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.
One of the most extraordinary achievements in molecular biology of the 20th century has been the advance in the knowledge of genome structure and function, such as the discovery of the double helical structure of DNA (1) and the sequencing of the human genome (2,3). Despite the immense significance these discoveries have played in understanding the evolution of genes and the underlying molecular basis for a myriad of human disorders, the key to understanding the mechanisms of physiological structure-function relationships of genes and their role in pathological disease remains at the level of RNA and protein. For example, recent work on the cystic fibrosis transmembrane conductance regulator (CFTR) gene identified transcriptional and posttranscriptional mechanisms of regulating transcript production and stability and their concomitant effects on translation in physiological and pathological conditions; information that could only have arisen from investigating both RNA and protein by in vivo and in vitro assays (4,5). Thus, scientific investigation at the level of the transcriptome and proteome is the next most significant challenge facing researchers today. However, one of the difficulties in achieving this goal for many species is the inability to obtain tissue for RNA or protein work. Various reasons may exist for this—some species are highly protected (e.g., the platypus); some tissue-specific genes are only expressed in tissues that are extremely difficult to obtain by noninvasive biopsy; and some tissues may be so degraded that RNA and protein are no longer viable (e.g., ancient tissues from preserved specimens). In all the above cases, only genomic DNA (gDNA) may be available to scientific researchers.
Since most eukaryotic genes contain noncoding introns that interrupt the flow of protein coding information present in exons, achieving the knowledge of functionality encoded at the level of mRNA and protein requires the laborious removal of all introns prior to any experimental procedure. A small number of earlier studies have attempted to address this issue with some limited success (6,7). Here, we report an efficient technique for the in vitro removal of multiple introns or any other regions of gDNA. Based on an overlapping fusion-PCR strategy, we name this technique SPLICE, for swift PCR for ligating in vitro constructed exons. This relatively simple and efficient technique results in the generation of a DNA fragment containing the coding information of any gene from the gDNA of any species that can be subsequently used by downstream procedures to assay a particular aspect of a gene's function.
As proof-of-principle, we tested the ability of the SPLICE technique to generate a single continuous piece of double-stranded coding DNA for the short-wavelength-sensitive 1 (SWS1) opsin gene from gDNA of the brown lemur, Eulemur fulvus (8).Materials and Methods
A schematic overview of the SPLICE strategy is shown in (Figure 1). A detailed account of all reagents, equipment, as well as a step-by-step guide for the SPLICE technique used in this report may be found in the Supplementary Material (available online at www.BioTechniques.com). The Supplementary Material also contains an in-depth troubleshooting guide to maximize the success of the SPLICE technique when applied to a particular gene of interest.Figure 1.
Prior to primer design, it is important to establish the boundaries for each DNA segment to be amplified (see Supplementary Material). In our report, a set of primers ((Table 1)) was designed to encompass exons to be amplified similar to that outlined in (Figure 2) and (Figure 3). All internal primers were 30 nucleotides in length to ensure specific binding to the target region of gDNA and encompass an exon-exon boundary or a fusion point between the two DNA sequences to be united. For forward internal primers, the first half of the oligonucleotide sequence corresponded to the last 15 nucleotides of an upstream exon, with the second half being identical to the first 15 nucleotides of a downstream exon. The reverse internal primers comprised the reverse complement sequence of the forward primer. In all cases, the fusion point between the two DNA sequences being joined was located at the center of each internal oligonucleotide. Depending on the requirements of the downstream assay, modifications may be included in some of the primers at this design stage. In this study, oligonucleotides were designed with the inclusion of restriction enzyme recognition sites in the two outer (sense and antisense) primers for ease of cloning, the addition of a Kozak consensus sequence upstream of the translation initiation codon in the outer forward primer to ensure efficient protein production in a downstream assay, and replacement of the translation termination codon with partial linker sequence that is inframe with a downstream reporter gene of interest.