Techniques for Nucleic Acid Purification from Plant, Animal, and Microbial Samples

Abstract

Efficient isolation of high-quality nucleic acid from sampled material is critical for the success of downstream analyses including PCR, sequencing, and molecular cloning. There are four principles that have primarily driven the innovation in nucleic acid isolation practices: efficiency of recovery, speed of processing, ease of the procedure, and the purity of the isolated product.

Nucleic acid isolation generally occurs via a two-step process: liberation of nucleic acid through cellular lysis followed by purification of the liberated nucleic acid from the lysate. Liberation can be achieved by utilizing any combination of chemical, enzymatic, and mechanical lysis methods. Purification of nucleic acid is typically performed using liquid- and solid-phase separation techniques. Phenol–chloroform extraction has been widely considered the gold standard since 1956, however, solid-phase extraction methods exist utilizing specific resins and inorganic matrices that, in the presence of certain chemical reagents, facilitate reversible nucleic acid binding.

Isolation of nucleic acids can be challenging depending on the nature of the sample. Samples like plants and microbes possess rigid cell walls that must be ruptured to ensure efficient nucleic isolation. Soil, stool, and plant samples can also contain polyphenolic compounds that can inhibit downstream analysis. Blood, plasma/serum, saliva, milk, and other biological fluids contain high levels of protein, which can inhibit nucleic acid separation. Many microbial species are refractory to most conventional lysis techniques but must be effectively lysed to ensure unbiased, accurate analysis. Qualification criteria for the isolation and purification of nucleic acids, and the obstacles presented by animal, plant, and microbial samples are described below.