Normalization of full-length-enriched cDNA.
Methods Mol Biol
; 729: 85-98, 2011.
Article
em En
| MEDLINE
| ID: mdl-21365485
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Biblioteca Gênica
/
DNA Complementar
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article