title: Normalization of full-length-enriched cDNA.
abstract: A well-recognized obstacle to efficient high-throughput analysis of cDNA libraries is the differential abundance of various transcripts in any particular cell type. Decreasing the prevalence of clones representing abundant transcripts before sequencing, using cDNA normalization, may significantly increase the efficacy of random sequencing and is essential for rare gene discovery. Duplex-specific nuclease (DSN) normalization allows the generation of normalized full-length-enriched cDNA libraries to permit a high gene discovery rate. The method is based on the unique properties of DSN from the Kamchatka crab and involves denaturation-reassociation of cDNA, degradation of the ds-fraction formed by abundant transcripts by DSN, and PCR amplification of the remaining ss-DNA fraction. The method has been evaluated in various plant and animal models.
authors.affiliation: Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia. katya@ibch.ru
authors.forename: Ekaterina A
authors.initials: EA
authors.lastname: Bogdanova
authors.forename: Ekaterina V
authors.initials: EV
authors.lastname: Barsova
authors.forename: Irina A
authors.initials: IA
authors.lastname: Shagina
authors.forename: Alexander
authors.initials: A
authors.lastname: Scheglov
authors.forename: Veronika
authors.initials: V
authors.lastname: Anisimova
authors.forename: Laura L
authors.initials: LL
authors.lastname: Vagner
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Lukyanov
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Shagin
mesh_terms: D000818:Animals; D033381:Anomura / Q000201:enzymology / Q000235:genetics; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics; D003854:Deoxyribonucleotides / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004720:Endonucleases / Q000378:metabolism; D015723:Gene Library; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis
chemical_list: D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D003854:Deoxyribonucleotides; D004720:Endonucleases