Simple and cost-effective restriction endonuclease analysis of human adenoviruses

Abstract

Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0 μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0 μg among the HAdV-3 (n=73) isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated.

Figure 1

Typical HAdV CPE. (a) Noninfected A549 cells, (b) A549 cells infected with HAdV-1, (c) A549 cells infected with HAdV-3, (d) A549 cells infected with HAdV-4, and (e) A549 cells infected with HAdV-37.

Figure 2

Picture of the REA. (a) REA pattern for BamHI and SmaI (fast digest). (b) REA pattern for BglII and HindIII. M shows Lambda DNA-HindIII digest marker. Numbers 1, 3, 4, and 37 show HAdV-1, -3, -4, and -37, respectively.