SPLICE: a technique for generating in vitro spliced coding sequences from genomic DNA.
We describe a rapid and cost-effective technique for the in vitro removal of introns and other unwanted regions from genomic DNA to generate a single sequence of continuous coding capacity, where tissues required for RNA extraction and complementary DNA synthesis are unavailable. Based on an overlapping fusion-PCR strategy, we name this procedure SPLICE (for swift PCR for ligating in vitro constructed exons). As proof-of-principle, we used SPLICE successfully to generate a single piece of DNA containing the coding region of a five-exon gene, the short-wavelength-sensitive 1 (SWS1) opsin gene, from genomic DNA extracted from the brown lemur, Eulemur fulvus, in only two short rounds of PCR. Where the genomic structure and sequence is known, this technique may be universally applied to any gene expressed in any organism to generate a practical unit for investigating the function of a particular gene of interest. In this report, we provide a detailed protocol, experimental considerations, and suggestions for troubleshooting.
Davies
Wayne L
WL
UCL Institute of Ophthalmology, London, UK. w.davies13@gmail.com
Carvalho
Livia S
LS
Hunt
David M
DM
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005091:Exons / Q000235:genetics*; D016678:Genome; D066298:In Vitro Techniques; D007438:Introns / Q000235:genetics*; D007900:Lemur; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D012326:RNA Splicing; D017299:Rod Opsins / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D017299:Rod Opsins