Extraction and amplification of fungal DNA from compost samples

One technique for the characterization of fungal communities in compost is denaturing gradient gel electrophoresis (DGGE) of specifically amplified ribosomal DNA (rDNA). A major challenge in applying DGGE to microbial communities in compost is recovering DNA that is suitable for PCR, as interfering humic compounds are often co-purified with DNA. Thus, any DNA extraction method must be evaluated with respect to PCR inhibition. DNA was extracted from two types of autoclaved compost using BIO 101's FastDNA® SPIN® kit for soil either alone or with an added cetyltrimethylammonium bromide (CTAB) step to assist in removal of humic compounds. The purity of the extracted compost DNA was then examined by PCR amplification of pure culture fungal DNA in the presence of varying amounts of compost DNA. Three PCR primer pairs, which have been used previously for the amplification of fungal rDNA, were used to amplify 1.7 kb and 0.5 kb regions of 18S rDNA and a 0.6 kb variable region of the large subunit rDNA. The additional CTAB step in the DNA extraction procedure was crucial in obtaining DNA suitable for PCR from cured compost. Furthermore, the amount of compost DNA that inhibited PCR was dependent upon the primer pair being used. Thus, for a given primer pair and compost medium, the maximum amount of compost DNA allowable for successful PCR amplification, and therefore for DGGE analysis of fungal communities, was determined.