Insect Molecular Biology (1998) 7(1), 95±99 SHORT NOTE Preparation and puri®cation of DNA from insects for AFLP analysis A. Reineke, P. Karlovsky and C. P. W. Zebitz Institute of Phytomedicine, University of Hohenheim, Stuttgart, Germany Abstract Analysis of ampli®ed fragment length polymorphism (AFLP) has the potential to become a powerful new DNA ®ngerprinting technique for studying genetic relationships and genetic diversity in arthropods. Since DNA of high quality is a crucial prerequisite for AFLP analysis we evaluated the applicability of six protocols (one fast and four complex methods with phenol-chloroform treatments as well as one CTAB- based method) for extracting DNA from insect material and three additional DNA puri®cation steps. The most rapid DNA isolation method did not produce DNA suitable for AFLP analysis. Among four complex methods tested, two protocols resulted in compara- tively low yields of DNA that was therefore not used as template for AFLP analysis. The other two complex methods with phenol treatments and a CTAB-based DNA extraction protocol provided DNA suitable for AFLP assay. An additional puri®cation of the DNA using spermine precipitation revealed a few extra bands in an AFLP gel that were masked in unpuri®ed DNA. Therefore spermine precipitation is recom- mended for AFLP templates. Keywords: DNA extraction, ampli®ed fragment length polymorphism, insects. Introduction The analysis of genetic variation using DNA ®nger- printing techniques has become an important approach in taxonomic, population genetic and evolu- tionary studies of a variety of insect species. The most frequently used DNA markers include restriction frag- ment length polymorphisms (RFLPs) of mitochondrial or nuclear DNA, DNA ®ngerprinting of 'microsatellite' or 'minisatellite' sequences, standard polymerase chain reaction (PCR), and random ampli®ed poly- morphic DNA (RAPD) analysis of nuclear DNA. Recently, a novel DNA ®ngerprinting technique called AFLP (ampli®ed fragment length polymorphism) was introduced by Vos et al. (1995). The AFLP techni- que is based on selective ampli®cation of a subset of DNA fragments generated by restriction endonu- cleases. As compared with RFLPs and RAPDs, the method exhibits a higher resolution and information content. Further advantages over classic techniques are a very low amount of genomic DNA necessary for the analysis (a critical point with RFLP) and a good reproducibility of the results (a notorious problem with RAPD; Meunier & Grimont, 1993; Muralidharan & Wakeland, 1993). In addition, AFLP markers are co- dominant, which is important for studies on diploid organisms such as plants and insects. A crucial prerequisite for AFLP assay is the comple- teness of the digestion of DNA by restriction endo- nucleases. An incomplete restriction digestion is considered a major technical problem because even a small fraction of partial digestion fragments may result in detectable bands after ampli®cation. These bands will be interpreted as false polymorphisms, thus con- fusing the results of the analysis. Even with an excess of restriction enzymes as used in AFLP, partial diges- tion may result from a contamination of DNA with inhibitors, e.g. negatively charged polysaccharides and phenols (Lin & Kuo, 1995). Received 12 March 1997; accepted 17 July 1997. Correspondence: Annette Reineke, University of Hohenheim, Institute of Phytomedicine, Department of Entomology, D-70593 Stuttgart, Germany. e-mail: areineke@uni-hohenheim.de. # 1998 Blackwell Science Ltd 95