Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers.
Anal Biochem
; 416(1): 141-3, 2011 Sep 01.
Article
en En
| MEDLINE
| ID: mdl-21530478
ABSTRACT
Directional cloning of complementary DNA (cDNA) primed by oligo(dT) is commonly achieved by appending a restriction site to the primer, whereas the second strand is synthesized through the combined action of RNase H and Escherichia coli DNA polymerase I (PolI). Although random primers provide more uniform and complete coverage, directional cloning with the same strategy is highly inefficient. We report that phosphorothioate linkages protect the tail sequence appended to random primers from the 5'â3' exonuclease activity of PolI. We present a simple strategy for constructing a random-primed cDNA library using the efficient, size-independent, and seamless In-Fusion cloning method instead of restriction enzymes.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Fosfatos
/
Biblioteca de Genes
/
ADN Complementario
/
Cartilla de ADN
Tipo de estudio:
Clinical_trials
Idioma:
En
Revista:
Anal Biochem
Año:
2011
Tipo del documento:
Article
País de afiliación:
Singapur