Recovery of upstream cDNA sequences by a PCR-based biotin-capture method.
The world-wide, large-scale sequencing efforts have generated an abundance of partial cDNA sequences, i.e., expressed sequence tags (ESTs), accessible in the public databases. To enable functional characterization of these partial cDNA sequences, general and robust methods for recovery of upstream full-coding cDNA sequences are needed. Here, a novel biotin- and PCR-assisted capture method was used directly on poly(A)+ RNA for the purpose of generating a full-coding sequence of a gene with only partially known sequence and for which a full-length clone of the gene was not found in existing cDNA libraries. The presented method involves linear extension by reverse transciptase from a biotinylated primer annealing in a region with known sequence. After capture of the generated single-stranded cDNA onto paramagnetic beads, unspecifically annealing primers, i.e., arbitrary primers, were used to generate cDNA fragments that could be amplified by PCR and thereafter directly sequenced without subcloning. By using the presented strategy, which is to be seen as a complement to rapid amplification of cDNA ends (RACE)-related methods, we were able to recover full-coding sequence versions of two potential splice variants of the target gene. The general applicability of the novel method for recovery and sequencing of cDNA sequences is discussed.
Gräslund
S
S
Kungliga Tekniska Högskolan, Stockholm, Sweden.
Larsson
M
M
Sterky
F
F
Uhlén
M
M
Lundeberg
J
J
Höög
C
C
Ståhl
S
S
D000818:Animals; D001710:Biotin; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis* / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D014644:Genetic Variation; D008280:Magnetics; D008297:Male; D051379:Mice; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods*; D012326:RNA Splicing; D012194:RNA-Directed DNA Polymerase; D017422:Sequence Analysis, DNA; D019809:Streptavidin; D013737:Testis / Q000737:chemistry
D017931:DNA Primers; D018076:DNA, Complementary; D001710:Biotin; D019809:Streptavidin; D012194:RNA-Directed DNA Polymerase