Unique sequence characteristics account for good DGGE separation of almost full-length 18S rDNAs.

Abstract

A major limiting factor for DGGE-based microbial community studies is that the fragments should not be much longer than 500bp for successful analysis. However, relatively high-resolution was achieved based on DGGE of the long 18S rDNA fragment (>1500bp), which might be surprising due to the known decrease in DGGE resolution of DNA molecules with large melted regions. A unique sequence characteristic was found in a specific region (ca. 275bp, named the NS1-end region) of 18S rDNAs, and fungal communities separated from Hong Qu glutinous rice wine brewing system was used to reveal the relationship between high resolution capacity and the unique sequence characteristics. The results showed that DGGE separation of the long 18S rDNA fragments depended on their NS1-end regions. The region is composed of a sequence-variable and short-length GC-poor region (ca. 160bp) and a GC-rich region (ca. 110bp), which contribute to the high resolution capacity achieved for DGGE of the long 18S rDNA fragments. Thus DGGE of the long 18S rDNA fragment is recommended as a target fragment for studies of fungal communities whose 18S rDNAs possess similar sequence characteristics. Good resolution and almost full-length 18S rDNA sequences can thus be obtained to provide more accurate and reliable analysis of fungal communities. Since more sequences are obtained directly from the PCR product through the long rDNA fragment approach, this is a convenient and effective approach for sequence-based analysis without using other complementary methods such as an rDNA clone library method.