DNA barcoding is a strong species identification tool for all animal taxa, and can easily be conducted when materials are under DNA friendly conditions. In contract, a full-length (659 bp) sequencing has been limited for the degraded DNAs extracted from old museum specimens. The initial challenges to retrieve the authentic DNA fragments from old museum specimens were attempted by obtaining short sequences (<300 bp) with the cloning process after PCR, making it both expensive and time-consuming. In this study, we employed a modified method to analyze the full-length DNA barcoding regions in 31~52 year-old butterfly specimens (301 dried specimens of 39 species) using direct sequencing after PCR with two different methods: 1) the successful PCR rates of 0 to 5.6% using four universal primer sets were too low to obtain authentic sequences and the cross-contamination was detected in almost all successful amplicons; 2) the success rates of PCR using specie-specific overlapping primer sets were distinctly high, reaching up to 75% with 98% authentic and 2% non-specific sequences. Thus, the result showed the method that using species-specific primer set per species yields the most effective success rates of both PCR and sequencing from degraded DNA without incorrect sequences.

In DNA barcoding, the DNA degradation of old museum specimens has been limited full-length (658bp) sequencing. The challenges associated with the retrieval and authentication of degraded DNA extracts from fossil and old museum specimens were principally limited to analyze the relatively short sequences (<300 bp). Furthermore, almost protocols in other to analyzed the degraded DNA contained the cloning process after PCR causing the time-consuming and the rising costs. To overcome these problematic circumstances, we tried a modified method to analyze full-length of DNA barcoding region in 30~60 year-old butterfly specimens (225 samples in 28 species), using direct sequencing after PCR with species-specific overlapping primer sets per each species. As a result, all of 28 species have been successfully analyzed, although 178 samples (79%) are completely generated barcoding sequences ranged from 640 to 658 bp and 47 samples (21%) are partially sequenced ranged from 100 to 500 bp. Thus, the result showed that the direct PCR sequencing using the overlapping primer sets per species appears to have great potential efficiency for analysis of degraded DNA without incorrect sequences.