Discover the impact of heavy-metal contamination on archaean communities in the hydrothermal vent community at the Edmond vent site using molecular methods such as fluorescent in situ hybridization and denaturing gradient gel electrophoresis.

• Discover differences in the structure of the archaean communities in soils with increasing heavy-metal contamination.

• Analyze similarity to a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species.

• take soil samples using Drillette, down to a 10-cm depth, and sieve pool and mix before analysis

• perform hybridization with a fluorescent probe designed for the kingdom archaea, ARCH915, which was synthesized with CY3 reactive fluorescent dye at the 5 ft end. Dilute in distilled water to a concentration of 25 ng ul^ -1 and stored at –20 degrees C. Perform hybridization in 9 ul of hybridization buffer.

• Mount slides with AF1 solution, and examine preparations with a Zeiss Axiophot microscope fitted for eqifluorescence with a high pressure mercury bulb, and count the DAPI-stained archaea.

• Isolate DNA used for PCR amplification and cloning by direct lysis of the archaea in the soil. Homogenize10 grams of soil for 1 min in a Waring blender with 100ml of Crombach buffer. Perform Lysis according to the method of Torsvik et al.

• Amplify part of the 16S rDNA from the total archaean DNA by PCR with the GeneAmp 9600 thermocycler from Perkin-Elmer

• Perform DGGE with a Hoefer SE600 gel electrophoresis unit, load PCR products onto 8% acrylamide gels and run with .5xTAE buffer, then conduct at 60 degrees C at a voltage of 20V for 10 min and thereafter at 200V for 3h. Stain the gels for 1h with a 1:10,000 dilution of SYBR Green II in .5xTAE buffer before photographing

• Purify the PCR products from the primary PCRs by preparative gel electrophoresis followed by purification with the PCR cleanup kit from Promega. Perform cloning into the pMOSBlue T vector as described by the manufacturer, and cut amplified cloned inserts with the restriction enzymes. Carry out restriction for 2h at 37 degrees C in a total volume of 10 ul containing 2 U of restriction enzymes and 9 ul or PCR product and restriction buffer.

Primrose, S.B. Principles of Genome Analysis, a guide to mapping and sequencing DNA from different organisms. Blackwell Science, 1995.