Exon amplification restriction ligation (EARL): an efficient strategy for direct sequencing of exons.

Gene polymorphisms and mutations have been correlated to different diseases and clinical syndromes. Therefore, screening for gene mutations is an appropriate approach to researching disease. The location of mutation hot spots is not known in new or rarely analyzed genes. To locate such hot spots, the screening of a complete gene sequence in a representative number of individuals is required. Sequencing the gene message (mRNA) after RT-PCR amplification does not depend on the knowledge of exon-intron boundaries. However, this approach needs an appropriate source for the isolation of fresh RNA (i.e., cells expressing the gene in a significant amount). Since the availability of intact mRNA is limited, the direct sequencing of exons after amplification from genomic DNA is the commonly used approach. Many human genes consist of exons with introns of several kilobases in size in between them, and often the exons are smaller than 200 bp. Therefore, sequencing of each single exon is a timeand cost-intensive approach, depending on the number of exons to be analyzed. In an ongoing research project, we screened the human P-selectin gene for polymorphisms. The gene consists of 17 exons, and literature data about the location of its polymorphisms is limited (2,3). Thus, we decided to screen all exons in 200 DNA samples, leading to 3400 sequencing reactions. By calculating the costs of reagents, chemicals, and plastics, the sequencing reaction was recognized as the most expensive step. Out of the 17 P-selectin exons, 14 are smaller than 200 bp, and four are even smaller than 100 bp. Sequencing such small PCR products is inefficient because the reagents are expensive. Furthermore, the read length of a sequence is mainly limited by the DNA sequencer system used and not by the sequencing reaction. Costa et al. (1) described a multiplex PCR approach followed by cycle sequencing to detect mutations in the FIX gene. The eight exons and the polyadenylation site are co-amplified within one PCR and are subsequently sequenced using one specific primer and dye terminators. The total cost for mutation screening is lower because only one PCR is required. However, each exon is sequenced separately, which does not reduce the number of sequencing reactions, the most cost-intensive step in the procedure. The standard procedure for direct sequencing of PCR products with fluorescently labeled primers and a LICOR sequencing system (LI-COR, Lincoln, NE, USA) includes re-amplification of the products using nested and tailed primers. The tails contain the binding site for standard sequencing primers, such as universal and reverse, which are then used in cycle sequencing reactions. Here, we describe an imBenchmarks