A simple method for obtaining DNA suitable for RAPD analysis from Azospirillum

PEDRAZA, RAÚL OSVALDO; DIAZ RICCI, JUAN CARLOS

Environmental Microbiology- Methods and Protocols

Humana Press

Lugar: New Jersey, USA; Año: 2004; p. 151 - 156

The Random Amplified Polymorphic DNA (RAPD) technique has increasingly been used in the last decade as a simple, low cost and time effective technique for the analysis of genomic polymorphism among related organisms. This method proved to be particularly useful in genomic fingerprinting and phylogenetic studies of microorganisms that present taxonomic problems or a large diversity within the taxon such as clinical and soil microorganisms (1-5). However, this technique presents persistent problems of reproducibility that have been attributed to a high sensitivity to subtle procedure changes and to the quality of the DNA used as template (6-8). For this reason, other techniques more time and budget demanding such as RFLP (Restriction Fragments Length Polymorphism) and AFLP (Amplified Fragment Length Polymorphism) have been used as valid and more reliable alternatives to assess genomic diversity (9, 10). Although DNA fingerprints obtained by RFLP and AFLP yield excellent results, often money limitation indicates that RAPD is the most convenient technique to use. Additionally, since the RAPD method is much faster and simpler than RFLP and AFLP, it is also well suited when large number of specimens or microbial isolates that require previous laborious manipulations have to be analyzed. Thus, any procedural simplification of tedious and time consuming steps are welcome, provided that the information quality does not decrease due to technical artifacts or loss of reproducibility; the latter is precisely the most sensitive issue of the RAPD technique attributed mainly to DNA quality. Hence, obtaining the DNA in such a quality that can be directly used in RAPD experiments is the most critical and time-consuming step required in this technique and has to be carefully adjusted and optimized for every case under study.