We originally became interested in the gene encoding the heat shock protein Hsp90 when studying genes involved in developmental arrest in the free-living nematode, Caenorhabditis elegans. Due to its relatively conserved sequence, Hsp90 has also been used as a phylogenetic marker in a wide range of organisms. Since there is great need for additional genes for strengthening nematode phylogeny, we sought to determine the usefulness of Hsp90 for this purpose.

Over the past several years, it has become apparent that C. elegans codon usage differs substantially from that of many plant-parasitic nematodes. To amplify the Hsp90 gene, we designed new PCR primers and cycling conditions useful for a wide variety of plant-parasitic nematode species.

PRIMER U831 U288 L1110B

F/R F F R

5'-> 3' SEQUENCE                                  AAYAARACMAAGCCNATYTGGAC GAYACVGGVATYGGNATGACYAA TCGCARTTCTCCATRTYTGGAC

PCR fully described in Skantar and Carta, 2005; modified from Skantar and Carta, 2000.

25 µl reactions:
500µM each primer
200µM dNTPs
1 unit Taq polymerase
1x Taq buffer
template ~20 ng genomic DNA or 1µl “nematode smash” extract

Cycling is a modified “step-down” PCR; instead of stepping down between cycles, there is a step down within each cycle.

1 cycle:o 45 cycleso  o  o    o 1 cycle:o

94oC 2 min 94oC 20s 65oC 5s 60oC 5s 55oC 5s 68oC 1 min 65oC 15 min

Skantar, A. M. and Carta, L. K. Multiple displacement amplification (MDA) of total genomic DNA from Meloidogyne spp. and comparison to crude DNA extracts in PCR of ITS1, 28S D2-D3 rDNA and Hsp90. Nematology 7 (2): 285-293. 2005.