A quick and effective in-house method of DNA purification from agarose gel suitable for sequencing

摘要

Sequencing of DNA fragments (e.g., ITS, 16S, 18S, particular genes, and molecular markers) is increasingly required in studies on microbial diversity, microbial genetic population and phylogeny, sequencing of alleles, and searching for SNPs, among others. The cost of obtaining these DNAs, in quantity and quality for sequencing, is high as it involves special kits to recover DNA from gel after PCR, or the cloning and purification of plasmids with commercial kits. Genetic population and other studies require the analyses of many samples, and therefore, the high cost represents an obstacle for carrying out such projects in countries where there is great biodiversity, such as the tropical and subtropical developing countries, where funds are limited. Modifying an already known method for DNA recovery from gel, the first in-house protocol of DNA recovery suitable for direct use in sequencing is presented herein. This protocol is broadly applicable on DNAs from all different living beings, e.g., bacteria, fungi, and plants. Its simplicity, speed, and low cost make this procedure amenable for high-throughput DNA sequencings as required in microbial population studies, development of molecular markers, molecular identification of strains in microbial collections, and others. Recovery of DNA fragments from agarose gel is one of the most common tasks in molecular biology laboratories. Therefore, its potential of applicability of the protocol presented here is enormous.