Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes

T-RFLP clone characterization (screening) was optimized for a fast and base pair-accurate characterization of clones from marine Archaea collected from the Eastern Mediterranean Sea. Because of the high sensitivity of T-RFLP f ingerprinting, a protocol was developed where 10 initial PCR cycles gave de tectable terminal fragments from clones. Additionally, forward and reverse primers for PCR were individually labeled and detected simultaneously to as sess the suitability of the forward and reverse fragments for T-RFLP screen ing. Based on independent restriction digests with the tetrameric restricti on enzymes HhaI, RsaI and HaeIII to characterize the 49 archaeal clones in our library, the clones were grouped into 13 T-RFLP operational taxonomic u nits (OTUs). Reverse fragments generally gave less heterogeneous fragments in size. The accuracy of T-RFLP screening was evaluated by sequencing repre sentative clones. Closely related clones (approximate to 97% similarity) co uld only be resolved with multiple restriction digests where forward and re verse fragments were included in the analysis. All fragments from the clone library were detected in the T-RFLP fingerprint from the complex archaeal community. We found representatives of marine group I, II and III Archaea. Thus, the recently discovered low abundant marine group III Archaea could b e clearly differentiated from the other clones in our library and comprised a considerable fraction of the clone library (approximate to 12%). Therefo re, our T-RFLP screening approach proved successful in characterizing novel archaeal sequences from the marine environment. (C) 2001 Published by Else vier Science B.V.