Minilibraries constructed from cDNA generated by arbitrarily primed RT-PCR: an alternative to normalized libraries for the generation of ESTs from nanogram quantities of mRNA.
The generation of expressed sequenced tags (ESTs) depends on the arbitrary selection of individual cDNA clones from libraries. The efficiency of this process reflects the clonal structure of the library used and can be significantly increased using size selected, directional, normalized cDNA libraries. This strategy, however, is not readily applicable when mRNA is limiting, as is the case in the study of complex microorganisms such as parasites, fetal tissues or tumor biopsies. We show here that the construction and systematic sequencing of minilibraries of cDNAs produced by arbitrarily primed PCR provides an alternative means of efficiently generating ESTs in situations where only nanogram quantities of RNA are available. This methodology greatly compensates for unequal message abundance, avoids the need for complex library construction, is equally applicable to the analysis of abundant or rare biological material and is ideally suited to multicenter programmes.
Dias Neto
E
E
Centro de Pesquisas René Rachou-FIOCRUZ, Belo Horizonte, Brazil.
Harrop
R
R
Correa-Oliveira
R
R
Wilson
R A
RA
Pena
S D
SD
Simpson
A J
AJ
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D016208:Databases, Factual; D015723:Gene Library; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics; D012550:Schistosoma mansoni / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D016324:Sequence Tagged Sites
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase