Revista de investigación forense

Assessing Tissue Lysis with Sodium Dodecyl Sulphate for DNA Extraction from Frozen Animal Tissue

Abstract

Mobolaji Temitope Akinwole and Isaac Adeyemi Babarinde

Background: Many molecular biology experiments start with DNA extraction. The quality and the quantity of the extracted DNA are very important for downstream analyses. Therefore, the DNA extraction process is very important and efforts should be aimed at constant improvement of the available protocols.

Objective: The first objective of this study is to compare various lysing agents that use Sodium Dodecyl Sulphate (SDS) in DNA extraction process. The second objective is to report detailed protocol for the extraction of DNA from frozen animal sample.

Methods: The efficiencies of four different lysing reagents involving SDS and a commercial lysing agent were compared. The reagents tested were ethylenediaminetetraacetic acid (EDTA), Sodium Chloride-Tris-EDTA (STE), Sodium chloride (NaCl) and tris acid (Tris). The four lysing agents investigated were SDS+EDTA+NaCl (SEN), SDS+EDTA+Tris (SET), SDS+STE+NaCl (SSN) and SDS+STE+Tris (STT). In addition, Qiagen (Q) lysing buffer was also included. The experiments were conducted in duplicates. The five lysing reagents were used to extract DNA from homogenized muscles and bones from the sternum of juvenile Japanese giant flying squirrel that had been frozen for four years.

Results: With the protocol presented here, gel electrophoresis, nanospec, bioanalyzer and PCR showed that all the five lysing reagents were able to extract DNA from the homogenized muscles and sternum bones of frozen rodent species. Large DNA molecules could be retrieved from all the lysing reagents. However, A260/A280 shows that SSN and STT had the purest extracted DNA molecules.

Conclusion: We present a protocol that successfully extracted DNA with good integrity from frozen animal sample. The comparison of five lysing reagents showed that although all the reagents successfully extracted DNA, the purest extracted DNA came from SDS+STE+NaCl (SSN) and SDS+STE+Tris (STT). Interestingly, DNA from all the reagents was successfully amplified with PCR suggesting that the impurities may not be of significant impacts to the downstream analyses.