Exploiting formalin-preserved fish specimens for resources of DNA barcoding.

Area of Science:

• Molecular Biology
• Genetics
• Museum Science

Background:

• Museum collections contain valuable formalin-fixed specimens for DNA barcoding.
• Extracting high-quality DNA and amplifying target genes from these samples is challenging.
• Existing methods often struggle with DNA degradation and PCR inhibition.

Purpose of the Study:

• To develop an optimized DNA extraction protocol for formalin-fixed samples.
• To improve DNA quantity and quality for reliable downstream applications.
• To minimize polymerase chain reaction (PCR) inhibition in extracts from old specimens.

Main Methods:

• A hot alkali pre-treatment combined with the cetyltrimethylammonium bromide (CTAB) method was used for DNA extraction.
• A semi-nested PCR-based method was developed to enhance gene amplification efficiency.
• The protocol was tested on formalin-preserved samples of varying ages.

Main Results:

• The developed protocol successfully recovered genomic DNA from formalin-fixed samples.
• High quantity and quality of DNA were achieved, suitable for sequencing.
• The COI gene was reliably amplified and sequenced, even from 23-year-old specimens.
• The method demonstrated effectiveness in minimizing PCR inhibition.

Conclusions:

• The hot alkali pre-treatment and CTAB method, coupled with semi-nested PCR, is a reliable protocol for DNA recovery from formalin-fixed samples.
• This method significantly enhances the utility of museum collections for DNA barcoding initiatives.
• The protocol enables successful genetic analysis of even aged, challenging specimens.