Bigdye technology

DNA was extracted from ethanol preserved tissue samples using a standard CTAB-chloroform/isoamylalcohol method. Novel microsatellite markers specific to H. trunculus were developed by construction of a microsatellite-enriched genomic library following methods outlined in refs28 (link)29 (link).
Individuals collected at two times from each of the three sites (temporal sample 1: n = 48 per site; temporal sample 2: n = 28–30 per site) were genotyped at four microsatellite loci (GenBank KJ765703-KJ765706) by electrophoresis of PCR products on an ABI3500 automated sequencer (Applied Biosystems, Foster City, California, USA), with allele sizes determined using GENEMAPPER30 (link). Mitochondrial DNA (mtDNA) was assessed by PCR amplification and sequencing of a 602bp fragment of the cytochrome oxidase I gene, which was amplified using species-specific primers HtrucF (5′-ATATGGTCAGGGCTTGTTGG-3′) and HtrucR (5′-CCTGCAGGATCAAAGAACG-3′) designed from GenBank sequences (EU391577). Sequencing was performed with the PCR primers using BigDye technology (Applied Biosystems). Sequences were edited, aligned and trimmed to a standard length (532bp) in BIOEDIT31 .

The PCR products with abnormal bands (polymorphisms) in the electrophoretic pattern of the SSCP were sent to Ruralex Fagos (Buenos Aires, Argentina) for sequencing. The sequencing was performed using the dideoxy method using the terminal chain method and Big Dye^® technology (Applied Biosystems, Foster City, CA, USA). The primers used for sequencing were the same as those used for the PCR reaction. The cycling conditions were as follows: 96°C for 5 min; 35 cycles of 30 sec at 94°C, 30 sec at 51°C and 4 min at 60°C; followed by a cycle of 10 min at 60°C. The amplification products were purified using a protocol based on MgCl₂/ethanol and run on an ABI 310 genetic analyzer (Applied Biosystems). The results were analyzed using ABI PRISM^® 3100-Avant and 3100 Data Collection v2.0 software (Applied Biosystems).